Date of Award
Doctor of Philosophy
Veterinary Microbiology and Preventive Medicine
Jeefrey J. Zimmerman
The use of oral fluid for the assessment of health and diagnosis of disease in humans and animals has a surprisingly long history. Early investigators attempted to evaluate metabolic diseases in humans by testing oral fluid for a variety of analytes, e.g., glycogen, crystal salts, and acid salts. This early work led to the conclusion that the "principals" present in serum were also present in saliva. As early as 1909, sensitive and specific agglutination of "Micrococcus melitensis" (Brucella melitensis) by oral fluid from patients diagnosed with Malta Fever was reported, thereby, indirectly demonstrating the presence of antibody in saliva. After these early reports, developments in oral fluid diagnostics were generally overshadowed by technical improvements in the detection of analytes in blood or serum. This began to change following a report of the detection of antibodies against human immunodeficiency virus (HIV) in oral fluid from patients with acquired immunodeficiency syndrome (AIDS). Because of this report and/or because of concurrent advances in diagnostic technology, oral fluid-based assays for a wide range of infectious and non-infectious diseases, drugs, hormones, and disease markers have been developed and implemented in the last two decades.
The objective of the first study was to determine whether PRRSV and/or anti-PRRSV antibodies were present in oral fluids at diagnostic levels. The level and duration of PRRSV and anti-PRRSV antibodies in serum and oral fluids was evaluated in three age groups of pigs (4, 8, or 12 weeks of age) inoculated with a type 2 (North American) PRRSV isolate. Serum, buccal swabs, and pen-based oral fluid samples were collected for 63 days following inoculation. Specimens were assayed for PRRSV by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), and for anti-PRRSV antibodies by enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody (IFA). PRRSV was detected by real-time qRT-PCR in serum for approximately 5 weeks and in oral fluids for approximately 4 weeks postinoculation. Pig age at the time of inoculation had no effect on the quantity or duration of virus in oral fluid samples. Low levels of anti-PRRSV antibody were detected in oral fluid samples by ELISA and IFA. Although the approach remains to be validated in the field, the results of this experiment suggest that pen-based oral fluid sampling could be an efficient, cost-effective approach to PRRSV surveillance in swine populations.
The objective of the second study was to validate the use of oral fluids to detect infections with porcine respiratory and reproductive syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) in three commercial swine herds. Oral-fluid and serum samples were collected from one barn on each of three PRRSV-infected finishing sites. Six pens per barn (20 to 30 pigs per pen) were sampled repeatedly, beginning when the pigs entered the facilities (3 weeks of age), and then at 5, 8, 12, and 16 weeks of age. Serum samples were tested using a commercial PRRS ELISA. Both serum and oral-fluid samples were tested for PRRSV by quantitative reverse-transcriptase polymerase chain reaction (PCR), and oral fluids were tested for PCV2 by qRT-PCR. Site One pigs seroconverted to PRRS at 12 weeks of age, and Site Two and Three pigs at 5 to 8 weeks of age. All individual serum samples tested PCR-negative for PRRSV in pigs 3 to 5 weeks old, while > 1 sample tested positive in pigs 8, 12, and 16 weeks old, with 77% agreement between oral-fluid and serum pen-level results. At all sites, > 1 oral-fluid sample tested PCR-positive for PCV2 beginning when pigs were 8 weeks old. Oral-fluid samples may be used to monitor PRRSV and PCV2 infections in commercial production systems. PRRS virus is detectable in oral fluids for 3 to 8 weeks, and PCV2 may be detectable for > 8 weeks. Sampling at 2- to 4-week intervals is recommended for surveillance of PRRSV and PCV2.
The objective of the third study was to evaluate the stability of porcine reproductive and respiratory syndrome virus (PRRSV) and anti-PRRSV antibodies in oral fluid as a function of time and temperature. A 4-liter pool of swine oral fluid was collected from 16-week old finisher pigs. To ensure uniform, quantifiable levels of virus and antibody over time, the pool was "spiked" with 4 ml of PRRSV isolate ISU-P containing 1 x 1012 RNA copies per ml and 10 ml of concentrated hyper-immune anti-PRRSV antibodies. The pool was divided into 3 equal portions: (1) no treatment; (2) chlorhexidine digluconate at 0.01% by volume; (3) isothiazolinone at 3 parts per million. Each treatment was run in triplicate at each of five temperatures (-20yC, 4yC, 10yC, 20yC, 30yC). Samples were removed at specific intervals (0 hr, 12 hr, 24 hr, 48 hr, 72 hr, 144 hr, 216 hr, and 288 hr), stored at -80yC, and then assayed for: (1) PRRSV RNA; (2) IgM, IgA, and IgG; (3) ELISA-detectable PRRSV-specific antibody; (4) culturable bacteria per ml. The results showed that the stability of anti-PRRSV antibody and PCR-detectable PRRSV was highly temperature-dependent, with antimicrobial treatment providing no improvements in stability at lower temperatures. In particular, both virus and antibody were stable at ≤ 10yC over 12 days of storage. Conventional serum storage protocols (freezing or refrigeration at 4yC) will preserve PRRSV and anti-PRRSV antibody in oral fluid diagnostic samples.
The objective of the fourth study was to evaluate the onset, level, and duration of quantitative polymerase chain reaction-detectable PCV2 and anti-PCV2 antibody in oral fluid was evaluated using samples collected from experimentally-inoculated pigs for 98 days post inoculation (DPI). Pigs (n = 24) were obtained at 3 weeks of age and randomly allocated to 4 treatment pens of 6 pigs each: (1) negative control group; (2) inoculated with PCV2a (strain ISU 40895) on DPI 0; (3) inoculated with PCV2a (strain ISU-40895) on DPI 0 and re-challenged at DPIs 35 and 70; (4) inoculated with PCV2a (ISU-40895), PCV2b (PVG4072), and PCV2a (ISU-4838) on DPIs 0, 35, and 70, respectively. Serum was collected from each animal and one oral fluid sample was collected from each pen (group) every other day from DPI 2 through DPI 14 and weekly through 98 DPI. Oral fluid samples were assayed for the presence of PCV2 by PCR, anti-PCV2 IgG antibody by enzyme linked immunosorbent assay (ELISA), and anti-PCV2 antibody isotypes (IgA, and IgM) by ELISA. Serum was assayed for anti-PCV2 IgG by ELISA. Anti-PCV2 antibodies (IgG, IgM, and IgA) were detected in oral fluid from experimentally inoculated pigs from 14 to 98 DPI. PCV2 was detected by PCR in oral fluid samples from all pens of inoculated pigs at 2 DPI. Thereafter, PCV2 was detected in oral fluid throughout DPI 98. Overall, the data indicated that PCV2 infection in swine populations can be efficiently monitored using oral fluid specimens.
John Rodger Prickett
Prickett, John Rodger, "Detection of viral pathogens of swine using oral fluid specimens" (2009). Graduate Theses and Dissertations. 11013.