Bronchoalveolar lavage fluid cytokine, cytology and IgE allergen in horses with equine asthma.

The pathophysiology of equine asthma (EA) is still not fully described, but the involvement of an allergic reaction is strongly suspected. This theory has led to the use of allergen-specific immunoglobulin (Ig)E tests to support a diagnosis of asthma. The objective of this descriptive study was to evaluate the correlation between four subgroups of EA (mastocytic mild equine asthma [MEA], neutrophilic MEA, mixed MEA, and severe equine asthma [SEA]), allergen specific IgE (measured in both serum and BALF) and mRNA expression of selected genes in bronchoalveolar lavage fluid (BALF). Serum and BALF were collected from 64 horses with a history of lower airway problems with or without poor performance. Differential cell counts from BALF were used to assign horses to one of four groups (mastocytic MEA; neutrophilic MEA, mixed MEA, and SEA). The expression of messenger RNA (mRNA) coding for IL4, IL5, IL8, IL10, TGFB, TNFA, toll-like receptor (TLR)4, IL1RA, IL1B, matrix metalloproteinase 8 (MMP8), TLR9, chemokine ligand 5 (CCL5) and cluster of differentiation (CD)14 in BALF were measured using reverse transcriptase (RT) quantitative PCR (qPCR). Allergen-specific IgE was measured in serum and BALF using an allergen-specific IgE ELISA test with the screening panel: house mites, storage mites, mould and pollen. As expected, the BALF neutrophil differential count correlated with mRNA expression of MMP-8 (r = 0.611, p < 0.001), TLR-4 (r = 0.540, p < 0.001), IL-1RA (r = 0.490, p < 0.001), IL-1ß (r = 0.463, p < 0.001) and IL-8 (r = 0.302, p = 0.015). Cytokine expression of IL-1ß (p = 0.014), MMP8 (p = 0.028) and IL-1RA (p = 0.037) was significantly higher in the SEA group compared to the MEA subgroups. The BALF mast cell count was correlated with allergen-specific IgE for insects (r = 0.370, p = 0.002) and pollen (r = 0.313, p = 0.011). Eosinophils in BALF were correlated with BALF mRNA expression of IL-4 (r = 0.340, p = 0.006) together with a significant correlation between BALF eosinophils and allergen-specific IgE for mites (r = 0.930, p < 0.001) and pollen in BALF (r = 0.837, p < 0.001). No correlation was found between allergen-specific IgE in serum and BALF for any of the allergen in the screening panel. Based on these results from allergen-specific IgE in horses with EA is not found in systemic circulation, and only the mastocytic and mixed subgroups of horses with EA had allergen-specific IgE in BALF. Further studies are needed to clarify the relationships identified here.

[Immunoglobulin concentration in equine colostrum and blood of newborn foals as well as clinically relevant IgG evaluation methods - An overview]. / Immunglobulinkonzentration im equinen Kolostrum und im Blut neugeborener Fohlen sowie klinisch relevante IgG-Bestimmungsmethoden.

Due to the special structure of the equine placenta, foals depend on an adequate intake of high-quality colostrum post natum in order to ensure the development of passive immunity. The quality of the colostrum is determined, among other things, by the IgG content. This may be evaluated in the colostrum by direct and indirect methods (density and refractive index). The density of the colostrum is measured by a colostrometer and should amount to at least 1060 g/l. Refractometry is suitable for assessing the relative density or refractive index. Good equine colostrum has a Brix value of at least 23 %. The IgG concentration in the blood of the foal may also be determined by direct and indirect methods. The SNAP®-Test is regarded as a direct semi-quantitative measurement method, with values > 800 mg/dl indicating an adequate IgG concentration. Furthermore, the radial immuno-diffusion test, the latex agglutination test, and the immunoturbimetry are direct methods that may be applied. Indirect methods include the zinc sulphate turbidity test, the glutaraldehyde coagulation test, as well as the measurement of total protein, globulin concentration and γ-glutamyl transferase activity.

Co-reactivity between related and unrelated environmental allergens in equine allergen-specific IgE serology testing in the UK.

BACKGROUND: Identification of environmental allergens in horses with allergic disease facilitates allergen avoidance and targeted immunotherapy. HYPOTHESIS/OBJECTIVES: To evaluate allergenic co-reactivity between 44 environmental allergens. ANIMALS: Horses with suspected allergic disease (n = 344) whose sera were submitted for environmental allergen testing. METHODS AND MATERIALS: Allergen-specific IgE serological assays were performed using 44 allergens divided into six taxonomically related groups: house dust/storage mites, moulds, insects, grass, tree and weed pollens. Using pairwise comparisons, odds ratios (ORs) were calculated for each environmental pair to determine if there was increased or decreased likelihood of a positive result for one allergen, given a positive result to another. The OR significance was set (using Holm-Bonferroni correction) at P < 0.00006 for all horses (n = 344) and P < 0.00005 for horses with at least one positive reaction (n = 239). Using one-way ANOVA with Tukey's post hoc tests (significance at P < 0.05), differences in mean log e ORs between three groups, taxonomically related allergens with a statistically significant association (related-associated), related allergens lacking a significant association (related-nonassociated) and unrelated allergens were tested. RESULTS: Statistically significant associations were found between both related and unrelated allergen pairs, the former being more frequent. For all horses (n = 344) and horses with at least one positive reaction (n = 239), co-reactivity ranged from 100% (grasses) to 0% (moulds). The weeds group was exceptional in having more co-reactions with another group (grasses). CONCLUSIONS AND CLINICAL IMPORTANCE: Co-reactivity was shown within and between certain related allergen groups. Further studies are required to determine whether this is the result of antigenic cross-reactivity.

Determination of irritant threshold concentrations of multiple tree, grass, weed and mould allergens for intradermal testing of horses residing in the southern USA.

BACKGROUND: Appropriate allergen threshold concentrations (TCs) for intradermal testing (IDT) have not been established in horses for many pollen and mould allergens. OBJECTIVES: To determine the TCs in non-allergic horses and describe the frequency of late phase reactions for 26 allergens, including trees, grasses, weeds and moulds in horses residing in the southern Unites States. ANIMALS: Twenty four clinically normal horses in the southern United States. METHODS: Threshold concentrations for different allergens were determined using IDT subjective measurements at 30 minutes. Delayed reactions were evaluated at 4 and 24 h. RESULTS: Threshold concentrations (all PNU/mL) were established for eight tree allergens (black willow 1,000, box elder 1,000, live oak 1,000, pecan 2,000, white ash 4,000, red oak 4,000, red mulberry 2,000 and green ash 2,000); two grass allergens (Johnson grass 250 PNU/mL and Kentucky blue grass 500 PNU/mL); two weeds (carelessweed 1,000 PNU/mL, great ragweed 500 PNU/mL) and one mould (Curvularia 8,000 PNU/mL). The TC was not determined due to excessive reactivity at the lowest concentration tested (1,000 PNU/mL) for bahia and perennial rye grass. Eleven other allergens did not meet the criteria to establish a TC when evaluated at 30 min due to lack of positive reactions. Multiple allergens caused positive reactions in ≥10% of horses at 4 h. Reactions at 24 h were rare with the exception of one horse. CONCLUSIONS AND CLINICAL IMPORTANCE: This study identified intradermal TC for multiple pollen and mould allergens in horses. These values may prove useful for optimizing allergen concentrations for IDT of allergic horses.

Quantification of equine immunoglobulin A in serum and secretions by a fluorescent bead-based assay.

Only few quantitative reports exist about the concentrations and induction of immunoglobulin A (IgA) in mucosal secretions of horses. Despite this, it is widely assumed that IgA is the predominant immunoglobulin on mucosal surfaces in the horse. Here, two new monoclonal antibodies (mAbs) against equine IgA, clones 84-1 and 161-1, were developed and characterized in detail. Both IgA mAbs specifically bound monomeric and dimeric equine IgA in different applications, such as Western blots and fluorescent bead-based assays. Cross-reactivity with other equine immunoglobulin isotypes was not observed. The new IgA mAb 84-1 was used in combination with the previously characterized anti-equine IgA mAb BVS2 for the development and validation of a fluorescent bead-based assay to quantify total IgA in equine serum and various secretions. The IgA assay's linear detection ranged from 64pg/ml to 1000ng/ml. For the quantification of IgA in serum or in secretions an IgA standard was purified from serum or nasal wash fluid (secretory IgA), respectively. The different standards were needed for accurate IgA quantification in the respective samples taking the different signal intensities of monomeric and dimeric IgA on the florescent bead-based assay into account. IgA was quantified by the bead-based assay established here in different equine samples of healthy adult individuals. In serum the median total IgA was 0.45mg/ml for Thoroughbred horses (TB, n=10) and 1.16mg/ml in Icelandic horses (ICH, n=12). In nasopharyngeal secretions of TB (n=7) 0.13mg/ml median total IgA was measured, and 0.25mg/ml for ICH (n=12). Saliva of ICH (n=6) contained a median of 0.15mg/ml, colostrum of Warmbloods (n=8) a median of 1.89mg/ml IgA. Compared to IgG1 and IgG4/7 quantified in the same samples, IgA appeared as the major immunoglobulin isotype in nasopharyngeal secretions and saliva while it is a minor isotype in serum and colostrum. The newly developed monoclonal antibodies against equine IgA and the resulting bead-based assay for quantification of total IgA can notably improve the evaluation of mucosal immunity in horses.

Cytokine levels in colostrum and in foals' serum pre- and post-suckling.

The purpose of this study is to investigate the presence of IL-4, IL-8, IL-13 and IFN-γ in equine colostrum and in foals' serum. Samples were obtained from 14 mares and their healthy foals. Soon after parturition, 10ml of colostrum was collected, filtered, centrifuged and frozen until assayed. Blood samples were obtained from each foal at birth (TO) and again after 24h (T24), after which they were frozen until assayed. Serum IgG was measured at 24h of age with an immunoturbidimetric quantitative method. Cytokine concentration was determined using commercially available ELISA tests. Statistical analyses revealed a significant difference in serum concentration of IL-4 at T0 and at T24 (p<0.05) and a significant correlation between the serum IL-4 at T24 and colostral IL-4. These results suggest the absorption of IL-4 from colostrum. The presence of IL-8 in the pre-suckle foal's serum may be due to an endogenous production. With the exception of two samples, there was no IL-13 detected in the foals' serum at birth and remained undetectable in 8/14 samples after 24h. This cytokine was also undetectable in four colostrum samples, where its concentration showed a wide range and a high standard deviation. IFN-γ was present in both the colostrum and in the foals serum at birth.

HORSE SPECIES SYMPOSIUM: Can the microbiome of the horse be altered to improve digestion?

Intensive management practices in the horse industry present a unique challenge to the microbiome of the large intestine. Common management practices such as high-concentrate diets, low forage quality, meal feeding, and confinement housing have an impact on intestinal function, specifically large intestinal fermentation. The microbiome of the equine large intestine is a complex and diverse ecosystem, and disruption of microbiota and their environment can lead to increased incidence of gastrointestinal disorder. Digestion in the horse can be improved through a variety of approaches such as feedstuff selection, forage quality, feeding management, and inclusion of digestive aids. These digestive aids, such as prebiotics and probiotics, have been used to improve digestibility of equine diets and stabilize the microbiome of the large intestine. Probiotics, or direct-fed microbials, have been widely used in horses for treatment and prevention of gastrointestinal disease. The introduction of these live, beneficial microorganisms orally into the intestinal tract has yielded variable results. However, it is difficult to compare data due to variations in choice of organism, dosage, and basal diet. Although there are still many unanswered questions about the mode of action of successful probiotics, evidence indicates competitive inhibition and enhanced immunity. Lactic acid bacteria such as , and and yeast have all successfully been used in the horse. Use of these products has resulted in improved fiber digestibility in horses offered both high-starch and high-fiber diets. When high-concentrate diets were fed, probiotic supplementation helped maintain cecal pH, decreased lactic acid concentrations, and enhanced populations of cellulolytic bacteria. Similarly, use of prebiotic preparations containing fructooligosaccharide (FOS) or mannanoligosaccharides have improved DM, CP, and NDF digestibility when added to high-fiber diets. Furthermore, use of FOS in horses reduced disruptions in colonic microbial populations after an abrupt change in diet and altered fecal VFA concentrations toward propionate and butyrate. Potential use of prebiotics and probiotics to create greater stability in the equine microbiome impacts not only the digestibility of feed but also the health of the horse.

Allergic sensitization is age-dependently associated with rhinitis, but less so with asthma.

BACKGROUND: Epidemiologic data describing the association between allergic sensitization and asthma and allergic rhinitis in adults are scarce. OBJECTIVE: To determine the prevalence and impact of specific sensitization to airborne allergens on asthma and allergic rhinitis among adults in relation to age. METHODS: A random population sample (age 21-86 years) was examined with structured interview and analysis of specific IgE to 9 common airborne allergens. Of those invited, 692 (68%) subjects participated in blood sampling. IgE level of 0.35 U/mL or more to the specific allergen was defined as a positive test result. RESULTS: Allergic sensitization decreased with increasing age, both in the population sample and among subjects with asthma and allergic rhinitis. In a multivariate model, sensitization to animal was significantly positively associated with asthma (odds ratio [OR], 4.80; 95% CI, 2.68-8.60), whereas sensitization to both animal (OR, 3.90; 95% CI, 2.31-6.58) and pollen (OR, 4.25; 95% CI, 2.55-7.06) was significantly associated with allergic rhinitis. The association between allergic sensitization and rhinitis was consistently strongest among the youngest age group, whereas this pattern was not found for asthma. The prevalence of allergic sensitization among patients with asthma decreased by increasing age of asthma onset, 86% with asthma onset at age 6 y or less, 56% at age 7 to 19 years, and 26% with asthma onset at age 20 years or more. CONCLUSIONS: Sensitization to animal was associated with asthma across all age groups; allergic rhinitis was associated with sensitization to both pollen and animal and consistently stronger among younger than among older adults. Early onset of asthma was associated with allergic sensitization among adults with asthma.

Effects of orally administered galacto-oligosaccharides on immunological parameters in foals: a pilot study.

BACKGROUND: In the first phase of life, in which the immune system is primed and the bacterial colonization of epithelial surfaces takes place, foals are highly susceptible to bacterial infections. Next to strategies to optimize maternally acquired immunity in individual foals, current research explores other options to modulate immune responses in foals. During the past decades, oligosaccharide supplements were developed to mimic beneficial properties of the oligosaccharides, which are present in colostrum and milk. In human infants and laboratory animal species, dietary supplementation with galacto-oligosaccharides (GOS) has been shown to result in prebiotic and immunomodulating effects, with long-term beneficial consequences for both defensive and allergic immune responses. As yet, no studies are published concerning the in vivo effects of GOS in horses. The current study was designed as a pilot study to investigate the effects of an orally applied, commercially available GOS product in a group of pony foals. The treatment and the control group consisted of six and four foals, respectively. Foals were treated during the first four weeks of life and subsequently followed up for another ten weeks. RESULTS: In peripheral blood mononuclear cells (PBMCs) derived from GOS-treated foals at day 28, a standardized lipopolysaccharide challenge resulted in significantly lower relative mRNA expression levels of the pro-inflammatory cytokines interferon-γ and interleukin-6 compared with PBMCs of control foals. In the 98-day period of investigation, no significant effects of the GOS supplement were observed on clinical and blood parameters for immunity and general health in these foals. CONCLUSIONS: Based on these first results, we can conclude that this dose regimen of GOS was well accepted by the foals and did not result in any detectable undesirable side effects. More clinical trials are required to confirm the attenuating effects of GOS treatment on equine pro-inflammatory immune responses and to implement this into practice.

Clinical parameters, intestinal function, and IGF1 concentrations in colostrum-deprived and colostrum-fed newborn pony foals.

Colostrum (COL) contains cytokines and growth factors that may enhance intestinal development in neonates. The hypothesis of this study was that besides providing immunoglobulins, COL is important for intestinal function and meconium release in foals. Newborn foals were either fed COL (n = 5) or an equal amount of milk replacer (MR, n = 7) during the first 24 hours of life. To ensure passive immunity, all foals received 1 L plasma. Postnatal development, meconium release, intestinal motility, white blood cell count, insulin-like growth factor 1, and intestinal absorptive function (xylose absorption test) were evaluated. Clinical findings and meconium release were not affected by feeding of COL or MR. Ultrasonography revealed a slightly larger jejunum and stomach in group COL versus MR (P < 0.05). The percentage of polymorphonuclear leucocytes was higher in foals of group MR versus group COL (P < 0.05) and the percentage of lymphocytes was lower in MR compared with COL foals (P < 0.05). Plasma insulin-like growth factor 1 concentration increased during the first 14 days after birth in both groups. A xylose absorption test on Day 5 revealed similar increases in plasma xylose concentrations after oral intake. In conclusion, feeding of COL versus MR was without effect on meconium release and intestinal absorptive function. Differences between foals fed COL and MR with regard to intestinal function are apparently without clinical relevance. In foals that have not received maternal COL, there is no major risk of intestinal problems if they are fed MR and provided with immunoglobulins by transfusion of plasma.

Analysis of soluble CD14 and its use as a biomarker in neonatal foals with septicemia and horses with recurrent airway obstruction.

Soluble CD14 (sCD14) binds bacterial lipopolysaccharide (LPS) and acts as an anti-inflammatory LPS-inhibitor in vivo. In humans, sCD14 is one of the soluble biomarkers used for various inflammatory diseases and conditions, however, sCD14 assays have not yet been evaluated in horses. Here, we developed and optimized a bead-based assay for the quantification of sCD14 in horses. The assay was then used to determine native sCD14 concentrations in serum from healthy and septic foals, in the colostrum of healthy mares and in plasma from adult horses with recurrent airway obstruction (RAO) and control horses. Healthy foals and adult horses had sCD14 concentrations in serum or plasma in the high ng/ml range. The concentration of sCD14 in colostrum samples from healthy mares was in the µg/ml range. Foals with septicemia and adult horses with RAO had significantly higher sCD14 concentrations in their circulation than the respective control groups. The findings suggest that sCD14 can become a valuable biomarker for neonatal septicemia, RAO and possibly also for other inflammatory diseases in horses. Further studies and larger samples numbers are required to determine normal sCD14 concentration ranges and those that are indicative of disease progression, severity or prognosis.

The effect of selenium supplementation on vaccination response and immune function in adult horses.

Selenium status has been reported to affect immune function across many different species. Yet few studies have focused on the effect of Se status on the equine immune system. This study examined the effect of Se supplementation on vaccination response and immune function in mature horses. Twenty-eight horses were blocked by age and sex and were randomly allocated to 1 of 4 dietary treatment groups: low Se (LS), adequate Se (AS), Se-yeast (SP), and sodium selenite (SS). For 35 wk, horses allocated to LS, SP, and SS received a low-Se diet (0.06 mg/kg DM) with the intention to lower Se stores, whereas AS received an adequate Se diet (0.12 mg/kg DM). A 29-wk repletion phase was as follows: LS and AS were kept on the diets fed during the depletion period, whereas SP and SS received the depletion diet plus their respective Se supplements to achieve a dietary Se concentration of 0.3 mg/kg DM. The Se status of the horses was monitored using whole blood Se and glutathione peroxidase (GSH-Px) activity as indicators. At wk 22 and 25 of the repletion phase, horses were vaccinated intramuscularly with 10 mg ovalbumin (OVA). Horses were also vaccinated against equine influenza at wk 25. Blood samples were collected for 7 wk after initial vaccination for serum separation and at 0, 3, and 5 wk postvaccination for peripheral blood mononuclear cell (PBMC) isolation and whole blood cytokine mRNA evaluation. At wk 22 of the repletion phase, both Se and GSH-Px were greater for SP and SS compared with AS and LS (P < 0.001). Serum vitamin E was similar between treatments. Response to OVA vaccination, evaluated as OVA-specific IgG production, cytokine mRNA expression of PBMC stimulated with OVA in vitro, and lymphocyte proliferation, was unaffected by Se status. Similarly, memory response to the influenza vaccine was not affected by Se status. However, decreased mRNA expression of selected cytokines was observed in PBMC stimulated with phorbol 12-myristate 13-acetate for LS compared with AS, SP, and SS (P < 0.05). Whole blood mRNA expression of IL-10 was greater for SS compared with LS, AS, and SP (P = 0.043). Although the OVA and influenza vaccination responses were unaffected by Se status, other measures of immune function did indicate that low Se status could adversely affect cell-mediated immunity.

Change of antibody levels to ferritin in the sera of foals after birth: possible passive transfer of maternal anti-ferritin autoantibody via colostrum and age-related anti-ferritin autoantibody production.

Antibody (immunoglobulin G (IgG), IgM or IgA) levels relative to ferritin in six foal sera (three male and three female) after birth (day 0 and 2, 6, 10, 20, 28, 36, 40, 52 and 56 weeks of age) were semi-quantitatively measured with normalization with antibody activity to ferritin in one adult horse serum. After addition of horse spleen ferritin to the serum sample, the complex formed between antibodies to ferritin in the serum and ferritin was co-immunoprecipitated using antibody to horse spleen ferritin. Antibody classes of the co-immnoprecipitate were detected with antibodies specific for horse IgG, IgM or IgA heavy chain. Six adult horse serum samples were found to have ferritin-binding activities in all immunoglobulin classes examined. Although ferritin antibody activities (IgG, IgM and IgA) were scant in the foal sera before sucking colostrum (day 0), their activities increased at 2 weeks of age. IgG antibodies showed a biphasic response and IgM antibody activity increased up to 40 weeks of age. Antibody (IgG, IgM and IgA) activities to ferritin in three colostrum samples were significantly higher than in adult horse serum samples. These results demonstrate that antibody to ferritin in foal serum is derived from colostrum after birth and is produced thereafter.

Preclinical assessment of a polyspecific antivenom against the venoms of Cerrophidion sasai, Porthidium nasutum and Porthidium ophryomegas: Insights from combined antivenomics and neutralization assays.

A polyspecific antivenom is used in Central America for the treatment of envenomings by viperid snakes. This antivenom is generated in horses hyperimmunized with a mixture of venoms from Bothrops asper, Crotalus simus and Lachesis stenophrys. The present study analyzed the ability of this antivenom to neutralize the venoms of three Central American viperid species of the 'Porthidium group', i.e. Porthidium nasutum, Porthidium ophryomegas and Cerrophidion sasai, formerly classified as Cerrophidion godmani. In addition, the immunorecognition of the components of these venoms was assessed by immunoaffinity antivenomics. The antivenom proved effective in neutralizing the lethal, hemorrhagic, myotoxic, phospholipase A(2) (PLA(2)) and proteinase activities of the three venoms, albeit exhibiting quantitative differences in the values of the Median Effective Doses (ED(50)). Excepting for certain low molecular mass bands corresponding to disintegrins, and some PLA(2)s and PI-metalloproteinases, Western blotting and immunoaffinity chromatography revealed immunorecognition of most Porthidium and Cerrophidion venom proteins. In agreement with in vivo neutralization assays, immobilized antivenom IgGs showed higher immunocapturing activity of toxins from both Porthidium taxa than from C. sasai. Overall our results demonstrate a significant paraspecific protection of the Costa Rican polyspecific antivenom against the three venoms sampled. They also stress the need to search for novel ways to enhance the immune response of horses against several weakly immunogenic venom components.

Correlation of serum IgG concentration in foals and refractometry index of the dam's pre- and post-parturient colostrums: an assessment for failure of passive transfer in foals.

The object of this study was to evaluate the usefulness of measuring the differences in the values of the serum total protein (DVSTP) concentration of foals and the refractometry index (DVRI) of the milk of dams before and after nursing of the colostrum for assessing failure of passive transfer (FPT) in foals. Serum samples from 31 foals were collected before the first nursing and other 1 to 6 times between 4 and 24 hr after birth. Paired colostrum and milk samples were collected from 14 of their dams at the same time. Serum samples were analyzed for IgG concentration using a single radial immunodiffusion (SRID) test (98 samples) and total protein concentration using a temperature-compensating refractometer (98 samples). Colostrum and milk samples were analyzed for refractometry index (RI) using a Brix refractometer (71 samples). DVSTP concentration and DVRI were significantly correlated with serum IgG concentration. The negative predictive values (NPVs) of DVSTP concentration for detecting serum IgG concentrations<400 mg/dl and<800 mg/dl were 98.2% and 91.3% when the cutoff value is set to 0.4 mg/dl and 0.8 mg/dl, respectively. Furthermore, the NPVs of DVRI for detecting serum IgG concentrations<400 mg/dl and<800 mg/dl were 97.3% and 96.3% when the cutoff value is set to 6% and 10%, respectively. The results suggest that measurement of DVRI is useful in assessing FPT as an initial "stall-side" screening test, because it is easy, inexpensive to perform and allows for rapid interpretation.

Intestinal barrier function in neonatal foals: options for improvement.

Gastrointestinal defence in the new-born is limited in comparison to adults, due to an immature epithelial barrier function and deficits in both innate and adaptive immune responses. Consequently, neonates (including foals) are at increased risk of disturbance to mucosal homeostasis during initial intestinal colonisation that may lead to excessive inflammation and bacterial translocation into the bloodstream, resulting in septicaemia. Bacterial recognition by Pattern Recognition Receptors (PRRs) and their downstream regulation of cytokine release have been shown to be pivotal for gastrointestinal mucosal homeostasis and the development of a functional intestinal barrier. Evidence suggests that selective PRR agonists limit the inflammatory responses and improve epithelial barrier function. Milk, and in particular colostrum, contain a broad array of oligosaccharides which seem to act as PRR agonists. This class of compounds forms a source for new dietary formulas that may orchestrate gut colonisation by the commensal flora in the early phase of life and so reduce the risks of inflammation and pathogen invasion.

Effects of selenium source on measures of selenium status and immune function in horses.

The effects of selenium (Se) supplementation and source on equine immune function have not been extensively studied. This study examined the effects of oral Se supplementation and Se source on aspects of innate and adaptive immunity in horses. Fifteen horses were assigned to 1 of 3 groups (5 horses/group): control, inorganic Se (sodium selenite), organic Se (Se yeast). Immune function tests performed included: lymphocyte proliferation in response to mitogen concanavalin A, neutrophil phagocytosis, antibody production after rabies vaccination, relative cytokine gene expression in stimulated lymphocytes [interferon gamma (IFNγ), interleukin (IL)-2, IL-5, IL-10, tumor necrosis factor alpha (TNFα)], and neutrophils (IL-1, IL-6, IL-8, IL-12, TNFα). Plasma, red blood cell Se, and blood glutathione peroxidase activity were measured. Plasma and red blood cell Se were highest in horses in the organic Se group, compared with that of inorganic Se or control groups. Organic Se supplementation increased the relative lymphocyte expression of IL-5, compared with inorganic Se or no Se. Selenium supplementation increased relative neutrophil expression of IL-1 and IL-8. Other measures of immune function were unaffected. Dietary Se content and source appear to influence immune function in horses, including alterations in lymphocyte expression of IL-5, and neutrophil expression of IL-1 and IL-8.

Les effets d'un supplément de sélénium (Se) ainsi que sa source sur la fonction immunitaire équine n'ont pas été étudiés à fond. On examina dans la présente étude les effets d'un supplément oral en Se et les sources de Se sur des éléments de l'immunité innée et adaptative de chevaux. Quinze chevaux ont été assignés à un de trois groupes (5 chevaux/groupe); témoin, Se inorganique (sélénite de sodium), et Se organique (Se provenant de levures). Les tests de fonctions immunitaires effectués étaient : prolifération lymphocytaire en réponse au mitogène concanaviline A, phagocytose par les neutrophiles, production d'anticorps après vaccination anti-rabique, expression relative des gènes des cytokines de lymphocytes stimulés [interferon gamma (IFNγ), interleukine (IL)-2, IL-5, IL-10, facteur de nécrose tumorale alpha (TNFα)], et de neutrophiles (IL-1, IL-6, IL-8, IL-12, TNFα). Le Se plasmatique et des globules rouges, ainsi que l'activité de la glutathion peroxydase ont été mesurés. Le Se plasmatique et des globules rouges étaient plus élevés chez les chevaux dans le groupe de Se organique, comparativement au groupe recevant le Se inorganique ou le groupe témoin. Un supplément de Se organique entraîna une augmentation d'expression relative d'IL-5 par les lymphocytes, comparativement au Se inorganique ou aucun Se. Un supplément de Se augmenta l'expression relative d'IL-1 et IL-8 par les neutrophiles. Les autres mesures des fonctions immunitaires n'étaient pas affectées. Le contenu et les sources de Se alimentaire semblent influencer les fonctions immunitaires des chevaux, incluant des altérations dans l'expression d'IL-5 par les lymphocytes, et l'expression d'IL-1 et IL-8 par les neutrophiles.(Traduit par Docteur Serge Messier).

Transfer of tumour necrosis factor-α via colostrum to foals.

This study aimed to determine whether TNF-α is transferred to equine neonates via colostrum and the relationship between TNF-α and IgG concentrations in the equine neonate. Colostrum, presuckle and postsuckle foal serum samples were collected from healthy mares and their foals. Equine TNF-α ELISA and IgG SRID kits were used to determine the concentrations of TNF-α and IgG, respectively. Statistical analysis was performed using the Spearman rank correlation. TNF-α concentrations in all presuckle foal serum were below the limit of detection in 15/16 foals and increased in postsuckle foal serum to a mean concentration of 7.7 x 10(4) pg/ml. TNF-α concentrations in postsuckle foal serum and colostrum showed significant correlation (rho=0.668; P=0.005). However, TNF-α and IgG concentrations in colostrum or postsuckle foal serum did not correlate (rho<-0.016; P>0.05). Ratios of TNF-α/IgG in colostrum or postsuckle foal serum showed significant correlation (rho=0.750; P=0.0008). These results indicate that TNF-α is transferred to the foal via colostrum absorption and may play a role in early immunity.

Gene expression analysis in the thalamus and cerebrum of horses experimentally infected with West Nile virus.

Gene expression associated with West Nile virus (WNV) infection was profiled in the central nervous system of horses. Pyrosequencing and library annotation was performed on pooled RNA from the CNS and lymphoid tissues on horses experimentally infected with WNV (vaccinated and naïve) and non-exposed controls. These sequences were used to create a custom microarray enriched for neurological and immunological sequences to quantitate gene expression in the thalamus and cerebrum of three experimentally infected groups of horses (naïve/WNV exposed, vaccinated/WNV exposed, and normal).From the sequenced transcriptome, 41,040 sequences were identified by alignment against five databases. 31,357 good sequence hits (e<10(-4)) were obtained with 3.1% of the sequences novel to the equine genome project. Sequences were compared to human expressed sequence tag database, with 31,473 equine sequences aligning to human sequences (69.27% contigs, 78.13% seed contigs, 80.17% singlets). This indicated a high degree of sequence homology between human and equine transcriptome (average identity 90.17%).Significant differences (p<0.05) in gene expression were seen due to virus exposure (9,020), survival (7,395), and location (7,649). Pathways analysis revealed many genes that mapped to neurological and immunological categories. Involvement of both innate and adaptive components of immunity was seen, with higher levels of expression correlating with survival. This was highlighted by increased expression of suppressor of cytokine signaling 3 in horses exposed to WNV which functions to suppress innate immunity. Pentraxin 3 was most increased in expression for all horses exposed to WNV.Neurological pathways that demonstrated the greatest changes in gene expression included neurotransmitter and signaling pathways. Decreased expression of transcripts in both the glutamate and dopamine signaling pathways was seen in horses exposed to WNV, providing evidence of possible glutamate excitotoxicity and clinical signs associated with decreased dopamine. Many transcripts mapped to non-infectious neurological disease functions, including mental disorders and degenerative neuropathies.

In vivo priming and ex vivo activation of equine neutrophils in black walnut extract-induced equine laminitis is not attenuated by systemic lidocaine administration.

Laminitis is a crippling disease of horses characterized by an inflammatory response in the tissue that suspends the axial skeleton within the hoof. Pain is a common feature of laminitic pathology and its management is an important component of the treatment regime for this disease. Systemic lidocaine administration is commonly utilized to manage pain in equine laminitis; however, the potential anti-inflammatory effects of this drug during the treatment of equine laminitis have not been investigated. Here, we sought to determine if lidocaine concentrations achieved in the plasma (therapeutic concentrations) of horses systemically administered lidocaine are capable of attenuating neutrophil activation and associated inflammation. To identify markers of activation, purified neutrophils were stimulated in vitro with LPS or recombinant equine IL-8 (reqIL-8) and surface expression of CD13 and CD18 was ascertained by immunofluorescent staining. Activation with LPS or reqIL-8 in vitro induced an elevated expression of CD13 as well as a putative conformational change in CD18 detected by elevated staining with a sub-saturating concentration of anti-CD18 mAb. Lidocaine attenuated the activation-induced changes in CD13 and CD18 expression only when used at 30-70 times therapeutic concentrations. For in vivo analyses, horses were administered black walnut extract (BWE) to induce laminitis and either systemic lidocaine (n=6) or saline (n=6) as a control. Whole blood was collected and incubated with or without reqIL-8. Following which, leukocytes were stained for CD13 and CD18. Protein was extracted from laminar tissue and subjected to gelatin zymography to measure matrix metalloproteinase-9 (MMP-9) accumulation. Results obtained show that changes in neutrophil size, granularity/complexity, CD13 surface expression and CD18 staining intensity occurred over time post BWE administration irrespective of lidocaine treatment in response to incubation alone or with 100 ng/ml of reqIL-8. The mean fluorescence intensities of neutrophils stained for either CD13 or CD18 did not differ between lidocaine treated and saline controls, nor did lamellar MMP-9 content measured by gelatin zymography. Thus, using changes in surface expression of CD13 and CD18 as markers of neutrophil activation in the horse we have shown that BWE treatment activates neutrophils in vivo and this is not affected by systemic administration of lidocaine at levels used to manage pain.