Degree Type

Dissertation

Date of Award

2013

Degree Name

Doctor of Philosophy

Department

Veterinary Microbiology and Preventive Medicine

First Advisor

Jeffrey J. Zimmerman

Abstract

Surveillance is the primary means to generate information on the presence and/or distribution of pathogens in animal populations. Fulfillment of this function is ultimately dependent on the program's ability to collect, test, aggregate, and analyze data from individual samples. The problem addressed in this dissertation is whether oral fluid specimens could be used to surveillance and monitor individually-based and pen-based for porcine reproductive and respiratory syndrome virus (PRRSV) infection. In particular, but not exclusively, the aim of this research was to evaluate the oral fluid emzyme-linked immunosorbent assay (ELISA) could be used to detect anti-PRRSV antibody isotypes. This problem was addressed in the logical series of experiments described below.

The objective of the Chapter 3 was to evaluate a commercial serum antibody ELISA performance that was modified to detect anti-PRRSV antibodies in pen-based oral fluid specimens. Experimental and field oral fluid samples from defined PRRSV infection status were used to derive the kinetics of detectable concentrations of antibody against PRRSV. IgM and IgA were readily detected in oral fluid specimens from populations in which PRRSV infection was synchronized among all individuals, but not in samples collected in commecial herds. In contrast, IgG was readily detected at diagnostically useful levels in both experimental and field samples for up to 126 days (duration of the study). Estimates of the IgG oral fluid ELISA performance were based on results from testing positive oral fluid samples (n = 492) from experimentally inoculated pigs (n = 251) and field samples (n = 241), and negative oral fluid samples (n = 367) from experimentally inoculated pigs (n = 84) and field samples (n = 283). ROC analysis estimated the diagnostic sensitivity and specificity of the assay as 94.7% (95% CI: 92.4, 96.5) and 100% (95% CI: 99.0, 100.0), respectively, at a sample:positive (S/P) ratio cutoff of ≥ 0.40. The results of this study suggest that the IgG oral fluid ELISA can provide efficient, cost-effective PRRSV monitoring in commercial herds and PRRSV surveillance in elimination programs.

Chapter 4 addressed the repeatability and reproducibility of oral fluid antibody ELISA. The precision of a PRRSV oral fluid antibody ELISA was evaluated by calculating reliability coefficients for assay repeatability (within laboratory) and assay reproducibility (between laboratories). Randomly ordered oral fluid samples from known (n = 39) and unknown (n = 224) PRRSV antibody status were tested in 12 diagnostic laboratories. Each laboratory tested the samples twice, first using an antibody ELISA kit and reagents provided to them (Phase 1) and then using an ELISA kit and reagents configured in their laboratory (Phase 2). Repeatability (within laboratory) reliability coefficients calculated using results from samples from known PRRSV antibody status ranged from 0.724 to 0.997 in Phase 1 and 0.953 to 0.998 in Phase 2. Reproducibility (between laboratories) reliability coefficients were calculated for three conditions: Case 1 - samples of unknown status (n = 224); Case 2 - samples from known status (n = 39), and Case 3 - all samples (n = 263). Among the three cases, reliability coefficients ranged from 0.937 to 0.964 in Phase 1 and 0.922 to 0.935 in Phase 2. For Case 3, it was estimated that 96.7% of the total variation in Phase 1 and 93.2% in Phase 2 could be attributed to the oral fluid samples themselves. Overall, the PRRSV oral fluid antibody ELISA was highly repeatable and reproducible. This study supported the routine use of this test in laboratories providing diagnostic service to pig producers.

The study objective outlined in Chapter 5 was to describe and contrast the kinetics of the humoral response in serum and oral fluid specimens during acute PRRSV infection. The study was done in three trials with 24 boars used in each trial. Boars were intramuscularly (IM) inoculated with a commercial modified live virus (MLV) vaccine (Trial 1), a Type 1 PRRSV field isolate (Trial 2), or a PRRSV Type 2 field isolate (Trial 3). Oral fluid samples were collected from individual boars on day post inoculation (DPI) -7 and daily for 21 DPI. Serum samples were collected from all boars on DPI -7, 0, 7, 14, 21 and from 4 randomly selected boars on DPIs 3, 5, 10, and 17. Thereafter, serum and oral fluid were assayed for PRRSV antibody using antibody isotype-specific ELISAs (IgM, IgA, IgG) adapted to serum or oral fluid. Statistically significant differences in viral replication and antibody responses were observed among the three trials in both serum and oral fluid specimens. Anti-PRRSV antibody isotype responses in serum (IgM, IgA, and IgG) were first detected in samples collected on DPI 7, 10, and 10, respectively. Oral fluid IgM, IgA, and IgG were detected in samples collected between DPI 3 to 10, 7 to 10, and 8 to 14, respectively. This study enhanced the scientific knowledge concerning the pig's the humoral immune response to PRRSV and provided a broader foundation for understanding, interpreting, and developing oral fluid antibody-based diagnostics.

The objective for the study described in Chapter 6 was to assess oral fluid samples use when collected from piglets prior to weaning as a method to monitor sow herd PRRSV antibody and infection status. Samples originated from four ~12,500 sow herds. All four herds were considered endemically infected with PRRSV based on historic diagnostic data. Oral fluid samples were collected from 600 litters prior to weaning and serum samples from their dams after weaning. All samples were randomized and tested for PRRSV antibodies (IgM, IgA, IgG and PRRS X3 Ab Test or PRRS Oral Fluids Ab Test) and PRRSV (RT-qPCR and sequencing). Results were analyzed for associations with sow parity, litter size, and farm by analysis of variance (ANOVA) and correlation analyses. No sow serum samples were positive for PRRSV by RT-qPCR, but 9 of 600 oral fluid samples were confirmed positive by PRRSV RT-qPCR testing at two laboratories. Two were successfully sequenced (ORF5) and identified as wild-type PRRSV. This study showed that piglet oral fluid samples are a useful and sensitive approach for monitoring PRRSV circulation in endemically infected and/or vaccinated herds, as well as for monitoring the PRRSV status in PRRSV negative herds.

Copyright Owner

Apisit Kittawornrat

Language

en

File Format

application/pdf

File Size

116 pages

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