Selenium supplementation restores innate and humoral immune responses in footrot-affected sheep.

Dietary selenium (Se) alters whole-blood Se concentrations in sheep, dependent upon Se source and dosage administered, but little is known about effects on immune function. We used footrot (FR) as a disease model to test the effects of supranutritional Se supplementation on immune function. To determine the effect of Se-source (organic Se-yeast, inorganic Na-selenite or Na-selenate) and Se-dosage (1, 3, 5 times FDA-permitted level) on FR severity, 120 ewes with and 120 ewes without FR were drenched weekly for 62 weeks with different Se sources and dosages (30 ewes/treatment group). Innate immunity was evaluated after 62 weeks of supplementation by measuring neutrophil bacterial killing ability. Adaptive immune function was evaluated by immunizing sheep with keyhole limpet hemocyanin (KLH). The antibody titer and delayed-type hypersensitivity skin test to KLH were used to assess humoral immunity and cell-mediated immunity, respectively. At baseline, FR-affected ewes had lower whole-blood and serum-Se concentrations; this difference was not observed after Se supplementation. Se supplementation increased neutrophil bacterial killing percentages in FR-affected sheep to percentages observed in supplemented and non-supplemented healthy sheep. Similarly, Se supplementation increased KLH antibody titers in FR-affected sheep to titers observed in healthy sheep. FR-affected sheep demonstrated suppressed cell-mediated immunity at 24 hours after intradermal KLH challenge, although there was no improvement with Se supplementation. We did not consistently prevent nor improve recovery from FR over the 62 week Se-treatment period. In conclusion, Se supplementation does not prevent FR, but does restore innate and humoral immune functions negatively affected by FR.

Higher whole-blood selenium is associated with improved immune responses in footrot-affected sheep.

We reported previously that sheep affected with footrot (FR) have lower whole-blood selenium (WB-Se) concentrations and that parenteral Se-supplementation in conjunction with routine control practices accelerates recovery from FR. The purpose of this follow-up study was to investigate the mechanisms by which Se facilitates recovery from FR. Sheep affected with FR (n = 38) were injected monthly for 15 months with either 5 mg Se (FR-Se) or saline (FR-Sal), whereas 19 healthy sheep received no treatment. Adaptive immune function was evaluated after 3 months of Se supplementation by immunizing all sheep with a novel protein, keyhole limpet hemocyanin (KLH). The antibody titer and delayed-type hypersensitivity (DTH) skin test to KLH were used to assess humoral immunity and cell-mediated immunity, respectively. Innate immunity was evaluated after 3 months of Se supplementation by measuring intradermal responses to histamine 30 min after injection compared to KLH and saline, and after 15 months of Se supplementation by isolating neutrophils and measuring their bacterial killing ability and relative abundance of mRNA for genes associated with neutrophil migration. Compared to healthy sheep, immune responses to a novel protein were suppressed in FR-affected sheep with smaller decreases in FR-affected sheep that received Se or had WB-Se concentrations above 250 ng/mL at the time of the immune assays. Neutrophil function was suppressed in FR-affected sheep, but was not changed by Se supplementation or WB-Se status. Sheep FR is associated with depressed immune responses to a novel protein, which may be partly restored by improving WB-Se status (> 250 ng/mL).

Melatonin, as an adjuvant-like agent, enhances platelet responsiveness.

Melatonin exerts immunomodulatory actions that enhance the magnitude and quality of immune responses specific for certain antigens; this has raised the possibility of using melatonin to design novel vaccine adjuvant systems. The present study investigated the effect of subcutaneous slow-release melatonin implants and subcutaneous melatonin injections on the responsiveness of circulating platelets in sheep after vaccination against Dichelobacter nodosus (A1 and C serotypes), the bacterium that causes ovine footrot, a major cause of lameness in sheep. The experiments were carried out in sheep from a farm located in an area of Mediterranean-type ecosystem. Plasma melatonin levels were determined by radioimmunoassay, sheep platelet aggregation was monitored using an aggregometer and Ca2+ mobilization was determined by spectrofluorimetry using fura-2. Administration of melatonin either by implants or subcutaneous injections increased plasma melatonin concentrations, an effect that was found to be greater and more sustained when melatonin was administered via implants. Vaccination per se, as well as melatonin, increased the percentage and rate of platelet aggregation and reduced the lag-time in response to the physiological agonist thrombin, an effect that was found to be significantly greater when melatonin was administered to vaccinated animals. Melatonin enhanced thrombin-evoked Ca2+ release and entry and further increased Ca2+ mobilization observed in platelets from vaccinated sheep. These observations suggest that the use of melatonin, as a novel adjuvant, induces beneficial effects on platelet function and haemostasis, and opens new perspectives for therapeutic manipulation of immune responses to vaccination.

Melatonin enhances the immune response to vaccination against A1 and C strains of Dichelobacter nodosus.

Melatonin has been shown to exert immunomodulatory properties with broad application in veterinary medicine. Here we have investigated the effect of exogenous melatonin in the improvement of the immune response to administration of an immune-preparation of two stumps of A1 and C strains of Dichelobacter nodosus in sheep. Subcutaneous administration of melatonin enhanced plasma levels of melatonin from days 42 to 120. Administration of melatonin to vaccinated animals enhanced both the titer of antibodies and serum IgG levels to A1 and C strains of D. nodosus compared to vaccinated animals not treated with melatonin. Our results suggest that melatonin increased the immune response to vaccination and open new perspectives in the design of prophylactic strategies.

The use of melatonin as a vaccine agent.

Molecules with immunomodulatory properties determine the magnitude and quality of immune responses specific for the coadministered antigen. Melatonin is considered a biological-response modifier of the immune system with broad application in veterinary medicine. In seasonally-breeding animals, the indolamine is able to improve reproductive performance. With the purpose of expanding new advantageous roles for melatonin, we investigated the effect of subcutaneous slow-release melatonin implants in the humoral response after a vaccination. We reported here a new feature of melatonin as an adjuvant-like system towards Dichelobacter nodosus (A1 and C serotypes)--the bacterium which cause ovine footrot--the most important cause of lameness in sheep. Antibody titres determined by both agglutination and ELISA techniques were substantially higher and were sustained for a longer duration than non-implanted animals. Remarkably, the effect of melatonin was completely dependent on the presence of aluminium hydroxide. The finding that melatonin enhances a defined immune response in vivo opens new perspectives for the improvement of Th2-biased immune responses by alum adjuvants.

Application of ELISA to the serological diagnosis of virulent ovine footrot.

The sensitivity and specificity of an ELISA that detects antibody in the serum of sheep against Dichelobacter nodosus was investigated. The sensitivity of the assay was determined in two trials in which sheep free of virulent footrot were exposed to a virulent strain of D. nodosus. In the first trial up to 96.6% of sheep that developed severe foot lesions were seropositive 2 weeks later. The proportion of seropositive sheep depended on the interval between exposure to D. nodosus and collection of blood samples and reached a maximum 6 weeks after exposure in the first trial and plateaued from 8 weeks after exposure in the second trial. Of sheep with severe lesions present for only 1 week, 40.0% became seropositive, while 98.3% of sheep with severe lesions present for 3 or more weeks became seropositive in Trial 1. Of the unexposed control sheep from the same flock, 93.3% remained seronegative. At least 75% of sheep that were vaccinated with commercial footrot vaccines remained seropositive for at least 24 months after last vaccination and 58.5% remained seropositive for at least 36 months. The specificity of the assay in flocks of sheep from different geographical regions was determined also. Overall, 95.2% of 2978 sheep free of virulent footrot were seronegative. However the rate of false positive serological reactions ranged from 1.6% to 11.3% between flocks and was higher in older sheep (7.0%) compared with sheep less than 1.5 years old (2.7%). Passively acquired colostral antibodies against D. nodosus were measured in 22 lambs and did not persist beyond 55 days mean age. Non-specific reactions with cross reactive antigens were proposed to explain age related changes in serological results. Applications of this ELISA in footrot control programmes are discussed.

Colostral transfer of Bacteroides nodosus antibodies in sheep.

Ovine contagious foot rot may cause lameness in sheep, resulting in decreased wool growth and low weight gain. Affected neonatal lambs are difficult to treat, and treatment is labor intensive; thus, a method of prevention is warranted. Vaccination of ewes with a multivalent vaccine in an oil adjuvant induced development of antibody to the somatic O antigen of Bacteroides nodosus, and this antibody was detected in serum of newborn lambs after consumption of colostrum from the vaccinated ewes. Antibody titers were determined in 48 unvaccinated ewe/lamb pairs, and in 50 once-vaccinated and 78 twice-vaccinated pairs. Serum and colostrum O-agglutinin titers to B nodosus were determined by a microtitration agglutination test. Lambs from vaccinated ewes had significantly (P less than 0.05) higher O-agglutinin titers than those from unvaccinated ewes, and double vaccination of ewes resulted in the highest potentially protective titers (greater than 1:2,400) in ewes and lambs.