2.3. Immunization of the ewesThe ew

2.3. Immunization of the ewes

The ewes followed standard immunization with 2 mL of Ovivac® P vet (Intervet) given subcutaneously 4 weeks prior to parturition. Ovivac® P contains toxoids of Clostridium tetani, C. perfringens, and C. septicum, and inactivated cells from C. chauvoei, Mannheimia haemolytica, and Pasteurella trehalosi. The production of antitoxins against different Clostridium spp. induced by vaccination represents an important immunoprophylactic means of reducing diseases in sheep production. Antitoxins neutralize the toxins generated in both the intestine and the blood of the animals. Here, we assayed the antibodies against tetanus toxoid as a parameter of antitoxin production. Antibodies against M. haemolytica protect the lambs against this important environmental pathogen.

The ewes received an experimental intramuscular immunization with 0.5 mL of Equilis® Reserquin vet (Intervet) vaccine against equine herpes and influenza virus (EIV) given 5 and 2 weeks before expected parturition. This vaccine contains inactivated herpes virus types 1 and 4, and inactivated influenza viruses A/Equi 1 and A/Equi 2. Antibodies against influenza virus hemagglutinin protect cells against infection with such virus, and hence we assayed these antibodies as a measure of disease resistance.

2.4. Immunization of newborn lambs

Two-week-old lambs were vacciNatEd by subcutaneous injection of 0.5 mL of diphtheria (Corynebacterium diphtheriae) toxoid vaccine (Difterivaccine, Statens Serum Institut (SSI), Denmark) and intracutaneous injection of live attenuated human tuberculosis vaccine (BCG, Mycobacterium bovis) (SSI, Denmark). This was done to study their ability to produce specific antitoxins and the cell-mediated immunity response.

2.5. Immunological parameters in the ewes

The Ig level was monitored by measuring IgG and IgM concentrations (mg/mL) in blood samples at the beginning of the feeding period and two weeks before parturition. This was done using a single radial immunodiffusion assay (Mancini et al., 1965) performed in agarose gel with rabbit polyclonal antibody directed against sheep IgG (Fc) or swine polyclonal antibody to sheep IgM (Fc) (both from Nordic Immunological Laboratories, the Netherlands).

The production of antibodies specific against M. haemolytica, tetanus toxoid, and EIV was assayed in blood samples at the beginning of the experiment, at the time of immunization, and at parturition. The antibodies against M. haemolytica were assessed using an ELISA technique ( Lyche et al., 2006) with M. haemolytica A2 and a horseradish peroxidase-linked donkey anti-sheep IgG (Sigma Aldrich, USA). The antibodies against tetanus toxoid were determined by a passive hemagglutination test ( Whist et al., 2003), and those against EIV were evaluated by a virus hemagglutination inhibition test ( Lyche et al., 2006).

2.6. Transfer of maternal immunity to the new-born lambs

Transfer of the maternal Ig was monitored by measuring IgG and IgM levels in serum samples collected from 1-, 4-, and 6-week-old lambs, as described above. IgG and IgM concentrations were also analyzed in fat-free colostrum collected within 6 h of parturition as far as practically possible. Colostrum was centrifuged for 15 min at 4 °C and RCF 2200g, and then placed on ice for 10 min, after which the aqueous phase was transferred to new tubes for further radial immunodiffusion analysis.

Transfer of maternal specific immunity was monitored in 1-, 2-, 4-, and 6-week-old lambs by measuring specific antibodies to M. haemolytica, tetanus toxoidin, and EIV as described above.

2.7. Immune status in the lambs

After immunization of the lambs, specific antibodies to C. diphtheriae toxoid were measured when the animals were 2, 4, and 6 week-old by passive hemagglutination test ( Whist et al., 2003).

The cell-mediated immunity to M. bovis was evaluated in 6 week-old lambs 4 week after immunization by analyzing IFN-γ production and antigen-induced lymphocyte proliferation in vitro, and by performing a skin test. The following mitogens and antigens were used: phytohemagglutinin (PHA), poke weed mitogen (PWM), concanavalin A (ConA), and bovine tuberculin purified protein (PPD). Production of IFN-γ in whole-blood cultures stimulated with the PPD antigen was assessed by an ELISA for bovine IFN-γ (Bovigam, Prionics, Switzerland). In short, the cultures were prepared from immunized lambs and them exposed to 20 µL of PPD (final concentration 10 or 40 µg mL−1) or incubated in medium alone as a negative control ( Storset et al., 2001). Proliferation of lymphocytes induced by the mitogens and antigens was assayed in vitro by purified lymphocyte culture ( Storset et al., 2001) modified for sheep blood. Heparinized (15 IU/mL) blood was centrifuged for 10 min at 900g. The buffy coat was collected and mixed with 6–7 mL of Hanks buffered saline solution (HBSS; DAA laboratories, Austria), and then placed on top of 3 mL of Lymfoprep® (Axis-Shield PoC AS, Oslo, Norway; 1.077 g/mL) in siliconized vials. Each vial was centrifuged for 30 min at 700g, after which the lymphocyte layer was transferred to a new siliconized vial and washed by centrifugation for 15 min at 700g with 10 ml Hanks buffered saline solution and then again for 10 min in the same manner. Thereafter, the supernatant was carefully removed, 9.8 mL of HBSS was added to the pellet (approx. vol. 0.2 mL), and the cells were counted. The cells were subsequently centrifuged for 10 min at 700g and then resuspended to 1×106 mononucleated cells/mL in RPMI 1640 cell culture medium (RPMI 1640, Gibco Division of Invitrogen, Paisley, UK) containing 10% fetal calf serum (FCS), 2 mM glutamine, 0.15% bicarboNatE, 1% antibiotics (penicillin and streptomycin). A 180 μL aliquot of this cell suspension was immediately dispensed into each 96 flat-bottom wells on a microtiter pate (Nunc, Roskilde, Denmark). The following mitogens and antigen (20 μL/well) were used (final concentration within parentheses): PHA (10 μg/mL; Wellcome, Beckenham, England); PWM (20 μg/mL; Gibco, Renfrewshire, Scotland); ConA (20 μg/mL; Pharmacia) and PPD (15 μg/mL; Veterinary Laboratories Agency, New Haw, Addlestone, Surrey, UK). RPMI 1640 medium was used as diluent and for controls. All assays were performed in triplicate cultures, and the plates were incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air for 4 days.

Lymphocyte proliferation was evaluated by assessing incorporation of [3H] thymidine added to the cultures. Briefly, after exposure to [3H] thymidine, the cells were collected on a filter plate and counted using a TopCount NXT Gamma Counter (Storset et al., 2001). The lymphocyte response was expressed as the difference in mean optical density (OD) between a stimulated culture and a control culture.

The skin tests were performed on 6- to 7-week-old lambs. The lambs were shaved on the left side of the thorax, and the test was performed by intradermal injection of 0.1 mL of each of the mitogens PHA (50 and 100 µg mL−1 in PBS solution), ConA (100 and 200 µg mL−1 in PBS solution), and PWM (50 and 100 µg mL−1 in PBS solution), and the antigen PPD (2500 and 1250 iu 0.1 mL−1). As a negative control, we used 0.1 mL 0.9% NaCl (Sonne et al., 2006). The skin reaction was measured 24 h after the injections as the diameter (mm) of the localized swelling (palpable raised area), and also according to height in mm (0=negative control to +4) and degree of erythema (0=negative control to +3).

2.8. Analysis of α-tocopherol

The concentration of α-tocopherol was measured in the diet, the colostrum, and plasma samples. Before analysis, the feed samples were freeze-dried and finely milled, and 1 g aliquot was suspended in a solution consisting of 24 mL of ethanol (960 mL/L), 9 mL of methanol, 10 mL of ascorbic acid in water (200 g/L) and 7 mL of KOH:water (1:1 w/v). This suspension was saponified for 30 min at 80 °C (boiling) in the dark and then cooled in cold water. Next, 2 mL of the saponified mixture was diluted with 0.5 mL of water, after which tocopherols were quantitatively extracted with two 5.0 mL portions of heptane. The samples of plasma and colostrum were thawed before analysis. The colostrum samples were also heated at 40 °C for 20 min and mixed thoroughly. To extract tocopherols, 1000 mg of milk or 500 µL of blood plasma was diluted with 0.5 mL of ascorbic acid solution (200 g/L), 0.5 mL of methanol, 2.0 mL of ethanol, and 0.5 mL KOH:water (1:1 w/v). This mixture was adjusted to a final volume of 5.5 mL with water and then subjected to saponification at 80 °C for 20 min. After cooling, tocopherol extraction was performed twice using two 5 mL portions of heptane each time. The two extracts were mixed, and 100 µL was injected into the HPLC system for α-tocopherol analysis. All solvents used were of HPLC grade. The HPLC was carried out on a 4.0×125 mm Perkin Elmer HS-5-Silica column using heptane that contained 2-propanol (3.0 mL/L) and was degassed with helium as the mobile phase. The flow rate was 3.0 mL/min, and the column was held at room temperature. Fluorescence detection was performed at excitation and emission wavelengths of 290 and 327 nm, respectively. Identification and quantification of α-tocopherol was achieved by comparison of retention time and peak areas with external standards (Merck, D-64293 Darmstadt, Germany), and an extinction coefficient of, and an extinction coefficient of an 1% solution in an 1 cm cuvette of 71.0 at 294 nm was used for α-tocopherol (Jensen and Nielsen, 1996).

2.9. Feed mineral and quality analysis

Feed and pasture samples were analyzed at the Dairy One, Inc. Forage Testing Laboratory (Ithaca, NY) with wet chemical procedures. Crude protein content was determined using AOAC Method 990.03 (AOAC, 1990). Heat-stable, α-amylase-treated, sodium sulfite NDF was determined using an ANKOM fiber analyzer (ANKOM Technology Corporation, Fairport, NY) based on procedures describe b