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.

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

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.

Vericiguat preserved cardiac function and mitochondrial quality in a rat model of mitral regurgitation.

AIMS: New drugs for heart failure (HF) that target restoring the impaired NO-sGC-cGMP pathway are being developed. We aimed to investigate the effects of vericiguat, an sGC stimulator, on cardiac function, blood pressure (BP), cardiac mitochondrial quality, and cardiac fibrosis in rat models of chronic mitral regurgitation (MR). MATERIALS AND METHODS: We surgically induced MR in 20 Sprague-Dawley rats and performed sham procedures on 10 rats (negative control). Four weeks post-surgery, we randomly divided the MR rats into two groups: MR group and MR + vericiguat group. Vericiguat (0.5 mg/kg, PO) was administered once a day via oral gavage for 8 weeks, while the sham and MR groups received equivalent volumes of drinking water instead. We took echocardiography and BP measurements at baseline (4 weeks post-surgery) and at the end of study (8 weeks after treatment). At the study end, all rats were euthanized and their hearts were immediately collected, weighed, and used for histopathology and mitochondrial quality assessments. KEY FINDINGS: Vericiguat preserved cardiac functions and structural remodeling in the MR rats, with significantly lower systolic BPs than baseline values (P < 0.05). Additionally, vericiguat significantly improved the mitochondrial quality by attenuating ROS production, depolarization and swelling when comparing the values in both groups (P < 0.05). The fibrosis area also significantly decreased in the MR + vericiguat group (P < 0.05). SIGNIFICANCE: Vericiguat demonstrated cardioprotective effects on cardiac function, BP, and fibrosis by preserving mitochondrial quality in rats with HF due to MR.

Sacubitril/valsartan mitigates cardiac remodeling, systolic dysfunction, and preserves mitochondrial quality in a rat model of mitral regurgitation.

Sacubitril/valsartan (SAC/VAL), an angiotensin receptor blocker-neprilysin inhibitor, has been widely used to treat several types of heart failure. Nevertheless, the effects of drugs in mitral regurgitation patients, from the molecular level to therapeutic effects, remain unclear. This study investigates the roles of SAC/VAL on cardiac function, mitochondrial quality, autophagy, mitophagy, and natriuretic peptides in a rat model of chronic mitral regurgitation. Male Sprague-Dawley rats underwent MR induction (n = 16) and sham surgeries (n = 8). Four weeks post-surgery confirmed MR rats were randomly divided into MR (n = 8) and SAC/VAL (n = 8) groups. The SAC/VAL group was administered SAC/VAL, whereas the MR and the sham rats received vehicle via oral gavage daily for 8 weeks. Cardiac geometry, function, and myocardial fibrosis were assessed by echocardiography and histopathology. Spectrophotometry and real-time PCR were performed to assess the pharmacological effects on mitochondrial quality, autophagy, mitophagy, and natriuretic peptides. MR rats demonstrated significant left heart dilation and left ventricular systolic dysfunction compared with the sham group, which could be significantly improved by SAC/VAL. In addition, SAC/VAL significantly reduced myocardial cardiac remodeling and fibrosis in MR rats. SAC/VAL improved the mitochondrial quality by attenuating mitochondrial reactive oxygen species production and mitochondrial depolarization compared with the MR group. Also, the upregulation of autophagy-related, mitophagy-related, and natriuretic peptide system gene expression in MR rats was attenuated by SAC/VAL treatment. In conclusion, this study demonstrated that SAC/VAL treatment could provide numerous beneficial effects in MR conditions, suggesting that this drug may be an effective treatment for MR.

E2 and Erns isotype-specific antibody responses in serum and oral fluid after infection with classical swine fever virus (CSFV).

Oral fluid sampling for the detection of classical swine fever virus infection provides a relatively inexpensive method for conducting active CSF surveillance. The purpose of this study was to detect CSFV nucleic acid and antibody in serum and oral fluid samples in a group of 10 pigs infected with the moderate CSFV strain, Paderborn. Based on clinical signs, outcome, and other results, pigs were placed into one of three disease outcome groups; Acute, Chronic and Recovered. Oral fluid and serum samples were analyzed for the presence of CSFV nucleic acid along with E2 and Erns surface protein-specific IgM, IgG and IgA responses. The results were summarized into a timeline of detection events beginning with the appearance of E2-IgM in serum (3 DPI) followed by CSFV nucleic acid in serum (6 DPI), CSFV nucleic acid in oral fluid (8 DPI), E2-IgG in serum (20 DPI), and E2-IgG in oral fluid (24 DPI). The results show that a combination of molecular and serological analyses of oral fluid can be incorporated into CSF surveillance.

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.

Detection of Aujeszky's disease virus DNA and antibody in swine oral fluid specimens.

Aujeszky's disease virus (ADV) continues to circulate in commercial swine populations in many regions and in feral swine populations in most parts of the world, that is, ADV continues to present a risk to pork producers everywhere. Current DIVA vaccines and assays are highly effective in the control and/or eradication of ADV, but detection of wild-type ADV infection relies on testing individual pig specimens, for example, serum or muscle exudate ("meat juice"). Oral fluid specimens have been shown to be highly effective for the surveillance of a variety of swine pathogens and could offer the means to improve the efficiency of ADV surveillance in the field. In this study, the temporal patterns of ADV DNA and antibody detection in oral fluid and serum specimens were established in ADV-inoculated pigs (n = 14) using gB and gE PCRs, virus neutralization (VN) and three commercial serum antibody ELISAs (gB bELISA, gI bELISA and ADV iELISA). ADV DNA was detected in oral fluid samples (20% to 100%) from 3 to 21 days postinoculation (DPI), but not in serum. ADV antibody was detected in oral fluid specimens at DPI ≥ 10 with the gB bELISA (36% to 79%) and ADV iELISA (29% to 100%), but not the gI bELISA. These results suggest that oral fluid could be used as an alternative to individual pig sampling for ADV surveillance using PCR- and/or antibody-based assays.

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.

Oral fluid samples used for PRRSV acclimatization program and sow performance monitoring in endemic PRRS-positive farms.

An effective gilt acclimatization program is one of the most important management strategies for controlling porcine reproductive and respiratory syndrome virus (PRRSV) infection. Recently, oral fluid samples have been used as alternative diagnostic samples for various swine diseases. This study utilized oral fluids for PRRSV monitoring during the gilt acclimatization period in PRRSV endemic farms. The study was performed in two selected commercial breeding herds (farm A and farm B). PRRSV RNA and PRRSV-specific antibodies were monitored using oral fluid and serum samples. Sow performance parameters related to PRRSV infection were recorded and assessed. After PRRSV exposure during acclimatization, viral RNA was demonstrated in oral fluids from 1 to 10 weeks post-exposure (WPE). PRRSV RNA was detected in serum at 1 and 4 WPE in farm A and at 1, 4, 8, and 12 WPE in farm B. Prolonged viremia of gilts from farm B was possibly due to re-infection (within the herd) and later, reproductive problems were found in the breeding herd. The correlation of PRRSV RNA concentration in oral fluids and serum was evident. The S/P ratio values of PRRSV antibodies in oral fluid samples were higher and had similar patterns of antibody responses to the serum samples. The results suggest that the use of oral fluid samples for PRRSV monitoring during gilt acclimatization in endemic farms is effective, convenient, practical, and economical and would be most beneficial when used with other parameters.

Porcine reproductive and respiratory syndrome virus (PRRSV) surveillance using pre-weaning oral fluid samples detects circulation of wild-type PRRSV.

Oral fluid samples collected from litters of piglets (n=600) one day prior to weaning were evaluated as a method to surveil for porcine reproductive and respiratory syndrome virus (PRRSV) infections in four sow herds of approximately 12,500 sow each. Serum samples from the litters' dam (n=600) were included for comparison. All four herds were endemically infected with PRRSV and all sows had been vaccinated ≥ 2 times with PRRSV modified-live virus vaccines. After all specimens had been collected, samples were randomized and assayed by PRRSV real-time reverse transcription polymerase chain reaction (RT-qPCR) and four PRRSV antibody ELISA assays (IgM, IgA, IgG, and Commercial Kit). All sow serum samples were negative by PRRSV RT-qPCR, but 9 of 600 oral fluid samples tested positive at two laboratories. Open reading frame 5 (ORF5) sequencing of 2 of the 9 positive oral fluid samples identified wild-type viruses as the source of the infection. A comparison of antibody responses in RT-qPCR positive vs. negative oral fluid samples showed significantly higher IgG S/P ratios in RT-qPCR-positive oral fluid samples (mean S/P 3.46 vs. 2.36; p=0.02). Likewise, sow serum samples from RT-qPCR-positive litter oral fluid samples showed significantly higher serum IgG (mean S/P 1.73 vs. 0.98; p<0.001) and Commercial Kit (mean S/P 1.97 vs. 0.98; p<0.001) S/P ratios. Overall, the study showed that pre-weaning litter oral fluid samples could provide an efficient and sensitive approach to surveil for PRRSV in infected, vaccinated, or presumed-negative pig breeding herds.

Effect of collection material and sample processing on pig oral fluid testing results.

The effect of sampling material, sample processing, and collection order on the detection of analytes in pig oral fluid specimens was evaluated. Oral fluid samples were collected from 104 pens of commercial wean-to-finish pigs using ropes made of three different materials. Processed (centrifuged and filtered) and unprocessed oral fluid samples were tested using commercial ELISAs for porcine reproductive and respiratory syndrome virus (PRRSV) antibodies and total IgM, IgA, and IgG. Unprocessed samples were tested for PRRSV nucleic acid and processed samples were assayed for PRRSV neutralizing antibodies. Analysis of the data using repeated measures ANOVA and Tukey-Kramer adjusted t tests found statistically significant, non-uniform, and assay-dependent effects of all three factors. Therefore, when testing oral fluid specimens, swine health specialists, veterinarians, and diagnosticians should be aware of the potential impact of these factors on specific analytes. For monitoring health and welfare parameters, oral fluid samples should be collected using cotton-based materials and undergo minimal post-collection processing.

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.

Probability of detecting Porcine reproductive and respiratory syndrome virus infection using pen-based swine oral fluid specimens as a function of within-pen prevalence.

Pen-based oral fluid sampling has proven to be an efficient method for surveillance of infectious diseases in swine populations. To better interpret diagnostic results, the performance of oral fluid assays (antibody- and nucleic acid-based) must be established for pen-based oral fluid samples. Therefore, the objective of the current study was to determine the probability of detecting Porcine reproductive and respiratory syndrome virus (PRRSV) infection in pen-based oral fluid samples from pens of known PRRSV prevalence. In 1 commercial swine barn, 25 pens were assigned to 1 of 5 levels of PRRSV prevalence (0%, 4%, 12%, 20%, or 36%) by placing a fixed number (0, 1, 3, 5, or 9) of PRRSV-positive pigs (14 days post PRRSV modified live virus vaccination) in each pen. Prior to placement of the vaccinated pigs, 1 oral fluid sample was collected from each pen. Thereafter, 5 oral fluid samples were collected from each pen, for a total of 150 samples. To confirm individual pig PRRSV status, serum samples from the PRRSV-negative pigs (n = 535) and the PRRSV vaccinated pigs (n = 90) were tested for PRRSV antibodies and PRRSV RNA. The 150 pen-based oral fluid samples were assayed for PRRSV antibody and PRRSV RNA at 6 laboratories. Among the 100 samples from pens containing ≥1 positive pig (≥4% prevalence) and tested at the 6 laboratories, the mean positivity was 62% for PRRSV RNA and 61% for PRRSV antibody. These results support the use of pen-based oral fluid sampling for PRRSV surveillance in commercial pig populations.

Detection of Porcine reproductive and respiratory syndrome virus (PRRSV) antibodies in oral fluid specimens using a commercial PRRSV serum antibody enzyme-linked immunosorbent assay.

The purpose of the present study was to evaluate the diagnostic performance of a commercial serum antibody enzyme-linked immunosorbent assay (ELISA) modified to detect anti-Porcine reproductive and respiratory syndrome virus (PRRSV) antibodies in pen-based oral fluid specimens. Experimental and field oral fluid samples of defined status in reference to exposure of swine with PRRSV were used to derive the kinetics of detectable concentrations of antibody against PRRSV. Immunoglobulin (Ig)M 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. 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). Receiver operating characteristic analysis estimated the diagnostic sensitivity and specificity of the assay as 94.7% (95% confidence interval [CI]: 92.4, 96.5) and 100% (95% CI: 99.0, 100.0), respectively, at a sample-to-positive ratio cutoff of ≥0.40. The results of the study suggest that the IgG oral fluid ELISA can provide efficient, cost-effective PRRSV monitoring in commercial herds and PRRSV surveillance in elimination programs.

Uni- or bi-ventricular hypertrophy and susceptibility to drug-induced torsades de pointes.

INTRODUCTION: Cardiac hypertrophy is an independent risk factor for torsades de pointes (TdP), a polymorphic ventricular tachycardia that is often drug-induced, that may evolve into ventricular fibrillation and sudden death. Therefore this study was designed to determine if right (RVH), left (LVH), or biventricular (BVH) hypertrophy increases susceptibility to drug-induced TdP. METHODS: Rabbits were separated into 4 groups: control or RVH, LVH, BVH (studied 8weeks after banding of one or both great arteries). ECGs were recorded continuously under anesthesia after baseline and after rabbits received escalating doses of torsadogens (dofetilide, clofilium and terfenadine) or non-torsadogens (cilobradine, diltiazem and vehicle). The following parameters were measured [RR, PQ, QRS and QT] or calculated [QTc (F), short term variability of QT interval]. RESULTS: Generally, torsadogenicity for the compounds tested was dofetilide>clofilium>terfenadine, and there was no TdP following cilobradine, diltiazem or vehicle. In general the susceptibility to TdP was RVH>BVH>LVH>control. Rabbits with RVH developed TdP much more prevalently than for those with either LVH or BVH (p<0.05). At the low dose of dofetilide, LVH was actually protective. CONCLUSION: Rabbits with any form of hypertrophy develop prolongation of QT, QTc and increased QT instability. Rabbits with any form of hypertrophy are more prone to arrhythmia than normals in response to known torsadogens.

Assessment of ECG interval and restitution parameters in the canine model of short QT syndrome.

INTRODUCTION: The short QT syndrome (SQTS) is characterized by a short QT interval resulting from accelerated ventricular repolarization, and may be associated with ventricular fibrillation but not torsades de pointes. There are abundant data on the adverse effects of long QT, but knowledge of SQTS is sparse. The aim of this study was to examine whether analyses of several ECG biomarkers (QT, QTcB, QTcF, QTcV, QT(btb), and QT(RR1000)) and dynamic restitution of the beat-to-beat QT-TQ relationship (TQ(min), %QT/TQ ratio>1, QT/TQ ratio(max)) can be used to assess ECG changes in conscious dogs. METHODS: Sling-trained dogs were infused with escalating concentration of levcromakalim (0, 1.0, 3.3, and 10.0 microg/kg/min), pinacidil (0, 3.3, 10.0, and 33.3 microg/kg/min), and nicorandil (0, 0.03, 0.1, and 0.3 mg/kg/min), drugs known to shorten QT. The RR, QT, QTcB, QTcF, QTcV, QT(RR1000), and TQ were measured before and after each concentration of the QT shortening test compounds. RESULTS: Levcromakalim, pinacidil, and nicorandil but not vehicle significantly shortened RR, QT, QT(btb), QT(RR1000), and TQ but not QTc(B,F,V). The QT-RR cloud also shifted to the lower bounds of the normal QT-RR boundary by the test compounds. The percentage of beats with a QT/TQ ratio>1 was significantly increased in a dose response manner with levcromakalim and pinacidil and the lower TQ interval boundary (5th percentile) was decreased when compared to baseline or vehicle. DISCUSSION: QT(btb), QT(RR1000), and dynamic beat-to-beat measurements of restitution constitute clinically applicable ECG biomarkers for assessment of changes associated with arrhythmogenic risk of ventricular fibrillation due to QT abbreviation.