Animals and serum samples
A total of 395 pigs (405 samples) were included in the study. The pigs consisted of 2 groups. Group 1 (n = 334) included 152 boars from four German boar studs, 67 boars from one Austrian boar stud, 35 fatteners from one German pig breeding farm and 57 sows and gilts as well as 23 nursery piglets from two Austrian pig breeding farms. All farms were classified as PRRSV negative (category IV according to Holtkamp et al. [12]). Group 2 included a total of 71 samples from the following pigs: a) 39 fatteners from one Austrian and one German PRRSV positive fattening farm, b) 12 nursery piglets injected with a PRRSV type 2 strain at pre-vaccine stage and c) 20 fatteners challenged with a highly pathogenic PRRSV type 2 strain. Ten of the pigs mentioned under c) were the same as in b) and used twice for sampling with a time lag of 28 days between both sampling times. A blood sample was taken from each pig. All blood samples, except of the pigs mentioned under b) and c) in group 2, were collected in the course of monitoring programs and not taken for the purpose of this study. Housing, animal care and experimental protocol of the pigs mentioned under b) and c) were approved by the local ethics committee (Agency of the Government in Lower Austria, Department of Agrarian Law). Blood samples were centrifuged for 10 minutes at 2400 g within 4 hours after sampling and serum was kept frozen at minus 20°C until analysis.
Collection and handling of oral fluid samples
Oral fluid was collected from the above mentioned pigs in two different ways while they were fixated for blood sampling or, in case of the boars, during semen collection:
1. Individual oral fluid samples were collected via cotton gauze swab. For this purpose, the swab was held into the mouth of the respective pig with a serrefine and the oral fluid was allowed to soak into the swab (Figure 1a). The swabs were stored in a 50-ml-falcon tube at minus 20°C until re-collection of the oral fluid and analysis. For the re-collection of the oral fluid, the swab was centrifuged for 10 minutes at 2500 g in a 50-ml-falcon tube with filter (Figure 1b).
2. Individual oral fluid samples were collected via GenoTubes (Figure 2a). The GenoTubes soaked with oral fluid were stored at room temperature up to four weeks until analysis.
Group 1: In 289 pigs oral fluid could be collected in both described ways. In 22 pigs (boars from Austria) only cotton gauze swabs were used and in another 23 pigs (nursery piglets from Austria) only GenoTubes were utilised. Group 2: Oral fluid samples were collected from 35 pigs both via cotton gauze swab and via GenoTube. In another 4 pigs only cotton gauze swabs and in 22 pigs (32 samples) only GenoTubes were collected.
Oral fluid collection was done on the same day that the blood samples were taken from the respective pigs.
Detection of PRRSV antibodies by ELISA
All serum samples were analysed with the IDEXX PRRS X3 Ab test for the presence of antibodies against PRRSV.
All oral fluid samples were analysed with the IDEXX PRRS OF ELISA, designed for detection of antibodies against PRRRSV in oral fluid. To test the reproducibility of results, samples of group 1 were tested in two different measures. The capacity of the foam swab of the GenoTube was measured experimentally. For this reason, 10 GenoTubes were dived into oral fluid for some seconds and the amount of fluid soaked into the swab was measured with weighing. The average was at approximate 200 μl with no considerable deviation. To reconstitute the dried oral fluid, the foam swab of the GenoTube was re-suspended in a 1.5 ml microcentrifuge tube with 400 μl of the dilution buffer of the ELISA kit (Figure 2b) which means a 1:2 dilution of the contained oral fluid as is required in manufacturer’s instructions. To remove the remaining oral fluid from the foam swab, the GenoTubes were centrifuged for 10 minutes at 2500 g after removing the SafeDry medium from the tube (Figure 2c). The gained fluid was added into the respective microcentrifuge tube.
All serum and oral fluid ELISAs were conducted according to the manufacturer’s instructions. A brief description of the IDEXX PRRSV OF ELISA is given in [1]. In both ELISAs, samples with sample-to-positive (S/P) ratios ≥0.4 (cut-off value) were considered positive for PRRSV antibodies.
Statistical analysis
The specificity of the IDEXX PRRS OF ELISA in oral fluid from cotton gauze swabs and GenoTubes compared to the IDEXX PRRS X3 Ab test in serum was estimated using group 1. The sensitivity of the IDEXX PRRS OF ELISA in oral fluid from cotton gauze swabs and GenoTubes compared to the IDEXX PRRS X3 Ab test in serum was tested using the samples from group 2. The correlation of S/P values of the ELISAs were tested in group 2 with the correlation coefficient after Spearman. Over all samples, the accuracy of the IDEXX PRRS OF ELISA in oral fluid from cotton gauze swabs and GenoTubes was calculated. In measure one, the agreement of the IDEXX PRRS OF ELISA in oral fluid from cotton gauze swabs and GenoTubes with the IDEXX PRRS X3 Ab test in serum was determined with the kappa coefficient (κ) and interpreted according to Landis and Koch [13].