Kinetics of the porcine reproductive and respiratory syndrome virus (PRRSV) humoral immune response in swine serum and oral fluids collected from individual boars

Background The object of this study was to describe and contrast the kinetics of the humoral response in serum and oral fluid specimens during acute porcine reproductive and respiratory syndrome virus (PRRSV) infection. The study involved three trials of 24 boars each. Boars were intramuscularly inoculated with a commercial modified live virus (MLV) vaccine (Trial 1), a Type 1 PRRSV field isolated (Trial 2), or a Type 2 PRRSV field isolate (Trial 3). Oral fluid samples were collected from individual boars on day post inoculation (DPI) -7 and 0 to 21. Serum samples were collected from all boars on DPI −7, 0, 7, 14, 21 and from 4 randomly selected boars on DPI 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. Results Statistically significant differences in viral replication and antibody responses were observed among the three trials in both serum and oral fluid specimens. PRRSV 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. Conclusions This study enhanced our knowledge of the PRRSV humoral immune response and provided a broader foundation for the development and application of oral fluid antibody-based diagnostics

Recommendations for pen-based oral-fluid collection in growing pigs

Sampling guidelines were developed by observing pigs during oral-fluid sample collection in commercial herds. Pigs with previous oral-fluid collection experience (“trained”) should be allowed 20 minutes access to the rope. Pigs with no prior experience (“untrained”) should be allowed 60 minutes. One collection is enough to train pigs

Influenza A virus antibody in porcine oral fluid and its diagnostic applications

Influenza A virus (IAV) is capable of infecting a wide variety of avian and mammalian species, including swine. The control of IAV in swine populations is complicated by the fact that the virus is endemic in contemporary herds and may circulate in any age group. The detection of IAV has historically been based on testing individual pig nasal swab (virus detection) or serum (antibody detection) specimens. While individual pig sampling is adequate for the diagnosis of clinical IAV infections, the collection of adequate numbers of individual pig samples is too costly and labor-intensive for routine influenza surveillance or large-scale ecological studies. Therefore, the general question addressed in this dissertation is whether oral fluid specimens could be used to surveil IAV infections as an alternative to individual animal sampling. More specifically, the aim of this research was to evaluate an IAV oral fluid antibody enzyme-linked immunosorbent assay (ELISA) for the detection of IAV nucleoprotein (NP) antibody and its use in surveillance of swine populations. This question was addressed in the logical series of experiments described below. The initial objective was to determine whether diagnostic levels of IAV NP antibodies could be detected in swine oral fluid specimens by adapting the serum ELISA protocol to the oral fluid matrix (Chapter 3). The NP antibody ELISA was selected because the NP is highly conserved among IAV subtypes. The procedure for performing the NP blocking ELISA on oral fluid was modified from the serum testing protocol by changing sample dilution, sample volume, incubation time, and incubation temperature. The detection of NP antibody was evaluated using pen-based oral fluid samples (n = 182) from pigs inoculated with either influenza A virus subtype H1N1 or H3N2 under experimental conditions and followed for 42 days post inoculation (DPI). NP antibodies in oral fluid were detected from DPI 7 to 42 in all inoculated groups, i.e., the mean sample-to-negative (S/N) ratio of influenza-inoculated pigs was significantly different (p The availability of serum and oral fluid NP ELISAs provided the tools necessary to describe the kinetics of IAV NP antibody (IgM, IgA, and IgG) in serum and oral fluid specimen from animals of defined IAV infection status (Chapter 4). A significant oral fluid IgM response was only detected in unvaccinated groups. The maximum oral fluid IgM response in these groups was detected at DPI 8, after which it rapidly declined. Oral fluid IgA was detected in both vaccinated and unvaccinated groups on DPI 6. Levels of oral fluid IgA remained relatively stable through DPI 42. Oral fluid IgG responses in both vaccinated and unvaccinated groups were detected by DPI 8 and remained stable through DPI 42. IgM responses in serum and oral fluid were highly correlated in unvaccinated groups (r = 0.810), as were serum and oral fluid IgG responses in both unvaccinated (r = 0.839) and vaccinated (r = 0.856) groups. In contrast, the correlation between serum and oral fluid IgA was weak (r ~ 0.3), regardless of vaccination status. The results from this study demonstrated that NP-specific IgM, IgA, and IgG antibody were detectable in serum and oral fluid and their ontogeny was influenced by vaccination status, the time course of the infection, and specimen type. The feasibility of IAV surveillance in the field was evaluated using pre-weaning oral fluid samples from litters of piglets in four ~12,500 sow, IAV-vaccinated, breeding herds (Chapter 5). All four herds were considered endemically infected with IAV based on historic diagnostic data. Oral fluid samples were collected from 600 litters prior to weaning and serum samples from their dams after weaning. Litter oral fluid samples were tested for IAV by virus isolation, quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), RT-PCR subtyping, and sequencing. Commercial NP ELISA kits and NP isotype-specific assays (IgM, IgA, and IgG) were used to characterize NP antibody in litter oral fluid and sow serum. All litter oral fluid specimens (n = 600) were negative by virus isolation. Twenty-five oral fluid samples were positive by qRT-PCR, based on screening (Laboratory 1) and confirmatory testing (Laboratory 2). No hemagglutinin (HA) and neuraminidase (NA) gene sequences were obtained, but matrix (M) gene sequences were obtained for all qRT-PCR-positive samples submitted for sequencing (n = 18). Genetic analysis revealed that all M genes sequences were identical (GenBank accession no. KF487544) and belonged to the triple reassortant influenza A virus M gene (TRIG M) previously identified in swine. The proportion of IgM- and IgA-positive samples was significantly higher in sow serum and litter oral fluid samples, respectively (p < 0.01). Consistent with the extensive use of IAV vaccine, no difference was detected in the proportion of IgG- and blocking ELISA-positive sow serum and litter oral fluids. This study supported the use of oral fluid sampling as a means to conduct IAV surveillance in pig populations and demonstrated the inapparent circulation of IAV in piglets.</p

Detection of classical swine fever virus (CSFV) E2 and Erns antibody (IgG, IgA) in oral fluid specimens from inoculated (ALD strain) or vaccinated (LOM strain) pigs

The objective of this study was to describe oral fluid and serum antibody (IgG, IgA) responses against classical swine fever virus (CSFV) E2 and Erns proteins in pigs (n = 60) inoculated with a moderately virulent field strain (ALD, n = 30) or a modified live virus vaccine strain (LOM, n = 30). Oral fluid (n = 1391) and serum (n = 591) samples were collected from individually-housed pigs between day post inoculation (DPI) −14 to 28. Testing revealed the synchronous appearance of E2- and Erns-specific IgG and IgA antibodies in serum and oral fluids over time, with E2 and Erns IgG ELISAs providing better diagnostic performance than the IgA ELISAs. Overall the data suggest the feasibility of large-scale, cost-effective screening of populations for CSFV using oral fluid samples. Given the historic issues of cross-reactivity among pestiviruses, future research should focus on the development of CSFV-specific testing platforms for the detection of E2 and/or Erns IgG in oral fluid, ideally to be used in combination with DIVA vaccines.This is a manuscript of an article published as Panyasing, Yaowalak, Roongroje Thanawongnuwech, Ju Ji, Luis Giménez-Lirola, and Jeffrey Zimmerman. "Detection of classical swine fever virus (CSFV) E2 and Erns antibody (IgG, IgA) in oral fluid specimens from inoculated (ALD strain) or vaccinated (LOM strain) pigs." Veterinary microbiology 224 (2018): 70-77. doi: https://doi.org/10.1016/j.vetmic.2018.08.024. © 2018 Elsevier. CC BY-NC-N

Effective surveillance for early classical swine fever virus detection will utilize both virus and antibody detection capabilities

Early recognition and rapid elimination of infected animals is key to controlling incursions of classical swine fever virus (CSFV). In this study, the diagnostic characteristics of 10 CSFV assays were evaluated using individual serum (n = 601) and/or oral fluid (n = 1417) samples collected from −14 to 28 days post inoculation (DPI). Serum samples were assayed by virus isolation (VI), 2 commercial antigen-capture enzyme-linked immunosorbent assays (ELISA), virus neutralization (VN), and 3 antibody ELISAs. Both serum and oral fluid samples were tested with 3 commercial real-time reverse transcription-polymerase chain reaction (rRT-PCR) assays. One or more serum samples was positive by VI from DPIs 3 to 21 and by antigen-capture ELISAs from DPIs 6 to 17. VN-positive serum samples were observed at DPIs ≥ 7 and by antibody ELISAs at DPIs ≥ 10. CSFV RNA was detected in serum samples from DPIs 2 to 28 and in oral fluid samples from DPIs 4 to 28. Significant differences in assay performance were detected, but most importantly, no single combination of sample and assay was able to dependably identify CSFV-inoculated pigs throughout the 4-week course of the study. The results show that effective surveillance for CSFV, especially low virulence strains, will require the use of PCR-based assays for the detection of early infections (<14 days) and antibody-based assays, thereafter.This is a manuscript of an article published as Panyasing, Yaowalak, Roongtham Kedkovid, Roongroje Thanawongnuwech, Apisit Kittawornrat, Ju Ji, Luis Giménez-Lirola, and Jeffrey Zimmerman. "Effective surveillance for early classical swine fever virus detection will utilize both virus and antibody detection capabilities." Veterinary microbiology 216 (2018): 72-78. doi: https://doi.org/10.1016/j.vetmic.2018.01.020. Posted with Permission. © 2018 Elsevier. CC BY-NC-ND

Pimobendan prevents cardiac dysfunction, mitigates cardiac mitochondrial dysfunction, and preserves myocyte ultrastructure in a rat model of mitral regurgitation

Abstract Background Pimobendan has been proven to delay the onset of congestive heart failure (CHF) in dogs with mitral regurgitation (MR); however, molecular underlying mechanisms have not been fully elucidated. This study aimed to investigate (1) the effects of pimobendan on cardiac function, cardiac mitochondrial quality and morphology, and cardiac ultrastructure in a rat model of chronic MR and (2) the direct effect of pimobendan on intracellular reactive oxygen species (ROS) production in cardiac cells. MR was surgically induced in 20 Sprague-Dawley rats, and sham procedures were performed on 10 rats. Eight weeks post-surgery, the MR rats were randomly divided into two groups: the MR group and the MR + pimobendan group. Pimobendan (0.15 mg/kg) was administered twice a day via oral gavage for 4 weeks, whereas the sham and MR groups received equivalent volumes of drinking water. Echocardiography was performed at baseline (8 weeks post-surgery) and at the end of the study (4 weeks after treatment). At the end of the study protocol, all rats were euthanized, and their hearts were immediately collected, weighed, and used for transmission electron microscopy and mitochondrial quality assessments. To evaluate the role of pimobendan on intracellular ROS production, preventive or scavenging properties were tested with H2O2-induced ROS generation in rat cardiac myoblasts (H9c2). Results Pimobendan preserved cardiac functions and structure in MR rats. In addition, pimobendan significantly improved mitochondrial quality by attenuating ROS production and depolarization (P < 0.05). The cardiac ultrastructure and mitochondrial morphology were significantly preserved in the MR + pimobendan group. In addition, pimobendan appeared to play as a ROS scavenger, but not as a ROS preventer, in H2O2-induced ROS production in H9c2 cells. Conclusions Pimobendan demonstrated cardioprotective effects on cardiac function and ultrastructure by preserving mitochondrial quality and acted as an ROS scavenger in a rat model of MR

Performance of a Differentiation of Infected from Vaccinated Animals (DIVA) Classical Swine Fever Virus (CSFV) Serum and Oral Fluid Erns Antibody AlphaLISA Assay

Classical swine fever virus (CSFV) is an OIE-listed disease that requires effective surveillance tools for its detection and control. The aim of this study was to develop and evaluate the diagnostic performance of a novel CSFV Erns IgG AlphaLISA for both serum and oral fluid specimens that would likewise be compatible with the use of CSFV E2 DIVA vaccines. Test performance was evaluated using a panel of well-characterized serum (n = 760) and individual (n = 528) or pen-based (n = 30) oral fluid samples from four groups of animals: (1) negative controls (n = 60 pigs); (2) inoculated with ALD strain wild-type CSFV (n = 30 pigs); (3) vaccinated with LOM strain live CSFV vaccine (n = 30 pigs); and (4) vaccinated with live CSFV marker vaccine on commercial farms (n = 120 pigs). At a cutoff of S/P ≥ 0.7, the aggregate estimated diagnostic sensitivities and specificities of the assay were, respectively, 97.4% (95% CI 95.9%, 98.3%) and 100% for serum and 95.4% (95% CI 92.9%, 97.0%) and 100% for oral fluid. The Erns IgG antibody AlphaLISA combined DIVA capability with solid diagnostic performance, rapid turnaround, ease of use, and compatibility with both serum and oral fluid specimens.This article is published as Panyasing, Yaowalak, Luis Gimenez-Lirola, Roongroje Thanawongnuwech, Phakawan Prakobsuk, Yanee Kawilaphan, Apisit Kittawornrat, Ting-Yu Cheng, and Jeffrey Zimmerman. "Performance of a Differentiation of Infected from Vaccinated Animals (DIVA) Classical Swine Fever Virus (CSFV) Serum and Oral Fluid Erns Antibody AlphaLISA Assay." Animals 13, no. 24 (2023): 3802. doi: https://doi.org/10.3390/ani13243802. Copyright: © 2023 by the authors. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)

Kinetics of the porcine reproductive and respiratory syndrome virus (PRRSV) humoral immune response in swine serum and oral fluids collected from individual boars

Background The object of this study was to describe and contrast the kinetics of the humoral response in serum and oral fluid specimens during acute porcine reproductive and respiratory syndrome virus (PRRSV) infection. The study involved three trials of 24 boars each. Boars were intramuscularly inoculated with a commercial modified live virus (MLV) vaccine (Trial 1), a Type 1 PRRSV field isolated (Trial 2), or a Type 2 PRRSV field isolate (Trial 3). Oral fluid samples were collected from individual boars on day post inoculation (DPI) -7 and 0 to 21. Serum samples were collected from all boars on DPI −7, 0, 7, 14, 21 and from 4 randomly selected boars on DPI 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. Results Statistically significant differences in viral replication and antibody responses were observed among the three trials in both serum and oral fluid specimens. PRRSV 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. Conclusions This study enhanced our knowledge of the PRRSV humoral immune response and provided a broader foundation for the development and application of oral fluid antibody-based diagnostics.This article is from BMC Veterinary Research 9 (2013); 61, doi: 10.1186/1746-6148-9-61. Posted with permission.</p

Recommendations for pen-based oral-fluid collection in growing pigs

Sampling guidelines were developed by observing pigs during oral-fluid sample collection in commercial herds. Pigs with previous oral-fluid collection experience (“trained”) should be allowed 20 minutes access to the rope. Pigs with no prior experience (“untrained”) should be allowed 60 minutes. One collection is enough to train pigs.This article is from Journal of Swine Health and Production 22 (2014): 138. Posted with permission.</p