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문제 정의
- A recent study demonstrated the usefulness of oral fluid samples for detection of Porcine reproductive and respiratory syndrome virus (PRRSV) and anti-PRRSV antibody (28). The present report describes a field-based longitudinal cohort study to assess the utility of pen-based oral fluid samples as an alternative to serum for herd PCV2 testing.
제안 방법
- A commercial sandwich ELISA (INGEZIM Circovirus IgG®/IgM®, INGENASA, Madrid, Spain), which is labeled for detection of both IgM and IgG specific for PCV2 in a differential manner (25), was utilized to test serum at 1:100 dilution by following the recommended manufacturer’s procedure.
- Three different ELISA formats were used in the current study. An indirect ELISA (hereafter, ISU ELISA), which was designed to detect IgG antibody specific for the capsid protein [ORF (open reading frame) 2 product] of PCV2 (24), was performed on serum at 1:100 dilution and on oral fluid samples at 1:100 and 1:5 dilutions. A commercial blocking ELISA (SERELISA® PCV2 Ab Mono Blocking, Synbiotics Corp.
- Fig 2. Anti-Porcine circovirus-2 IgG antibody responses in serum (A, C, E) and oral fluid samples (B, D, F) as determined by indirect fluorescent antibody test (A, B), ISU ELISA (C, D), and SERELISA (E, F). While serum samples were diluted 1:100 or 1:10 for ISU ELISA and SERELISA, respectively, as recommended by the manufacturer or developer, oral fluid samples were diluted 1:5 for testing.
- INGEZIM ELISA was not used because of its low sensitivity in detecting anti-PCV2 antibody in serum. As overall ELISA results correlated better with IFA results at the lower dilution of oral fluid samples, which was also the case for the serum samples, all results described below for oral fluid samples were from the assays using the modified procedure (i.e., low dilution).
- Nucleic acid sequences of ORF2 were aligned and analyzed using Lasergene® MegAlign software (DNASTAR Inc., Madison, WI, USA).
- Despite these advantages, a few disadvantages have been identified with using oral fluid samples: a) a relatively low concentration of antibody compared to serum; b) the tendency of rapid antibody or agent degradation by bacterial growth and proteolytic enzyme breakdown; and c) lack of standardized antibody detection procedure (9,11). To circumvent these difficulties, modifications (e.g., pretreatment of samples, and different dilution of sample) need to be made in test procedures to optimize the analytic and diagnostic performance of the tests. In the current study, oral fluid samples were tested at a lower dilution (1:5) than recommended (i.
대상 데이터
- A total of 90 samples (18 pens × 5 sampling points) of each type (i.e., oral fluid or pooled serum) were collected for the study.
- On 3 finishing sites that were historically known through laboratory diagnostic testing to be exposed to PCV2, pens of approximately 20-30 pigs each were monitored for PCV2 infection using oral fluid and serum samples. Six pens at each of the 3 sites were selected to be monitored. Samples (serum and oral fluid) were collected when pigs entered the facilities at 3 weeks of age, and subsequently at 5, 8, 12, and 16 weeks of age.
데이터처리
- , Cary, NC, USA). The repeated measurements of the level of antibody and viral genome in serum and oral fluid samples were analyzed with repeated measures ANOVA. In addition, the performance of the different serologic tests was compared by calculating % agreement with IFA results and kappa (κ) value as follows:
성능/효과
- At 16 weeks of age, the level of viremia ranged from 2 × 102 to 8 × 106 FFU equivalent/ml when pooled serum samples were tested. Based on the squencing data, all of the viruses circulating in all 3 sites were PCV2 of North Aerican genotype (PCV2a or PCV2 genotype 2), and their sequence homologies ranged from 99.6% to 100%.
- None of the study pens, with the exception of 1 pen each in sites 1 and 2, was seropositive for PCV2 at 12 weeks by IFA and SERELISA. However, all pens in site 1 and 3-5 pens in sites 2 and 3 became seropositive for PCV2 at 16 weeks by IFA and ISU ELISA, whereas no site was seropositive for PCV2 by SERELISA, suggesting different levels of sensitivity among the tests.
- In contrast, INGEZIM ELISA (designed to detect both IgG and IgM in a differential manner) was inefficient for detecting PCV2- specific IgG as compared to the other serologic assays employed in this study since only 6 of the 90 samples were determined to be positive for anti-PCV2 IgG by this assay (Table 2, Fig 3A). The assay determined all except 6 pens in site 2 to be positive for IgM specific for PCV2 (Table 2).
- 296), which indicated a fair level of agreement. Much less agreement in test results (49%) was observed between ISU ELISA and SERELISA, which could be attributed to the higher sensitivity of ISU ELISA. IFA and ISU ELISA represented a 76% agreement in test results (κ = 0.
- The lowest agreements in test results were observed between IFA and INGEZIM ELISA (49% agreement, κ = 0.099) and between ISU ELISA and INGEZIM ELISA (31% agreement, κ = 0.059).
- In contrast, results of SERELISA were not greatly influenced by the dilution factor. The recommended dilution (1:10) for SERELISA on serum samples slightly reduced the sensitivity of the test, as 92% agreement was observed between IFA and SERELISA.
- Using a lower dilution, a good agreement in test results was observed between IFA and SERELISA (93% agreement; κ = 0.87) and between IFA and ISU ELISA (88% agreement, κ = 0.75; Table 5).
- With such a modification, IFA, SERELISA, and ISU ELISA showed a relatively good agreement regardless of whether they were performed on serum (49-76% agreement; κ = 0.19-0.47) or oral fluid samples (88-93%; κ = 0.75-0.87).
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