Vaccine preparation and immunization
C. perfringens toxin preparation (P4039, Sigma-Aldrich, Bornem, Belgium) was either used as native toxin or treated with formaldehyde to generate a formaldehyde toxoid. Inactivation was obtained by adding a combination of 0.4 % formaldehyde solution (Sigma-Aldrich) and 0.05 M L-lysine (Sigma-Aldrich) and incubation at 37 °C for two days. The addition of 0.05 M L-lysine has previously been shown to preserve the antigenicity of alpha toxin during toxoid formation [19]. Inactivation of alpha toxin was confirmed by spotting 5 μl drops on 2 % egg yolk Columbia agar plates (Oxoid, Wesel, Germany), followed by incubation for 16 h at 37 °C [29]. Native and formaldehyde inactivated toxins were formulated with the adjuvant Quil A (Brenntag Biosector, Frederikssund, Denmark) at a final concentration of 350 μg antigen and 750 μg Quil A in 1.5 ml phosphate buffered saline (PBS) per animal and filter-sterilized using a 0.2 μm filter. A standard formalin inactivated multivalent commercial vaccine was used according to the manufacturer’s instructions (Covexin 10®, Zoetis, Louvain-la-Neuve, Belgium).
For immunization six 2-months old male Holstein Friesian calves were used. The calves were purchased from a local tradesman which collects dairy calves from herds in Eastern Flanders. They were housed on straw and received water and hay at libitum, and concentrates adjusted to the body weight.
For each antigen, two calves were immunized subcutaneously in the neck. The calves received a primer vaccination at the age of two months, with booster immunizations 14 and 28 days later. No strong adverse reactions were observed. Although no fever (>39.5 °C) was induced, all calves experienced a mild hyperthermia for two days following the vaccination. As described in the drug information leaflet of the commercial vaccine, localized swelling occurred at the site of injection. This effect was more pronounced in the calves vaccinated with the commercial formaldehyde inactivated clostridial vaccine (7–10 cm diameter) as compared to the calves vaccinated with either native toxins or the L-lysine protected, formaldehyde inactivated toxins (0–6 cm diameter). Blood samples were taken before primer vaccination and two weeks after the final booster vaccination.
SDS-PAGE and Western Blot
The proteins present in the toxin preparation were visualized on a 12 % SDS-PAGE followed by Coomassie Briliant Blue staining (Sigma-Aldrich). For the Western Blot analysis, 16 μl of cell-free supernatants of the C. perfringens strain JIR325 (10x concentrated using Vivaspin, Sartorium Stedim Biotech GmbH, Goettingen, Germany) or 6 μg of the C. perfringens toxin preparation were loaded on a 12 % SDS-PAGE. The proteins from the gel were transferred to nitrocellulose membranes of 0.45 μm pore size. Non-specific binding to the blots was blocked with 5 % skimmed milk powder in PBS, followed by overnight incubation at 4 °C with a 1/500 dilution of the immune sera collected two weeks after the final booster immunization. For this incubation step, the sera of the 2 animals that were vaccinated with a given vaccine preparation were pooled. Blots were washed with 0.1 % tween 20 in PBS and incubated for 1 h at room temperature with horseradish peroxidase-labelled rabbit-anti-bovine IgG (Sigma-Aldrich). Blots were developed with CN/DAB substrate kit (Thermo Fisher Scientific, Rockford, USA). The test was performed in triplicate. The specific immunoreactive protein bands were identified in the parallel-run Coomassie stained gel followed by MALDI analysis.
Enzyme-linked immunosorbent assay
The immune response following vaccination was also measured by ELISA using serum samples two weeks after the final booster immunization.
Alpha toxin-specific antibody levels were determined by the end-point dilution method using a blocking ELISA (Clostridium perfringens alpha toxin serological ELISA kit, Bio-X Diagnostics, Jemelle, Belgium). For each ELISA, sera were used at a dilution 1:50 and assays were performed in duplicate. The specific antibody level of the immune serum was expressed as the percent inhibition (% inhib) by means of the following formula: % inhib = [(OD neg – OD sample)/OD neg] * 100.
Perfringolysin O-specific antibody levels were measured using an indirect ELISA. Briefly, 96-well microtitre plates (Nunc MaxiSorp, Thermo Fisher Scientific) were coated with 20 μg recombinant perfringolysin O [30]. Non-specific binding was blocked with 1 % (w/v) bovine serum albumin (Sigma-Aldrich) in PBS. Two-fold dilutions of the sera ranging from a dilution of 1:50 to 1:51200 were added to the plates (100 μl of each dilution/well; in duplicate) and incubated for 2 h at 37 °C. Plates were washed with 0.1 % (v/v) Tween 20 in PBS and incubated for 1 h 30 min at 37 °C with horseradish peroxidase-labelled rabbit-anti-bovine IgG (Sigma-Aldrich). Bound conjugate was detected using the substrate 3,3′,5,5′-Tetramethylbenzidine (TMB) (Sigma-Aldrich). The reaction was blocked with H2SO4 and the absorbance was measured at 450 nm using a microplate reader (Multiscan MS, Thermo Labsystems, Helsinki, Finland). The end-point titer is expressed as the reciprocal of the last dilution that gave a reading of 0.1U above background (precolostral neonatal bovine serum).
Intestinal loop model
To study the protection against C. perfringens-induced necrosis provided by the antisera from calves vaccinated with the vaccine preparations, four intestinal loop experiments were performed. Intestinal loop experiments were performed according to a previously described protocol using 4 healthy male Holstein Friesian calves, purchased from a local tradesman which collects dairy calves from herds in Eastern Flanders [20]. Briefly, the calves were anesthetized and the small intestine was exteriorized. Per calf 80 intestinal loops of approximately 10 cm were ligated in the jejunum and a 5 cm space was left between the loops. Only half of the loops were injected, thus each time leaving one intervening loop to avoid leakage between sampled loops. For each vaccine preparation individual pre- and post-vaccination sera of 2 calves were used in two intestinal loop experiments. Intestinal loops were inoculated with 20 ml of a wild-type strain (JIR325) in combination with 10 ml of 25 % commercial milk replacer suspended in sterile NaCl solution, resulting in a total volume of 30 ml which was the same across all treatments and control loops. Prior to inoculation pre- or post-immune serum derived from calves immunized with the different vaccine preparations was added to the NaCl solution containing milk replacer, to obtain a final concentration of 6 % serum (v/v). In each calf five intestinal loops per test serum were injected. Also an equal number of control loops without addition of serum were injected either with C. perfringens (positive control) or with sterile bacterial growth medium (negative control). After injection of the loops, the abdomen was closed and the calves were maintained under anesthesia. At 5-h post-inoculation intestinal biopsy samples were taken, after which the animals were euthanized. Samples were fixed in 4 % phosphate buffered formaldehyde. They were embedded in paraffin wax, sectioned and stained with hematoxylin-eosin. The sections were evaluated in a blinded manner by a board certified pathologist for presence of tissue necrosis (0 = absence of necrosis, 1 = necrotic lesions present).
In vitro neutralization and cytotoxicity tests
Neutralization of alpha toxin activity on egg yolk lipoproteins in vitro
The alpha toxin activity was determined by its effect on egg yolk lipoproteins as previously described [31]. Therefore, fresh egg yolk was centrifuged (10,000 × g for 20 min at 4 °C) and diluted 1:10 in PBS. The ability of the sera to neutralize the alpha toxin activity was assessed by pre-incubating a two-fold dilution series of the sera (two wells per dilution) with a constant amount of alpha toxin (10 μg/ml recombinant alpha toxin in PBS solution) for 30 min at 37 °C prior to the addition of 10 % egg yolk emulsion. Recombinant alpha toxin was expressed in E. coli using the pBAD TOPO® TA Expression Kit (Invitrogen, Paisley, UK) followed by purification onto a Ni-sepharose column (His Gravitrap, GE Healthcare, Buckinghamshire, UK). After incubation of the plates at 37 °C for 1 h, the A620 was determined. Alpha toxin activity was indicated by the development of turbidity which results in an increase in absorbance. The inhibitory capacity of the antiserum was determined by applying a Hill function to the concentration-response data (GraphPad Prism 5, GraphPad Software, San Diego, CA, USA) and expressed as the dilution that inhibited 50 % of the alpha toxin activity. The test was performed in duplicate.
Neutralization of perfringolysin O activity in vitro
Perfringolysin O (PFO) activity was determined by measuring the hemolysis of horse erythrocytes using a doubling dilution assay as previously described [32]. The PFO titer is the reciprocal of the last dilution which showed complete hemolysis. Similar to the inhibition of the alpha toxin activities, the ability of sera to neutralize the PFO activity was assessed by pre-incubating a two-fold dilution series of the sera (two wells per dilution) with a constant amount of perfringolysin O (2 μg/ml recombinant perfringolysin O). Recombinant perfringolysin O was produced as previously described [33]. The inhibitory capacity of the antiserum was expressed as the highest dilution that inhibited perfringolysin O induced hemolysis. The test was performed in duplicate.
Endothelial cell cytotoxicity assay
Primary bovine umbilical vein endothelial cells (BUVEC) were isolated from umbilical cord veins by an adaptation of the method of Jaffe et al. as performed previously [10, 34]. The toxicity of C. perfringens supernatant towards cultured bovine endothelial cells has been reported previously [10]. The ability of the antisera to neutralize the C. perfringens cytotoxicity towards BUVECs was determined using a Neutral Red Uptake assay (NRU) [35]. Therefore, a two-fold dilution series of the sera (100 % - 0.4 %) prepared in serum free cell culture medium was pre-incubated for 30 min at 37 °C with an equal amount of C. perfringens supernatant. Cells were treated with 100 μl of these supernatant-serum mixtures. The inhibitory capacity of the antiserum was expressed as the highest dilution that yielded 80 % cell viability. As a positive control, cells were treated with C. perfringens supernatant which was pre-incubated for 30 min with serum free cell culture medium. Untreated cells, incubated with serum free cell culture medium served as a negative control. The test was performed in duplicate.
Statistical analysis
The 20 loops tested for each condition provided enough statistical power to detect a 40 % reduction in the development of necrotic lesions in the intestinal loop assay (95 % confidence, 80 % power) (Winepiscope 2.0).
The protective effect of the antisera in the intestinal loop assay as compared to the pre-immune sera and the untreated control loops were determined by a Fisher’s exact test (GraphPad Prism 5 software). Differences between groups were considered significant at p < 0.05.
