Serum samples
Cattle with known Erysipelothrix exposure
Two adult Angus feedlot cattle, part of the Iowa State University teaching herd and located in Ames, Iowa, USA were vaccinated with a commercial vaccine licensed for pigs (MaGESTic® 7 with SPUR®; Intervet Inc, DE, USA, serial number: 0784A009A) twice with 14 days interval by injecting 2 ml of the vaccine intramuscular into the neck area. Blood samples were collected before immunization (day 0) and at days 14, 28 and 42 post vaccination (dpv), centrifuged and the serum was collected and used for further analysis. The two cattle remained in the teaching herd after the experiment ended. In addition, 10 archived serum samples from gnotobiotic calves from an unrelated study (kindly provided by Dr Geraldine Taylor, Pirbright Institute, Surrey, United Kingdom) were used as controls.
Field samples
A total of 200 US feedlot cattle serum samples submitted for routine diagnostics were obtained from the Veterinary Diagnostic Laboratory at Iowa State University (kindly provided by Dr David Bauma and 370 serum samples were obtained from Scottish dairy cattle through the Scottish Agricultural College (SAC) (kindly provided by Dr Jill Thomson). All animals were at least one year old (adults), healthy and had no clinical signs. The serum samples corresponded to 23 US farms with an average of 8.6 animals per farm (min=1, max=15, median=10) and 70 Scottish farms with an average of 5.3 animals per farm (min=1, max=15, median=3).
Serological assays
ELISA and FMIA development
The recombinant protein rSpaA415, based on the major surface protective antigen A (SpaA) [6, 21] was used as antigen in both ELISA and FMIA. The optimal dilution of the serum sample and regents was determined by a checkerboard titration in both assays.
For the FMIA, 18 µg of the polypeptide rSpaA415 was coupled to 5 million of microbeads (bead region 45, Luminex Corp., TX, USA) using a 2 step carbodiimide reaction [21]. All the incubations were carried out for 30 min at room temperature, in the dark and under continuous shaking. Washing steps were performed using a magnetic separator and 200 μl of washing buffer composed of PBS with 0.05% of Tween 20, pH 7.4 (PBST).
Coupled beads were diluted in blocking buffer (StabilGuard; Surmodics, MN, USA) to a final concentration of 2500 beads per well and 50 μl of this solution were incubated with 50 μl of each serum sample diluted 1:800 in assay buffer (PBST containing 10% goat serum). The plate then was incubated and washed three times with washing buffer. Following this, 50 μl of a 20,000 dilution of a biotin conjugated goat anti-bovine IgG (Jackson Immuno Research, Cambridge, UK) was added to the wells and incubated for another 30 min. After three washings, 50 μl of streptavidin R-phycoerythrin conjugate (SAPE; MOSS, MD, USA) at 1:100 dilution in assay buffer were added to the wells. Finally, after the last incubation and washing steps, beads were re-suspended in 100 μl of assay buffer and analysed using a MAGPIX® reader system (Luminex corp., TX, USA).
The in-house ELISA was performed in 96 well plates (MaxiSorpTM; Nunc, NY, USA) coated with 0.3 µg/ml of rSpaA415 polypeptide and blocked using PBS with 10% chicken serum (Biowest, MO, USA) for 2 h at room temperature. The samples were diluted at 1:800 with PBS containing 10% rabbit serum (Biowest, MO, USA) and 100 μl was added to each well of the plate and left to incubate for 30 min at 37 °C. After washing the plates, 100 μl of a 1:30,000 dilution of peroxidase-conjugated rabbit anti-bovine (Jackson Immuno Research, Cambridge, UK) was added to each well and left to incubate for 30 min at 37 °C. After the final washing, 100 μl of tetramethylbenzidine-hydrogen peroxide (TMB) was added to each well as a substrate (KPL, MD, USA) and left for 15 min for colour to develop. The peroxidase reaction was stopped by adding 50 μl of 1% HCl solution (KPL, MD, USA) into each well. The optical density was then read at 450 nm using a spectrophotometer.
Growth agglutination test
The growth agglutination test was conducted to determine the agglutinating antibody titres of the sera as described elsewhere [3, 17-18]. Briefly, 50 μl of each serum sample was incubated for 1 h at 37 °C with 50 μl of 0.2 M 2-Mercaptoethanol (Sigma Aldrich, MO, USA). Two-fold dilutions (1:2 to 1: 2048) of the serum were made in brain heart infusion broth supplemented with 0.1% Tween 80 (BHI-T80), 0.3% Tris-HCl (pH 8.0), kanamycin (100 µg/ml) and gentamicin (50 µg/ml) in a 96 well plate. Following this, 50 μl of the E. rhusiopathiae strain E1-6P (serotype 1a), diluted 1:10 after an 18 h incubation in BHI-T80, was added to each of the wells. The agglutination was read after incubation at 37 °C for 18 h. The titres were expressed as the reciprocal of the number of the highest dilution of serum that showed agglutination.
Data analysis
Statistical evaluations were performed with the package SPSS (v.19). Statistical significance was set at a p value of <0.05. The level of agreement between tests was determined using Cohen’s Kappa and the percentage of agreement. Pearson coefficient was used to measure the level of correlation between tests. Kappa coefficients (k) with values between 0-0.01, 0.02-0.2, 0.21-0.4, 0.41-0.6, 0.61-0.8 and 0.81-1 were interpreted as no agreement, slight, fair, moderate, substantial and almost perfect agreement respectively [29]. Marginal homogeneity of paired data was tested by McNemar’s Chi-square test [30]. Assay results were expressed as Median Fluorescent Intensity (MFI) for FMIA and Optical Density (OD) and S/P value calculated as the (average OD sample/OD positive reference control) x 100 for the ELISA. The inter-assay variance was calculated using the coefficient of variance (CV) for a positive and a negative. CV with values <10%, 10%-15%, 15%-20%, > 20% were considered excellent, good, acceptable and excessive. A set of field samples (n=163) were chosen based on MFI and OD values and all available controls (n=18) and GAT was used as reference test to classify samples as positive or negative and MFI and S/P value were analysed in a ROC curve to generate an optimised cut-off [31].