{"id":{"repo_id":"iastate","oai_identifier":"oai:dr.lib.iastate.edu:20.500.12876/YvkA6Roz"},"canonical_url":"https://search.dev.ndltd.org/etd/iastate/oai:dr.lib.iastate.edu:20.500.12876/YvkA6Roz","repository":{"repo_id":"iastate","name":"Iowa State University","base_url":"https://dr.lib.iastate.edu/server/oai/request"},"display":{"title":"Assessment of Tonsil-Oral-Scrubbing (TOSc) to detect PRRSV RNA in sows and application of TOSc for PRRSV dynamics in breeding herds","abstract":"Porcine reproductive and respiratory syndrome virus (PRRSV) continues to pose a significant threat to the global swine industry. One major problem for PRRSV control and elimination in the breeding herd is the persistence of PRRSV at herd level, increasing time to stability (Linhares et al., 2014; Osemeke et al., 2022), which is defined as when there is diagnostic evidence of a sustained lack of viremia in pigs at weaning for 13 consecutive weeks according to the American Association of Swine Veterinarians (AASV) (Holtkamp, 2021). There are several well-established sample types including processing fluid (PF) (López et al., 2022), serum (Almeida et al., 2021), and family oral fluids (FOF) (Almeida et al., 2021) for PRRSV surveillance in the breeding herd. These samples majorly target suckling piglets and miss most of the breeding herd. As the sows constitute one of the major sources of virus transmission to the piglets (Cano et al., 2009; Kang et al., 2010; Pileri et al., 2016), it is crucial to establish strategies to monitor and characterize the PRRSV dynamics in the sow population to have a holistic picture of PRRSV activity in the whole herd. Currently, the most commonly used sample types for sows in the US are serum and tonsil scraping. These require snaring the sows and are labor-intensive and animal welfare unfriendly to obtain (Allende et al., 2000; Horter et al., 2002). Retrieving oral fluids (OF) from individual sows under field conditions is challenging because not all sows willingly respond to the rope (Pepin et al., 2014). A novel Tonsil-Oral-Scrubbing (TOSc) sampling method was recently developed, which retrieve fluids within seconds from the sow’s oral and tonsillar area without the necessity of snaring (Li et al., 2024). That study reported PRRSV RNA detection rate in TOSc samples similar to that of tonsil scraping samples and significantly higher than serum in acutely infected sows. However, as a newly developed sample type, it is crucial to characterize the composition of TOSc samples, to optimize the best practice of TOSc collection process , and to evaluate the suitability of TOSc samples for detecting PRRSV RNA in sows across different conditions before applying TOSc samples to investigate the PRRSV dynamics in the sows. This dissertation compared TOSc samples with other commonly used sample types for PRRSV RNA detection in sows over time; determined best practice for collecting TOSc samples; characterized the TOSc sample composition, and provided a visualization of the TOSc sample collection process; evaluated the effects of different processing procedures on PRRSV RNA detection of TOSc samples in the lab; and then investigated PRRSV vertical transmission and PRRSV dynamics in sows over time and by employing TOSc-based monitoring in breeding herds. Specifically, chapter 2 compared TOSc with serum, OF, and tonsil scraping to detect PRRSV RNA by real-time reverse transcription polymerase chain reaction (RT-rtPCR) from each of the 61 conveniently selected sows at 30-, 60-, and 90-days post live virus inoculation (LVI) with a wild-type PRRSV in one breeding herd. TOSc and tonsil scraping showed a similar decreasing PRRSV RNA detection, while the detection rates for OF and serum remained relatively constant over time. Moreover, the median Ct values of TOSc and tonsil scraping were numerically lower than OF and serum at all sampling points. Also, based on RT-rtPCR results of TOSc and tonsil scraping, most PRRSV RNA-positive sows turned negative by 90 days post-LVI, but a small proportion remained positive. However, small proportions of previously negative sows turned positive at 60- and 90-days post LVI, showing an intermittent mode of PRRSV RNA detection for both sample types in sows. Chapter 3 investigated the effect of specific TOSc collection factors on the PRRSV RNA results (detection rates and Ct values). Those factors include whether the sow was snared or not snared at TOSc collection (“snared” vs. “not snared”); whether the sow was laying down or standing at collection (“laying down” vs. “standing”); and type of collectors used for TOSc collection (“TOSc prototype” vs. “Spiral-headed AI catheter (SHAC)”). The effect for each factor was assessed in three independent studies following the same design: TOSc was collected twice from each studied sow, once with the baseline level for a factor (“not snared”, or “standing”, or “TOSc prototype”), and another time followed by the other level of the paired factor (“snared”, “laying down”, or “SHAC”, correspondingly). Results showed that “not snared” TOSc samples had numerically higher PRRSV RNA detection rate (60.7% vs. 52.5%, p=0.11), significantly lower median Ct values (31.9 vs. 32.3, p<0.01), and significantly higher sample volumethan “snared” samples (1.8 mL vs. 1.2 mL, p<0.01); “laying down” TOSc samples did not differ statistically (60.7% vs. 60.7%) in the PRRSV RNA detection rate, obtained numerically lower median Ct values (30.9 vs 31.3, p=0.19), but took 40% less collection time compared to “standing” TOSc samples; samples collected using the “TOSc prototype” had numerically higher PRRSV RNA detection rate (91.7% vs. 88.3%, p=0.27) and significantly lower median Ct values (32.8 vs. 34.5, p<0.01) than that from “SHAC”. Chapter 4 used a endoscope to visualize the TOSc collection process and histopathology to characterize TOSc sample composition. The video recording showed that after the endoscope-TOSc assembly passed the hard palate, it reached the tonsils of the soft palate. The tongue was pressing the collector towards the tonsils after swallow reflex activation. Histopathology revealed the presence of squamous mucosal cells and small clusters of immune cells, including lymphocytes, plasma cells, eosinophils and suspect macrophages in TOSc samples. Chapter 5 evaluated the effect of pooling and freeze-thaw on the probability of PRRSV RNA detection and Ct values in TOSc samples by RT-rtPCR. To test the effect of pooling on PRRSV RNA detection, 22 known RT-rtPCR-positive TOSc samples from PRRSV endemic herds were categorized into 3 groups by Ct values and diluted (pooled) with RT-rtPCR-negative TOSc samples from PRRSV negative farms at ratios of 1:3, 1:5, 1:8 and 1:10, respectively. To test the freeze-thaw effect on PRRSV RNA detection, 90 TOSc samples were conveniently collected from endemic herds. Each TOSc sample was mildly vortexed and equally aliquoted into 2 replicates after arrival at the laboratory. One aliquot was submitted for testing for PRRSV RNA by RT-rtPCR (non-freeze-thaw group), while the other aliquot was frozen at -20℃ overnight and thawed at -4℃ for 8 hours (freeze-thaw group) before submission and testing. The probability of PRRSV RNA detection decreased, and mean Ct values increased in TOSc samples with increasing dilution ratios, and the ranges of mean Ct value changes for each category were 1.4-1.8, 2.2-2.6, 2.4-2.9, and 2.8-3.6 after dilution at ratio of 1:3, 1:5, 1:8 and 1:10, respectively, compared with undiluted groups. The mean probability of PRRSV RNA detection for category A (29 < Ct < 32) remained 99% after all dilution levels, while that for category B (32 < Ct < 35) decreased to 95%, and 84% at dilution ratio of 1:8 and 1:10 respectively. For category C (35 < Ct < 38), there were significant drops in probability of detection since dilution ratio of 1:3 compared with undiluted samples (Tukey test, p<0.05). While the non-freeze-thaw group showed numerically higher probability of PRRSV RNA detection than the freeze-thaw group, the difference was not statistically significant (Tukey test, p=0.12). However, the non-freeze-thaw group showed significantly lower mean Ct values than the freeze-thaw group with a mean difference of 0.24 (Signed rank test, p<0.001). Chapter 6 described the PRRSV RT-rtPCR results (detection rate and Ct values) for each set of sows and piglet specimens within 12 hours post farrowing and investigated the probability and risk ratios for producing PRRSV-positive or PRRSV-negative live piglets based on RT-rtPCR results from sow TOSc and stillborn TF samples. At around 90 days post live-virus inoculation (LVI), 555 sows were sampled using TOSc 2 weeks pre-farrowing and tested for PRRSV RNA. From these, 59 PRRSV-positive sows, and 88 PRRSV-negative sows matched by parity were conveniently selected. TOSc from sows, blood swabs from live pigletS, and tongue fluid (TF) and serum from dead piglet were collected individually from all study litters within 12 hours post-farrowing. The pre-farrowing TOSc samples had significantly higher PRRSV positivity than TF, serum and blood swab pools, while dead piglet serum had significantly lower mean Ct values than all other sample types. TOSc PRRSV RNA postive sows had around 25% probability to produce positive live piglets while the PRRSV negative sows had around 90% probability to produce negative live piglets on the litter level. Pre-farrow TOSc, post-farrow TOSc, and serial TOSc PRRSV positive sows were 1.78 times, 2.75 times, 3.13 times more likely to produce positive live piglets than TOSc negative sows, respectively. Pre-farrow TOSc, post-farrow TOSc, and serial TOSc PRRSV negative sows were 1.14 times, 1.28 times and 2.29 times more likely to produce negative live pigletsthan TOSc positive sows, respectively. Chapter 7 characterized the dynamics of PRRSV RNA positivity in sows and associated risk factors using TOSc samples over a 24-week period. This field study was conducted in a commercial breed-to-wean farm on a plan to eliminate PRRSV by herd closure and using LVI for whole herd exposure after an outbreak. Pre- and post-LVI, periodic TOSc samplings at four-week intervals were performed on 275 conveniently selected sows with evenly distributed parity categories (Parity 0 (P0), parity 1-2 (P1-P2), and parity 3 and above (P3+)) and production phases categories (wean-breed, early gestation, middle gestation, late gestation, and farrowing) until 24 weeks post-LVI. Overall, PRRSV RNA positivity declined over time from the first sampling point- 2 days before LVI until the end of the trial. At 16 weeks post-LVI, the PRRSV RNA positivity reached 4.43%, below the defined threshold of a low prevalence scenario of 10% positivity and remained low prevalence until the end of the study. In the low prevalence scenario, there was a rebound of positivity, which was defined as an increase of mean positivity compared to the previous sampling point, to 8.00% at 24 weeks post-LVI. Parity demonstrated a significant effect on PRRSV RNA positivity in sows. At a high prevalence scenario (＞10% positivity), both P0 and P1-P2 groups constantly showed significantly higher PRRSV RNA positivity than the P3+ group (p＜0.05 for all pairwise comparisons). The P0 positivity was consistently numerically higher than P1-P2. The production phase effect was evident when the herd reached low prevalence (＜10% positivity); sows in farrowing had consistently higher PRRSV RNA positivity than sows in other production phases. The mean positivity increased to 28.56% and 34.49% in the farrowing group at 20 weeks post-LVI and 24 weeks post-LVI, respectively. To conclude, this dissertation establishes TOSc as an easy and practical sampling method for PRRSV RNA monitoring by RT-rtPCR in sows, offering advantages in collection ease, animal welfare, and diagnostic performance. The findings provide evidence-based guidance for optimizing TOSc use and offer new insights into PRRSV dynamics within the breeding herds. Thus, TOSc sampling can enhance surveillance strategies and support more targeted PRRSV control and elimination efforts, by identifying individual sows harboring PRRSV RNA in their oral-tonsilar area.","abstract_html":"Porcine reproductive and respiratory syndrome virus (PRRSV) continues to pose a significant threat to the global swine industry. One major problem for PRRSV control and elimination in the breeding herd is the persistence of PRRSV at herd level, increasing time to stability (Linhares et al., 2014; Osemeke et al., 2022), which is defined as when there is diagnostic evidence of a sustained lack of viremia in pigs at weaning for 13 consecutive weeks according to the American Association of Swine Veterinarians (AASV) (Holtkamp, 2021). There are several well-established sample types including processing fluid (PF) (López et al., 2022), serum (Almeida et al., 2021), and family oral fluids (FOF) (Almeida et al., 2021) for PRRSV surveillance in the breeding herd. These samples majorly target suckling piglets and miss most of the breeding herd. As the sows constitute one of the major sources of virus transmission to the piglets (Cano et al., 2009; Kang et al., 2010; Pileri et al., 2016), it is crucial to establish strategies to monitor and characterize the PRRSV dynamics in the sow population to have a holistic picture of PRRSV activity in the whole herd. Currently, the most commonly used sample types for sows in the US are serum and tonsil scraping. These require snaring the sows and are labor-intensive and animal welfare unfriendly to obtain (Allende et al., 2000; Horter et al., 2002). Retrieving oral fluids (OF) from individual sows under field conditions is challenging because not all sows willingly respond to the rope (Pepin et al., 2014). A novel Tonsil-Oral-Scrubbing (TOSc) sampling method was recently developed, which retrieve fluids within seconds from the sow’s oral and tonsillar area without the necessity of snaring (Li et al., 2024). That study reported PRRSV RNA detection rate in TOSc samples similar to that of tonsil scraping samples and significantly higher than serum in acutely infected sows. However, as a newly developed sample type, it is crucial to characterize the composition of TOSc samples, to optimize the best practice of TOSc collection process , and to evaluate the suitability of TOSc samples for detecting PRRSV RNA in sows across different conditions before applying TOSc samples to investigate the PRRSV dynamics in the sows. This dissertation compared TOSc samples with other commonly used sample types for PRRSV RNA detection in sows over time; determined best practice for collecting TOSc samples; characterized the TOSc sample composition, and provided a visualization of the TOSc sample collection process; evaluated the effects of different processing procedures on PRRSV RNA detection of TOSc samples in the lab; and then investigated PRRSV vertical transmission and PRRSV dynamics in sows over time and by employing TOSc-based monitoring in breeding herds. Specifically, chapter 2 compared TOSc with serum, OF, and tonsil scraping to detect PRRSV RNA by real-time reverse transcription polymerase chain reaction (RT-rtPCR) from each of the 61 conveniently selected sows at 30-, 60-, and 90-days post live virus inoculation (LVI) with a wild-type PRRSV in one breeding herd. TOSc and tonsil scraping showed a similar decreasing PRRSV RNA detection, while the detection rates for OF and serum remained relatively constant over time. Moreover, the median Ct values of TOSc and tonsil scraping were numerically lower than OF and serum at all sampling points. Also, based on RT-rtPCR results of TOSc and tonsil scraping, most PRRSV RNA-positive sows turned negative by 90 days post-LVI, but a small proportion remained positive. However, small proportions of previously negative sows turned positive at 60- and 90-days post LVI, showing an intermittent mode of PRRSV RNA detection for both sample types in sows. Chapter 3 investigated the effect of specific TOSc collection factors on the PRRSV RNA results (detection rates and Ct values). Those factors include whether the sow was snared or not snared at TOSc collection (“snared” vs. “not snared”); whether the sow was laying down or standing at collection (“laying down” vs. “standing”); and type of collectors used for TOSc collection (“TOSc prototype” vs. “Spiral-headed AI catheter (SHAC)”). The effect for each factor was assessed in three independent studies following the same design: TOSc was collected twice from each studied sow, once with the baseline level for a factor (“not snared”, or “standing”, or “TOSc prototype”), and another time followed by the other level of the paired factor (“snared”, “laying down”, or “SHAC”, correspondingly). Results showed that “not snared” TOSc samples had numerically higher PRRSV RNA detection rate (60.7% vs. 52.5%, p=0.11), significantly lower median Ct values (31.9 vs. 32.3, p&lt;0.01), and significantly higher sample volumethan “snared” samples (1.8 mL vs. 1.2 mL, p&lt;0.01); “laying down” TOSc samples did not differ statistically (60.7% vs. 60.7%) in the PRRSV RNA detection rate, obtained numerically lower median Ct values (30.9 vs 31.3, p=0.19), but took 40% less collection time compared to “standing” TOSc samples; samples collected using the “TOSc prototype” had numerically higher PRRSV RNA detection rate (91.7% vs. 88.3%, p=0.27) and significantly lower median Ct values (32.8 vs. 34.5, p&lt;0.01) than that from “SHAC”. Chapter 4 used a endoscope to visualize the TOSc collection process and histopathology to characterize TOSc sample composition. The video recording showed that after the endoscope-TOSc assembly passed the hard palate, it reached the tonsils of the soft palate. The tongue was pressing the collector towards the tonsils after swallow reflex activation. Histopathology revealed the presence of squamous mucosal cells and small clusters of immune cells, including lymphocytes, plasma cells, eosinophils and suspect macrophages in TOSc samples. Chapter 5 evaluated the effect of pooling and freeze-thaw on the probability of PRRSV RNA detection and Ct values in TOSc samples by RT-rtPCR. To test the effect of pooling on PRRSV RNA detection, 22 known RT-rtPCR-positive TOSc samples from PRRSV endemic herds were categorized into 3 groups by Ct values and diluted (pooled) with RT-rtPCR-negative TOSc samples from PRRSV negative farms at ratios of 1:3, 1:5, 1:8 and 1:10, respectively. To test the freeze-thaw effect on PRRSV RNA detection, 90 TOSc samples were conveniently collected from endemic herds. Each TOSc sample was mildly vortexed and equally aliquoted into 2 replicates after arrival at the laboratory. One aliquot was submitted for testing for PRRSV RNA by RT-rtPCR (non-freeze-thaw group), while the other aliquot was frozen at -20℃ overnight and thawed at -4℃ for 8 hours (freeze-thaw group) before submission and testing. The probability of PRRSV RNA detection decreased, and mean Ct values increased in TOSc samples with increasing dilution ratios, and the ranges of mean Ct value changes for each category were 1.4-1.8, 2.2-2.6, 2.4-2.9, and 2.8-3.6 after dilution at ratio of 1:3, 1:5, 1:8 and 1:10, respectively, compared with undiluted groups. The mean probability of PRRSV RNA detection for category A (29 &lt; Ct &lt; 32) remained 99% after all dilution levels, while that for category B (32 &lt; Ct &lt; 35) decreased to 95%, and 84% at dilution ratio of 1:8 and 1:10 respectively. For category C (35 &lt; Ct &lt; 38), there were significant drops in probability of detection since dilution ratio of 1:3 compared with undiluted samples (Tukey test, p&lt;0.05). While the non-freeze-thaw group showed numerically higher probability of PRRSV RNA detection than the freeze-thaw group, the difference was not statistically significant (Tukey test, p=0.12). However, the non-freeze-thaw group showed significantly lower mean Ct values than the freeze-thaw group with a mean difference of 0.24 (Signed rank test, p&lt;0.001). Chapter 6 described the PRRSV RT-rtPCR results (detection rate and Ct values) for each set of sows and piglet specimens within 12 hours post farrowing and investigated the probability and risk ratios for producing PRRSV-positive or PRRSV-negative live piglets based on RT-rtPCR results from sow TOSc and stillborn TF samples. At around 90 days post live-virus inoculation (LVI), 555 sows were sampled using TOSc 2 weeks pre-farrowing and tested for PRRSV RNA. From these, 59 PRRSV-positive sows, and 88 PRRSV-negative sows matched by parity were conveniently selected. TOSc from sows, blood swabs from live pigletS, and tongue fluid (TF) and serum from dead piglet were collected individually from all study litters within 12 hours post-farrowing. The pre-farrowing TOSc samples had significantly higher PRRSV positivity than TF, serum and blood swab pools, while dead piglet serum had significantly lower mean Ct values than all other sample types. TOSc PRRSV RNA postive sows had around 25% probability to produce positive live piglets while the PRRSV negative sows had around 90% probability to produce negative live piglets on the litter level. Pre-farrow TOSc, post-farrow TOSc, and serial TOSc PRRSV positive sows were 1.78 times, 2.75 times, 3.13 times more likely to produce positive live piglets than TOSc negative sows, respectively. Pre-farrow TOSc, post-farrow TOSc, and serial TOSc PRRSV negative sows were 1.14 times, 1.28 times and 2.29 times more likely to produce negative live pigletsthan TOSc positive sows, respectively. Chapter 7 characterized the dynamics of PRRSV RNA positivity in sows and associated risk factors using TOSc samples over a 24-week period. This field study was conducted in a commercial breed-to-wean farm on a plan to eliminate PRRSV by herd closure and using LVI for whole herd exposure after an outbreak. Pre- and post-LVI, periodic TOSc samplings at four-week intervals were performed on 275 conveniently selected sows with evenly distributed parity categories (Parity 0 (P0), parity 1-2 (P1-P2), and parity 3 and above (P3+)) and production phases categories (wean-breed, early gestation, middle gestation, late gestation, and farrowing) until 24 weeks post-LVI. Overall, PRRSV RNA positivity declined over time from the first sampling point- 2 days before LVI until the end of the trial. At 16 weeks post-LVI, the PRRSV RNA positivity reached 4.43%, below the defined threshold of a low prevalence scenario of 10% positivity and remained low prevalence until the end of the study. In the low prevalence scenario, there was a rebound of positivity, which was defined as an increase of mean positivity compared to the previous sampling point, to 8.00% at 24 weeks post-LVI. Parity demonstrated a significant effect on PRRSV RNA positivity in sows. At a high prevalence scenario (＞10% positivity), both P0 and P1-P2 groups constantly showed significantly higher PRRSV RNA positivity than the P3+ group (p＜0.05 for all pairwise comparisons). The P0 positivity was consistently numerically higher than P1-P2. The production phase effect was evident when the herd reached low prevalence (＜10% positivity); sows in farrowing had consistently higher PRRSV RNA positivity than sows in other production phases. The mean positivity increased to 28.56% and 34.49% in the farrowing group at 20 weeks post-LVI and 24 weeks post-LVI, respectively. To conclude, this dissertation establishes TOSc as an easy and practical sampling method for PRRSV RNA monitoring by RT-rtPCR in sows, offering advantages in collection ease, animal welfare, and diagnostic performance. The findings provide evidence-based guidance for optimizing TOSc use and offer new insights into PRRSV dynamics within the breeding herds. Thus, TOSc sampling can enhance surveillance strategies and support more targeted PRRSV control and elimination efforts, by identifying individual sows harboring PRRSV RNA in their oral-tonsilar area.","abstract_has_math":false,"creators":["Li, Peng"],"institution":"Iowa State University","degree_name":"Doctor of Philosophy","degree_level":"dissertation","degree_discipline":"Veterinary science","degree_department":"Department of Veterinary Diagnostic and Production Animal Medicine","school":null,"contributors":[],"advisors":["Linhares, Daniel C. L.","Almeida, Marcelo N.","Silva, Gustavo S.","Holtkamp, Derald J.","Trevisan, Giovani"],"committee_chairs":[],"committee_members":[],"year":2025,"date_issued":"2025-05","date_published":"2025-05","updated_at":"2026-07-24T02:38:34Z","subjects":[],"languages":["en"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.31274/td-20251215-116"],"render_values":[{"text":"https://doi.org/10.31274/td-20251215-116","href":"https://doi.org/10.31274/td-20251215-116","code":true}]}]},"links":{"outbound_url":"https://dr.lib.iastate.edu/handle/20.500.12876/YvkA6Roz","outbound_label":"Repository record","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Linhares, Daniel C. L.","Almeida, Marcelo N.","Silva, Gustavo S.","Holtkamp, Derald J.","Trevisan, Giovani"]},{"key":"dc:contributor.department","label":"Department","values":["Department of Veterinary Diagnostic and Production Animal Medicine"]},{"key":"dc:creator","label":"Author","values":["Li, Peng"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2025-06-25T22:43:53Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2025-06-25T22:43:53Z"]},{"key":"dc:date.issued","label":"Date","values":["2025-05"]},{"key":"dc:type","label":"Dc Type","values":["dissertation"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Veterinary science"]},{"key":"thesis:degree_level","label":"Degree Level","values":["dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Doctor of Philosophy"]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["Iowa State University"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["en"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.31274/td-20251215-116"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://dr.lib.iastate.edu/handle/20.500.12876/YvkA6Roz"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Porcine reproductive and respiratory syndrome virus (PRRSV) continues to pose a significant threat to the global swine industry. One major problem for PRRSV control and elimination in the breeding herd is the persistence of PRRSV at herd level, increasing time to stability (Linhares et al., 2014; Osemeke et al., 2022), which is defined as when there is diagnostic evidence of a sustained lack of viremia in pigs at weaning for 13 consecutive weeks according to the American Association of Swine Veterinarians (AASV) (Holtkamp, 2021). There are several well-established sample types including processing fluid (PF) (López et al., 2022), serum (Almeida et al., 2021), and family oral fluids (FOF) (Almeida et al., 2021) for PRRSV surveillance in the breeding herd. These samples majorly target suckling piglets and miss most of the breeding herd. As the sows constitute one of the major sources of virus transmission to the piglets (Cano et al., 2009; Kang et al., 2010; Pileri et al., 2016), it is crucial to establish strategies to monitor and characterize the PRRSV dynamics in the sow population to have a holistic picture of PRRSV activity in the whole herd. Currently, the most commonly used sample types for sows in the US are serum and tonsil scraping. These require snaring the sows and are labor-intensive and animal welfare unfriendly to obtain (Allende et al., 2000; Horter et al., 2002). Retrieving oral fluids (OF) from individual sows under field conditions is challenging because not all sows willingly respond to the rope (Pepin et al., 2014). A novel Tonsil-Oral-Scrubbing (TOSc) sampling method was recently developed, which retrieve fluids within seconds from the sow’s oral and tonsillar area without the necessity of snaring (Li et al., 2024). That study reported PRRSV RNA detection rate in TOSc samples similar to that of tonsil scraping samples and significantly higher than serum in acutely infected sows. However, as a newly developed sample type, it is crucial to characterize the composition of TOSc samples, to optimize the best practice of TOSc collection process , and to evaluate the suitability of TOSc samples for detecting PRRSV RNA in sows across different conditions before applying TOSc samples to investigate the PRRSV dynamics in the sows. This dissertation compared TOSc samples with other commonly used sample types for PRRSV RNA detection in sows over time; determined best practice for collecting TOSc samples; characterized the TOSc sample composition, and provided a visualization of the TOSc sample collection process; evaluated the effects of different processing procedures on PRRSV RNA detection of TOSc samples in the lab; and then investigated PRRSV vertical transmission and PRRSV dynamics in sows over time and by employing TOSc-based monitoring in breeding herds. Specifically, chapter 2 compared TOSc with serum, OF, and tonsil scraping to detect PRRSV RNA by real-time reverse transcription polymerase chain reaction (RT-rtPCR) from each of the 61 conveniently selected sows at 30-, 60-, and 90-days post live virus inoculation (LVI) with a wild-type PRRSV in one breeding herd. TOSc and tonsil scraping showed a similar decreasing PRRSV RNA detection, while the detection rates for OF and serum remained relatively constant over time. Moreover, the median Ct values of TOSc and tonsil scraping were numerically lower than OF and serum at all sampling points. Also, based on RT-rtPCR results of TOSc and tonsil scraping, most PRRSV RNA-positive sows turned negative by 90 days post-LVI, but a small proportion remained positive. However, small proportions of previously negative sows turned positive at 60- and 90-days post LVI, showing an intermittent mode of PRRSV RNA detection for both sample types in sows. Chapter 3 investigated the effect of specific TOSc collection factors on the PRRSV RNA results (detection rates and Ct values). Those factors include whether the sow was snared or not snared at TOSc collection (“snared” vs. “not snared”); whether the sow was laying down or standing at collection (“laying down” vs. “standing”); and type of collectors used for TOSc collection (“TOSc prototype” vs. “Spiral-headed AI catheter (SHAC)”). The effect for each factor was assessed in three independent studies following the same design: TOSc was collected twice from each studied sow, once with the baseline level for a factor (“not snared”, or “standing”, or “TOSc prototype”), and another time followed by the other level of the paired factor (“snared”, “laying down”, or “SHAC”, correspondingly). Results showed that “not snared” TOSc samples had numerically higher PRRSV RNA detection rate (60.7% vs. 52.5%, p=0.11), significantly lower median Ct values (31.9 vs. 32.3, p<0.01), and significantly higher sample volumethan “snared” samples (1.8 mL vs. 1.2 mL, p<0.01); “laying down” TOSc samples did not differ statistically (60.7% vs. 60.7%) in the PRRSV RNA detection rate, obtained numerically lower median Ct values (30.9 vs 31.3, p=0.19), but took 40% less collection time compared to “standing” TOSc samples; samples collected using the “TOSc prototype” had numerically higher PRRSV RNA detection rate (91.7% vs. 88.3%, p=0.27) and significantly lower median Ct values (32.8 vs. 34.5, p<0.01) than that from “SHAC”. Chapter 4 used a endoscope to visualize the TOSc collection process and histopathology to characterize TOSc sample composition. The video recording showed that after the endoscope-TOSc assembly passed the hard palate, it reached the tonsils of the soft palate. The tongue was pressing the collector towards the tonsils after swallow reflex activation. Histopathology revealed the presence of squamous mucosal cells and small clusters of immune cells, including lymphocytes, plasma cells, eosinophils and suspect macrophages in TOSc samples. Chapter 5 evaluated the effect of pooling and freeze-thaw on the probability of PRRSV RNA detection and Ct values in TOSc samples by RT-rtPCR. To test the effect of pooling on PRRSV RNA detection, 22 known RT-rtPCR-positive TOSc samples from PRRSV endemic herds were categorized into 3 groups by Ct values and diluted (pooled) with RT-rtPCR-negative TOSc samples from PRRSV negative farms at ratios of 1:3, 1:5, 1:8 and 1:10, respectively. To test the freeze-thaw effect on PRRSV RNA detection, 90 TOSc samples were conveniently collected from endemic herds. Each TOSc sample was mildly vortexed and equally aliquoted into 2 replicates after arrival at the laboratory. One aliquot was submitted for testing for PRRSV RNA by RT-rtPCR (non-freeze-thaw group), while the other aliquot was frozen at -20℃ overnight and thawed at -4℃ for 8 hours (freeze-thaw group) before submission and testing. The probability of PRRSV RNA detection decreased, and mean Ct values increased in TOSc samples with increasing dilution ratios, and the ranges of mean Ct value changes for each category were 1.4-1.8, 2.2-2.6, 2.4-2.9, and 2.8-3.6 after dilution at ratio of 1:3, 1:5, 1:8 and 1:10, respectively, compared with undiluted groups. The mean probability of PRRSV RNA detection for category A (29 < Ct < 32) remained 99% after all dilution levels, while that for category B (32 < Ct < 35) decreased to 95%, and 84% at dilution ratio of 1:8 and 1:10 respectively. For category C (35 < Ct < 38), there were significant drops in probability of detection since dilution ratio of 1:3 compared with undiluted samples (Tukey test, p<0.05). While the non-freeze-thaw group showed numerically higher probability of PRRSV RNA detection than the freeze-thaw group, the difference was not statistically significant (Tukey test, p=0.12). However, the non-freeze-thaw group showed significantly lower mean Ct values than the freeze-thaw group with a mean difference of 0.24 (Signed rank test, p<0.001). Chapter 6 described the PRRSV RT-rtPCR results (detection rate and Ct values) for each set of sows and piglet specimens within 12 hours post farrowing and investigated the probability and risk ratios for producing PRRSV-positive or PRRSV-negative live piglets based on RT-rtPCR results from sow TOSc and stillborn TF samples. At around 90 days post live-virus inoculation (LVI), 555 sows were sampled using TOSc 2 weeks pre-farrowing and tested for PRRSV RNA. From these, 59 PRRSV-positive sows, and 88 PRRSV-negative sows matched by parity were conveniently selected. TOSc from sows, blood swabs from live pigletS, and tongue fluid (TF) and serum from dead piglet were collected individually from all study litters within 12 hours post-farrowing. The pre-farrowing TOSc samples had significantly higher PRRSV positivity than TF, serum and blood swab pools, while dead piglet serum had significantly lower mean Ct values than all other sample types. TOSc PRRSV RNA postive sows had around 25% probability to produce positive live piglets while the PRRSV negative sows had around 90% probability to produce negative live piglets on the litter level. Pre-farrow TOSc, post-farrow TOSc, and serial TOSc PRRSV positive sows were 1.78 times, 2.75 times, 3.13 times more likely to produce positive live piglets than TOSc negative sows, respectively. Pre-farrow TOSc, post-farrow TOSc, and serial TOSc PRRSV negative sows were 1.14 times, 1.28 times and 2.29 times more likely to produce negative live pigletsthan TOSc positive sows, respectively. Chapter 7 characterized the dynamics of PRRSV RNA positivity in sows and associated risk factors using TOSc samples over a 24-week period. This field study was conducted in a commercial breed-to-wean farm on a plan to eliminate PRRSV by herd closure and using LVI for whole herd exposure after an outbreak. Pre- and post-LVI, periodic TOSc samplings at four-week intervals were performed on 275 conveniently selected sows with evenly distributed parity categories (Parity 0 (P0), parity 1-2 (P1-P2), and parity 3 and above (P3+)) and production phases categories (wean-breed, early gestation, middle gestation, late gestation, and farrowing) until 24 weeks post-LVI. Overall, PRRSV RNA positivity declined over time from the first sampling point- 2 days before LVI until the end of the trial. At 16 weeks post-LVI, the PRRSV RNA positivity reached 4.43%, below the defined threshold of a low prevalence scenario of 10% positivity and remained low prevalence until the end of the study. In the low prevalence scenario, there was a rebound of positivity, which was defined as an increase of mean positivity compared to the previous sampling point, to 8.00% at 24 weeks post-LVI. Parity demonstrated a significant effect on PRRSV RNA positivity in sows. At a high prevalence scenario (＞10% positivity), both P0 and P1-P2 groups constantly showed significantly higher PRRSV RNA positivity than the P3+ group (p＜0.05 for all pairwise comparisons). The P0 positivity was consistently numerically higher than P1-P2. The production phase effect was evident when the herd reached low prevalence (＜10% positivity); sows in farrowing had consistently higher PRRSV RNA positivity than sows in other production phases. The mean positivity increased to 28.56% and 34.49% in the farrowing group at 20 weeks post-LVI and 24 weeks post-LVI, respectively. To conclude, this dissertation establishes TOSc as an easy and practical sampling method for PRRSV RNA monitoring by RT-rtPCR in sows, offering advantages in collection ease, animal welfare, and diagnostic performance. The findings provide evidence-based guidance for optimizing TOSc use and offer new insights into PRRSV dynamics within the breeding herds. Thus, TOSc sampling can enhance surveillance strategies and support more targeted PRRSV control and elimination efforts, by identifying individual sows harboring PRRSV RNA in their oral-tonsilar area."]},{"key":"dc:format.mimetype","label":"Dc Format Mimetype","values":["PDF"]},{"key":"dc:title","label":"Title","values":["Assessment of Tonsil-Oral-Scrubbing (TOSc) to detect PRRSV RNA in sows and application of TOSc for PRRSV dynamics in breeding herds"]}]}],"canonical_facts":{"dc:contributor.advisor":["Linhares, Daniel C. L.","Almeida, Marcelo N.","Silva, Gustavo S.","Holtkamp, Derald J.","Trevisan, Giovani"],"dc:contributor.department":["Department of Veterinary Diagnostic and Production Animal Medicine"],"dc:creator":["Li, Peng"],"dc:date.accessioned":["2025-06-25T22:43:53Z"],"dc:date.available":["2025-06-25T22:43:53Z"],"dc:date.issued":["2025-05"],"dc:description.abstract":["Porcine reproductive and respiratory syndrome virus (PRRSV) continues to pose a significant threat to the global swine industry. One major problem for PRRSV control and elimination in the breeding herd is the persistence of PRRSV at herd level, increasing time to stability (Linhares et al., 2014; Osemeke et al., 2022), which is defined as when there is diagnostic evidence of a sustained lack of viremia in pigs at weaning for 13 consecutive weeks according to the American Association of Swine Veterinarians (AASV) (Holtkamp, 2021). There are several well-established sample types including processing fluid (PF) (López et al., 2022), serum (Almeida et al., 2021), and family oral fluids (FOF) (Almeida et al., 2021) for PRRSV surveillance in the breeding herd. These samples majorly target suckling piglets and miss most of the breeding herd. As the sows constitute one of the major sources of virus transmission to the piglets (Cano et al., 2009; Kang et al., 2010; Pileri et al., 2016), it is crucial to establish strategies to monitor and characterize the PRRSV dynamics in the sow population to have a holistic picture of PRRSV activity in the whole herd. Currently, the most commonly used sample types for sows in the US are serum and tonsil scraping. These require snaring the sows and are labor-intensive and animal welfare unfriendly to obtain (Allende et al., 2000; Horter et al., 2002). Retrieving oral fluids (OF) from individual sows under field conditions is challenging because not all sows willingly respond to the rope (Pepin et al., 2014). A novel Tonsil-Oral-Scrubbing (TOSc) sampling method was recently developed, which retrieve fluids within seconds from the sow’s oral and tonsillar area without the necessity of snaring (Li et al., 2024). That study reported PRRSV RNA detection rate in TOSc samples similar to that of tonsil scraping samples and significantly higher than serum in acutely infected sows. However, as a newly developed sample type, it is crucial to characterize the composition of TOSc samples, to optimize the best practice of TOSc collection process , and to evaluate the suitability of TOSc samples for detecting PRRSV RNA in sows across different conditions before applying TOSc samples to investigate the PRRSV dynamics in the sows. This dissertation compared TOSc samples with other commonly used sample types for PRRSV RNA detection in sows over time; determined best practice for collecting TOSc samples; characterized the TOSc sample composition, and provided a visualization of the TOSc sample collection process; evaluated the effects of different processing procedures on PRRSV RNA detection of TOSc samples in the lab; and then investigated PRRSV vertical transmission and PRRSV dynamics in sows over time and by employing TOSc-based monitoring in breeding herds. Specifically, chapter 2 compared TOSc with serum, OF, and tonsil scraping to detect PRRSV RNA by real-time reverse transcription polymerase chain reaction (RT-rtPCR) from each of the 61 conveniently selected sows at 30-, 60-, and 90-days post live virus inoculation (LVI) with a wild-type PRRSV in one breeding herd. TOSc and tonsil scraping showed a similar decreasing PRRSV RNA detection, while the detection rates for OF and serum remained relatively constant over time. Moreover, the median Ct values of TOSc and tonsil scraping were numerically lower than OF and serum at all sampling points. Also, based on RT-rtPCR results of TOSc and tonsil scraping, most PRRSV RNA-positive sows turned negative by 90 days post-LVI, but a small proportion remained positive. However, small proportions of previously negative sows turned positive at 60- and 90-days post LVI, showing an intermittent mode of PRRSV RNA detection for both sample types in sows. Chapter 3 investigated the effect of specific TOSc collection factors on the PRRSV RNA results (detection rates and Ct values). Those factors include whether the sow was snared or not snared at TOSc collection (“snared” vs. “not snared”); whether the sow was laying down or standing at collection (“laying down” vs. “standing”); and type of collectors used for TOSc collection (“TOSc prototype” vs. “Spiral-headed AI catheter (SHAC)”). The effect for each factor was assessed in three independent studies following the same design: TOSc was collected twice from each studied sow, once with the baseline level for a factor (“not snared”, or “standing”, or “TOSc prototype”), and another time followed by the other level of the paired factor (“snared”, “laying down”, or “SHAC”, correspondingly). Results showed that “not snared” TOSc samples had numerically higher PRRSV RNA detection rate (60.7% vs. 52.5%, p=0.11), significantly lower median Ct values (31.9 vs. 32.3, p<0.01), and significantly higher sample volumethan “snared” samples (1.8 mL vs. 1.2 mL, p<0.01); “laying down” TOSc samples did not differ statistically (60.7% vs. 60.7%) in the PRRSV RNA detection rate, obtained numerically lower median Ct values (30.9 vs 31.3, p=0.19), but took 40% less collection time compared to “standing” TOSc samples; samples collected using the “TOSc prototype” had numerically higher PRRSV RNA detection rate (91.7% vs. 88.3%, p=0.27) and significantly lower median Ct values (32.8 vs. 34.5, p<0.01) than that from “SHAC”. Chapter 4 used a endoscope to visualize the TOSc collection process and histopathology to characterize TOSc sample composition. The video recording showed that after the endoscope-TOSc assembly passed the hard palate, it reached the tonsils of the soft palate. The tongue was pressing the collector towards the tonsils after swallow reflex activation. Histopathology revealed the presence of squamous mucosal cells and small clusters of immune cells, including lymphocytes, plasma cells, eosinophils and suspect macrophages in TOSc samples. Chapter 5 evaluated the effect of pooling and freeze-thaw on the probability of PRRSV RNA detection and Ct values in TOSc samples by RT-rtPCR. To test the effect of pooling on PRRSV RNA detection, 22 known RT-rtPCR-positive TOSc samples from PRRSV endemic herds were categorized into 3 groups by Ct values and diluted (pooled) with RT-rtPCR-negative TOSc samples from PRRSV negative farms at ratios of 1:3, 1:5, 1:8 and 1:10, respectively. To test the freeze-thaw effect on PRRSV RNA detection, 90 TOSc samples were conveniently collected from endemic herds. Each TOSc sample was mildly vortexed and equally aliquoted into 2 replicates after arrival at the laboratory. One aliquot was submitted for testing for PRRSV RNA by RT-rtPCR (non-freeze-thaw group), while the other aliquot was frozen at -20℃ overnight and thawed at -4℃ for 8 hours (freeze-thaw group) before submission and testing. The probability of PRRSV RNA detection decreased, and mean Ct values increased in TOSc samples with increasing dilution ratios, and the ranges of mean Ct value changes for each category were 1.4-1.8, 2.2-2.6, 2.4-2.9, and 2.8-3.6 after dilution at ratio of 1:3, 1:5, 1:8 and 1:10, respectively, compared with undiluted groups. The mean probability of PRRSV RNA detection for category A (29 < Ct < 32) remained 99% after all dilution levels, while that for category B (32 < Ct < 35) decreased to 95%, and 84% at dilution ratio of 1:8 and 1:10 respectively. For category C (35 < Ct < 38), there were significant drops in probability of detection since dilution ratio of 1:3 compared with undiluted samples (Tukey test, p<0.05). While the non-freeze-thaw group showed numerically higher probability of PRRSV RNA detection than the freeze-thaw group, the difference was not statistically significant (Tukey test, p=0.12). However, the non-freeze-thaw group showed significantly lower mean Ct values than the freeze-thaw group with a mean difference of 0.24 (Signed rank test, p<0.001). Chapter 6 described the PRRSV RT-rtPCR results (detection rate and Ct values) for each set of sows and piglet specimens within 12 hours post farrowing and investigated the probability and risk ratios for producing PRRSV-positive or PRRSV-negative live piglets based on RT-rtPCR results from sow TOSc and stillborn TF samples. At around 90 days post live-virus inoculation (LVI), 555 sows were sampled using TOSc 2 weeks pre-farrowing and tested for PRRSV RNA. From these, 59 PRRSV-positive sows, and 88 PRRSV-negative sows matched by parity were conveniently selected. TOSc from sows, blood swabs from live pigletS, and tongue fluid (TF) and serum from dead piglet were collected individually from all study litters within 12 hours post-farrowing. The pre-farrowing TOSc samples had significantly higher PRRSV positivity than TF, serum and blood swab pools, while dead piglet serum had significantly lower mean Ct values than all other sample types. TOSc PRRSV RNA postive sows had around 25% probability to produce positive live piglets while the PRRSV negative sows had around 90% probability to produce negative live piglets on the litter level. Pre-farrow TOSc, post-farrow TOSc, and serial TOSc PRRSV positive sows were 1.78 times, 2.75 times, 3.13 times more likely to produce positive live piglets than TOSc negative sows, respectively. Pre-farrow TOSc, post-farrow TOSc, and serial TOSc PRRSV negative sows were 1.14 times, 1.28 times and 2.29 times more likely to produce negative live pigletsthan TOSc positive sows, respectively. Chapter 7 characterized the dynamics of PRRSV RNA positivity in sows and associated risk factors using TOSc samples over a 24-week period. This field study was conducted in a commercial breed-to-wean farm on a plan to eliminate PRRSV by herd closure and using LVI for whole herd exposure after an outbreak. Pre- and post-LVI, periodic TOSc samplings at four-week intervals were performed on 275 conveniently selected sows with evenly distributed parity categories (Parity 0 (P0), parity 1-2 (P1-P2), and parity 3 and above (P3+)) and production phases categories (wean-breed, early gestation, middle gestation, late gestation, and farrowing) until 24 weeks post-LVI. Overall, PRRSV RNA positivity declined over time from the first sampling point- 2 days before LVI until the end of the trial. At 16 weeks post-LVI, the PRRSV RNA positivity reached 4.43%, below the defined threshold of a low prevalence scenario of 10% positivity and remained low prevalence until the end of the study. In the low prevalence scenario, there was a rebound of positivity, which was defined as an increase of mean positivity compared to the previous sampling point, to 8.00% at 24 weeks post-LVI. Parity demonstrated a significant effect on PRRSV RNA positivity in sows. At a high prevalence scenario (＞10% positivity), both P0 and P1-P2 groups constantly showed significantly higher PRRSV RNA positivity than the P3+ group (p＜0.05 for all pairwise comparisons). The P0 positivity was consistently numerically higher than P1-P2. The production phase effect was evident when the herd reached low prevalence (＜10% positivity); sows in farrowing had consistently higher PRRSV RNA positivity than sows in other production phases. The mean positivity increased to 28.56% and 34.49% in the farrowing group at 20 weeks post-LVI and 24 weeks post-LVI, respectively. To conclude, this dissertation establishes TOSc as an easy and practical sampling method for PRRSV RNA monitoring by RT-rtPCR in sows, offering advantages in collection ease, animal welfare, and diagnostic performance. The findings provide evidence-based guidance for optimizing TOSc use and offer new insights into PRRSV dynamics within the breeding herds. Thus, TOSc sampling can enhance surveillance strategies and support more targeted PRRSV control and elimination efforts, by identifying individual sows harboring PRRSV RNA in their oral-tonsilar area."],"dc:format.mimetype":["PDF"],"dc:identifier.doi":["https://doi.org/10.31274/td-20251215-116"],"dc:identifier.uri":["https://dr.lib.iastate.edu/handle/20.500.12876/YvkA6Roz"],"dc:language.iso":["en"],"dc:title":["Assessment of Tonsil-Oral-Scrubbing (TOSc) to detect PRRSV RNA in sows and application of TOSc for PRRSV dynamics in breeding herds"],"dc:type":["dissertation"],"thesis:degree_discipline":["Veterinary science"],"thesis:degree_level":["dissertation"],"thesis:degree_name":["Doctor of Philosophy"],"thesis:institution_name":["Iowa State University"]},"updated_at":"2026-07-24T02:38:34Z"}