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.

Reactivity of Porcine Epidemic Diarrhea Virus Structural Proteins to Antibodies against Porcine Enteric Coronaviruses: Diagnostic Implications.

The development of porcine epidemic diarrhea virus (PEDV) antibody-based assays is important for detecting infected animals, confirming previous virus exposure, and monitoring sow herd immunity. However, the potential cross-reactivity among porcine coronaviruses is a major concern for the development of pathogen-specific assays. In this study, we used serum samples (n = 792) from pigs of precisely known infection status and a multiplex fluorescent microbead-based immunoassay and/or enzyme-linked immunoassay platform to characterize the antibody response to PEDV whole-virus (WV) particles and recombinant polypeptides derived from the four PEDV structural proteins, i.e., spike (S), nucleocapsid (N), membrane (M), and envelope (E). Antibody assay cutoff values were selected to provide 100% diagnostic specificity for each target. The earliest IgG antibody response, mainly directed against S1 polypeptides, was observed at days 7 to 10 postinfection. With the exception of nonreactive protein E, we observed similar antibody ontogenies and patterns of seroconversion for S1, N, M, and WV antigens. Recombinant S1 provided the best diagnostic sensitivity, regardless of the PEDV strain, with no cross-reactivity detected against transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV), or porcine deltacoronavirus (PDCoV) pig antisera. The WV particles showed some cross-reactivity to TGEV Miller and TGEV Purdue antisera, while N protein presented some cross-reactivity to TGEV Miller. The M protein was highly cross-reactive to TGEV and PRCV antisera. Differences in the antibody responses to specific PEDV structural proteins have important implications in the development and performance of antibody assays for the diagnosis of PEDV enteric disease.

DETECTION OF SARS-COV-2 NEUTRALIZING ANTIBODIES IN RETROPHARYNGEAL LYMPH NODE EXUDATES OF WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS) FROM NEBRASKA, USA.

Disease surveillance testing for emerging zoonotic pathogens in wildlife is a key component in understanding the epidemiology of these agents and potential risk to human populations. Recent emergence of SARS-CoV-2 in humans, and subsequent detection of this virus in wildlife, highlights the need for developing new One Health surveillance strategies. We used lymph node exudate, a sample type that is routinely collected in hunter-harvested white-tailed deer (WTD, Odocoileus virginianus) for surveillance of chronic wasting disease, to assess anti-SARS-CoV-2 neutralizing antibodies. A total of 132 pairs of retropharyngeal lymph nodes collected from Nebraska WTD harvested in Nebraska, US, in 2019 (pre-SARS-CoV-2 pandemic) and 2021 (post-SARS-CoV-2 pandemic) were tested for SARS-CoV-2 with reverse transcription PCR. Thereafter, exudates obtained from these same lymph nodes were tested for SARS-CoV-2 neutralizing antibodies using a surrogate virus neutralization test. Neutralizing antibodies were detected in the exudates with high diagnostic specificity (100% at proposed cutoff of 40% inhibition). Application of this testing approach to samples collected for use in other disease surveillance activities may provide additional epidemiological data on SARS-CoV-2 exposure, and there is further potential to apply this sample type to detection of other pathogens of interest.

Comparison of Two Diagnostic Assays for the Detection of Serum Neutralizing Antibody to Porcine Epidemic Diarrhea Virus.

Lactogenic immunity is important for the protection of piglets against many pathogens including porcine epidemic diarrhea virus. Circulating neutralizing antibodies levels in sow sera may help determine if a detectable immune response could confer protection to piglets. Neutralizing antibodies can be detected through various diagnostic assays. This study evaluated the diagnostic characteristics of two neutralizing antibody assays for porcine epidemic diarrhea virus neutralizing antibodies in serum of challenged gilts. Four treatment groups, control, non-vaccinated, vaccinated prior to challenge, and vaccinated following challenge, were comprised of 20 gilts. Serum sample were collected from each gilt prior to and following challenge with porcine epidemic diarrhea virus. Samples were evaluated for the presence of neutralizing antibodies via a fluorescent focus neutralization assay and a high-throughput neutralization assay. Diagnostic sensitivity and specificity for the fluorescent focus neutralization and high-throughput neutralization assays for this study were optimized at a cutoff of a dilution of 80 and 80% fluorescent reduction respectively and demonstrated moderate agreement based off the kappa statistic. The focus fluorescent neutralization and high-throughput neutralization assays can be used to monitor the status of neutralizing antibodies within animals or a population of animals. The high-throughput assay has advantages over the focus fluorescent assay in that it has a higher specificity at the indicated cut-off and the nature of the results allows for more discrimination between individual results.

Detection of Mannheimia haemolytica-Specific IgG, IgM and IgA in Sera and Their Relationship to Respiratory Disease in Cattle.

Mannheimia haemolytica is one of the major causes of bovine respiratory disease in cattle. The organism is the primary bacterium isolated from calves and young cattle affected with enzootic pneumonia. Novel indirect ELISAs were developed and evaluated to enable quantification of antibody responses to whole cell antigens using M. haemolytica A1 strain P1148. In this study, the ELISAs were initially developed using sera from both M. haemolytica-culture-free and clinically infected cattle, then the final prototypes were tested in the validation phase using a larger set of known-status M. haemolytica sera (n = 145) collected from feedlot cattle. The test showed good inter-assay and intra-assay repeatability. Diagnostic sensitivity and specificity were estimated at 91% and 87% for IgG at a cutoff of S/P ≥ 0.8. IgM diagnostic sensitivity and specificity were 91% and 81% at a cutoff of sample to positive (S/P) ratio ≥ 0.8. IgA diagnostic sensitivity was 89% whereas specificity was 78% at a cutoff of S/P ≥ 0.2. ELISA results of all isotypes were related to the diagnosis of respiratory disease and isolation of M. haemolytica (p-value < 0.05). These data suggest that M. haemolytica ELISAs can be adapted to the detection and quantification of antibody in serum specimens and support the use of these tests for the disease surveillance and disease prevention research in feedlot cattle.

Detection of pseudorabies virus antibody in swine serum and oral fluid specimens using a recombinant gE glycoprotein dual-matrix indirect ELISA.

Pseudorabies (Aujeszky disease) virus (PRV) was eliminated from domestic swine in many countries using glycoprotein E (gE)-deleted vaccines and serum antibody gE ELISAs, but PRV continues to circulate in some regions and in most feral swine populations in the world. We created a dual-matrix (serum and oral fluid) indirect IgG gE ELISA (iELISA) and evaluated its performance using samples from 4 groups of 10 pigs each: negative control (NC), vaccination (MLV), PRV inoculation (PRV), and vaccination followed by challenge (MLV-PRV). All serum and oral fluid samples collected before PRV challenge and all NC samples throughout the study were negative for gE antibodies by commercial blocking ELISA (bELISA) and our iELISA. Nasal swab samples from 9 of 10 animals in the PRV group were gB quantitative PRC (qPCR) positive at 2 days post-inoculation (dpi). The oral fluid iELISA detected a significant S/P response in the PRV (p = 0.03) and MLV-PRV (p = 0.01) groups by 6 dpi. ROC analyses of serum bELISA (n = 428), serum iELISA (n = 426), and oral fluid iELISA (n = 247) showed no significant differences in performance (p > 0.05). Our data support the concept of PRV surveillance based on oral fluid samples tested by an indirect gE ELISA.

Evaluation of colonization, variable lipoprotein-based serological response, and cellular immune response of Mycoplasma hyorhinis in experimentally infected swine.

Mycoplasma hyorhinis (Mhr) is a commensal of the upper respiratory tract that can be shed by nasal secretions and transmitted by direct contact in neonatal and nursery pigs. Lesions associated with Mhr infection include polyserositis and arthritis; however, systemic Mhr disease pathogenesis is not well characterized. This study aimed to investigate the immunopathogenesis and bacterial dissemination pattern of Mhr using single and multiple inoculation approaches in a caesarian-derived colostrum-deprived (CDCD) pig model. Animals in three treatment groups were inoculated once (Mhr 1; n = 12) or four (Mhr 2; n = 8) times with Mhr or sham-inoculated (NC group; n = 3) nasally and by tonsillar painting. Inoculum consisted of a triple cloned Mhr field isolate (4.5 × 107 CFU/mL) in Friis medium. Clinical signs were evaluated daily during the study. Serum and oral fluid antibody (IgA and IgG) response and cellular immune response were assessed using a recombinant chimeric VlpA-G-based indirect ELISA and by ELISpot, respectively. The presence of Mhr in oral fluids, nasal and oropharyngeal swabs were evaluated by qPCR. At 6 wpi, pigs were euthanized and evaluated for gross lesions consistent with Mhr and bacterial colonization in tonsils by qPCR. No clinical signs or gross lesions consistent with Mhr-associated disease were observed throughout the study. For Mhr 2 group, the presence of IgA and IgG in serum and oral fluids were detected at 2 and 4 weeks post-inoculation (wpi), respectively, while in Mhr 1, only IgA was detected in oral fluids at 6 wpi. The proportion of animals shedding Mhr in nasal secretions varied from 20 to 40 % in the Mhr 1 and 62.5-100% in the Mhr 2 group. However, the proportion of animals shedding Mhr in oropharyngeal swabs was consistent through the study (60 %) in Mhr 1 and fluctuated from 20 % to 87.5 % in Mhr 2 group. The lack of clinical signs and the presence of Mhr specific humoral response and bacterial colonization indicates that the multiple inoculation experimental model may mimic subclinical natural infection in the field. In addition, the humoral and transient cellular response did not result in bacterial clearance. Based on these results, animals would have to be exposed multiple times to mount a detectable immune response.

Pseudorabies (Aujeszky's disease) virus DNA detection in swine nasal swab and oral fluid specimens using a gB-based real-time quantitative PCR.

In this study, the detection of PRV DNA in nasal swab (n = 440) and oral fluid (n = 1,545) samples collected over time from experimentally PRV vaccinated and/or PRV inoculated pigs (n = 40) was comparatively evaluated by real-time PCR. Serum samples (n = 440) were tested by PRV gB/gE blocking ELISAs (Pseudorabies Virus gB Antibody Test Kit and Pseudorabies Virus gpI Antibody Test Kit, IDEXX Laboratories, Inc., Westbrook, ME) to monitor PRV status over time. Following exposure to a gE-deleted modified live vaccine (Ingelvac® Aujeszky MLV, Boehringer Ingelheim, Ridgefield, CT) and/or a wild-type virus (3 CR Ossabaw), PRV gB DNA was detected in oral fluid specimens in a pattern similar to that of nasal swabs. For quantitative analyses, PRV PCR quantification cycle (Cq) results were re-expressed as "efficiency standardized Cqs (ECqs)" as a function of PCR efficiency using plate-specific positive amplification controls. ROC analyses of the PRV gB PCR ECqs results showed a similar performance of the PRV gB PCR for nasal swab and oral fluid specimens (area under the ROC curve = 85 % vs 83 %) and, based on an ECq cutoff of 0.01 a diagnostic specificity of 100 % and diagnostic sensitivities for oral fluid and nasal swab specimens of 53 % (95 % CI: 43 %, 62 %) and 70 % (95 % CI: 55 %, 83 %), respectively. Thus, the results described herein demonstrated the detection of PRV gB DNA in swine oral fluid and supported the use of this specimen in PRV diagnosis and surveillance.

Detection of porcine epidemic diarrhea virus-neutralizing antibody using high-throughput imaging cytometry.

Porcine epidemic diarrhea virus (PEDV) is an emerging porcine coronavirus that causes a tremendous economic burden on the swine industry. The assessment of PEDV-neutralizing antibody levels provides a valuable tool to assess and predict herd immunity. We evaluated the performance of a PEDV imaging cytometry-based high-throughput neutralization test (HTNT) and compared the HTNT to a fluorescent focus neutralization (FFN) assay using serum samples from pigs of known PEDV infection status (n = 159). Estimates of diagnostic sensitivity and specificity for HTNT and FFN assays derived from receiver-operator characteristic (ROC) curve analyses showed that both PEDV FFN and HTNT provided excellent diagnostic performance. However, in the laboratory, imaging cytometry provided an objective and semi-automated approach that removed human subjectivity from the testing process and reduced the read-time of a 96-well plate to < 4 min. In addition, imaging cytometry facilitated the rapid collection and long-term storage of test images and data for further evaluation or client consultation. For PEDV and other pathogens, imaging cytometry could provide distinct advantages over classic virus neutralization or FFN assays for the detection and quantitation of neutralizing antibody.

Detection of pseudorabies virus antibody in swine oral fluid using a serum whole-virus indirect ELISA.

We evaluated the detection of pseudorabies virus (PRV) antibodies in swine oral fluid. Oral fluid and serum samples were obtained from 40 pigs allocated to 4 treatment groups (10 pigs/group): negative control (NC); wild-type PRV inoculation (PRV 3CR Ossabaw; hereafter PRV); PRV vaccination (Ingelvac Aujeszky MLV; Boehringer Ingelheim; hereafter MLV); and PRV vaccination followed by PRV inoculation at 21 d post-vaccination (MLV-PRV). Using a serum PRV whole-virus indirect IgG ELISA (Idexx Laboratories) adapted to the oral fluid matrix, PRV antibody was detected in oral fluid samples from treatment groups PRV, MLV, and MLV-PRV in a pattern similar to serum. Vaccination alone produced a low oral fluid antibody response (groups MLV and MLV-PRV), but a strong anamnestic response was observed following challenge with wild-type virus (group PRV). Analyses of the oral fluid PRV indirect IgG ELISA results showed good binary diagnostic performance (area under ROC curve = 93%) and excellent assay repeatability (intra-class correlation coefficient = 99.3%). The demonstrable presence of PRV antibodies in swine oral fluids suggests the possible use of oral fluids in pseudorabies surveillance.

Evaluation of the Serologic Cross-Reactivity between Transmissible Gastroenteritis Coronavirus and Porcine Respiratory Coronavirus Using Commercial Blocking Enzyme-Linked Immunosorbent Assay Kits.

This study compared the performances of three commercial transmissible gastroenteritis virus/porcine respiratory coronavirus (TGEV/PRCV) blocking enzyme-linked immunosorbent assays (ELISAs) using serum samples (n = 528) collected over a 49-day observation period from pigs inoculated with TGEV strain Purdue (n = 12), TGEV strain Miller (n = 12), PRCV (n = 12), or with virus-free culture medium (n = 12). ELISA results were evaluated both with "suspect" results interpreted as positive and then as negative. All commercial kits showed excellent diagnostic specificity (99 to 100%) when testing samples from pigs inoculated with virus-free culture medium. However, analyses revealed differences between the kits in diagnostic sensitivity (percent TGEV- or PRCV-seropositive pigs), and all kits showed significant (P < 0.05) cross-reactivity between TGEV and PRCV serum antibodies, particularly during early stages of the infections. Serologic cross-reactivity between TGEV and PRCV seemed to be TGEV strain dependent, with a higher percentage of PRCV-false-positive results for pigs inoculated with TGEV Purdue than for TGEV Miller. Moreover, the overall proportion of false positives was higher when suspect results were interpreted as positive, regardless of the ELISA kit evaluated.IMPORTANCE Current measures to prevent TGEV from entering a naive herd include quarantine and testing for TGEV-seronegative animals. However, TGEV serology is complicated due to the cross-reactivity with PRCV, which circulates subclinically in most swine herds worldwide. Conventional serological tests cannot distinguish between TGEV and PRCV antibodies; however, blocking ELISAs using antigen containing a large deletion in the amino terminus of the PRCV S protein permit differentiation of PRCV and TGEV antibodies. Several commercial TGEV/PRCV blocking ELISAs are available, but performance comparisons have not been reported in recent research. This study demonstrates that the serologic cross-reactivity between TGEV and PRCV affects the accuracy of commercial blocking ELISAs. Individual test results must be interpreted with caution, particularly in the event of suspect results. Therefore, commercial TGEV/PRCV blocking ELISAs should only be applied on a herd basis.

Early detection and differential serodiagnosis of Mycoplasma hyorhinis and Mycoplasma hyosynoviae infections under experimental conditions.

Mycoplasma hyorhinis (MHR) and Mycoplasma hyosynoviae (MHS) are common opportunistic pathogens in the upper respiratory tract and tonsils of swine. The identification of the specific species involved in clinical cases using conventional diagnostic methods is challenging. Therefore, a recombinant chimeric polypeptide based on the seven known variable lipoproteins (A-G) specific of MHR and a cocktail of surface proteins detergent-extracted from MHS cultures were generated and their suitability as antemortem biomarkers for serodiagnosis of MHR- and MHS-infection were evaluated by ELISA. M. hyorhinis and MHS ELISA performance, evaluated using serum samples collected over a 56-day observation period from pigs inoculated with MHR, MHS, M. hyopneumoniae, M. flocculare, or Friis medium, varied by assay, targeted antibody isotype, and cutoffs. The progressions of MHR and MHS clinical diseases were evaluated in relation to the kinetics of the isotype-specific antibody response in serum and bacterial shedding in oral fluids during the observation period. In pigs inoculated with MHR, bacterial DNA was detected in one or more of the 5 pens at all sampling points throughout the study, IgA was first detected at DPI 7, one week before the first clinical signs, with both IgA and IgG detected in all samples collected after DPI 14. The peak of MHS shedding (DPI 8) coincided with the onset of the clinical signs, with both IgA and IgG detected in all serum samples collected ≥ DPI 14. This study demonstrated, under experimental conditions, that both ELISAs were suitable for early detection of specific antibodies against MHR or MHS. The diagnostic performance of the MHR and MHS ELISAs varied depending on the selected cutoff and the antibody isotype evaluated. The high diagnostic and analytical specificity of the ELISAs was particularly remarkable. This study also provides insights into the infection dynamics of MHR-associated disease and MHS-associated arthritis not previously described.

Historical and contemporary aspects of maternal immunity in swine.

Maternal immunity plays a pivotal role in swine health and production because piglets are born agammaglobulinemic and with limited cell-mediated immunity, i.e. few peripheral lymphoid cells, immature lymphoid tissues, and no effector and memory T-lymphocytes. Swine do not become fully immunologically competent until about 4 weeks of age, which means that their compromised ability to respond to infectious agents during the first month of life must be supplemented by maternal immune components: (1) circulating antibodies derived from colostrum; (2) mucosal antibodies from colostrum and milk; and (3) immune cells provided in mammary secretions. Because maternal immunity is highly effective at protecting piglets against specific pathogens, strengthening sow herd immunity against certain diseases through exposure or vaccination is a useful management tool for ameliorating clinical effects in piglets and delaying infection until the piglets' immune system is better prepared to respond. In this review, we discuss the anatomy and physiology of lactation, the immune functions of components provided to neonatal swine in mammary secretion, the importance of maternal immunity in the prevention and control of significant pathogens.

A review of foot-and-mouth disease virus (FMDV) testing in livestock with an emphasis on the use of alternative diagnostic specimens.

Foot-and-mouth disease virus (FMDV) remains an important pathogen of livestock more than 120 years after it was identified, with annual costs from production losses and vaccination estimated at €5.3-€17 billion (US$6.5-US$21 billion) in FMDV-endemic areas. Control and eradication are difficult because FMDV is highly contagious, genetically and antigenically diverse, infectious for a wide variety of species, able to establish subclinical carriers in ruminants, and widely geographically distributed. For early detection, sustained control, or eradication, sensitive and specific FMDV surveillance procedures compatible with high through-put testing platforms are required. At present, surveillance relies on the detection of FMDV-specific antibody or virus, most commonly in individual animal serum, vesicular fluid, or epithelial specimens. However, FMDV or antibody are also detectable in other body secretions and specimens, e.g., buccal and nasal secretions, respiratory exhalations (aerosols), mammary secretions, urine, feces, and environmental samples. These alternative specimens offer non-invasive diagnostic alternatives to individual animal sampling and the potential for more efficient, responsive, and cost-effective surveillance. Herein we review FMDV testing methods for contemporary and alternative diagnostic specimens and their application to FMDV surveillance in livestock (cattle, swine, sheep, and goats).

Detection of porcine epidemic diarrhea virus (PEDV) IgG and IgA in muscle tissue exudate ("meat juice") specimens.

The diagnostic performance of porcine epidemic diarrhea virus (PEDV) IgG and IgA ELISAs was evaluated using paired serum and meat juice samples collected from PEDV-negative (n = 50) and PEDV-inoculated pigs (n = 87). Serum samples were tested by PEDV (IgG, IgA) ELISAs using a procedure performed routinely at the Iowa State University-Veterinary Diagnostic Laboratory (ISU-VDL). Serum samples were tested using PEDV serum IgG and IgA ELISA procedures as routinely performed at the Iowa State University-Veterinary Diagnostic Laboratory (ISU-VDL). Serum samples were diluted 1:50 and conjugate concentrations were 1/20,000 for IgG and 1/3000 for IgA. Meat juice samples were tested using the serum PEDV IgG and IgA ELISAs, with modifications, i.e., meat juice samples were diluted 1:25 and conjugate concentrations were 1/40,000 for IgG and 1/10,000 for IgA. Receiver operator characteristic (ROC) curve analyses were used to estimate diagnostic sensitivities and specificities over a range of sample-to-positive (S/P) cutoffs. Consistent with previous reports, this study showed that the PEDV IgG and IgA meat juice ELISAs provided excellent diagnostic performance and suggest that meat juice recovered from samples collected at slaughter could be used in routine PEDV surveillance.

Effects of sample handling on the detection of porcine reproductive and respiratory syndrome virus in oral fluids by reverse-transcription real-time PCR.

We evaluated effects of handling procedures on detection of porcine reproductive and respiratory syndrome virus (PRRSV) in oral fluids (OFs) by reverse-transcription real-time PCR (RT-rtPCR). The experiments were conducted using a composite sample of PRRSV-positive OF collected from 5-wk-old pigs vaccinated 15 d earlier with a modified-live PRRSV vaccine. Five pre-extraction sample-handling steps and all combinations thereof were evaluated: 1) thaw temperature (4°C or 25°C); 2) sample diluent (1:1 dilution with nuclease-free water or guanidinium thiocyanate-phenol); 3a) sonication of the sample (yes or no); 3b) temperature (4°C or 25°C) at which step 3a was conducted; and 4) temperature at which the sample was maintained after step 3b and until RNA extraction was initiated (4°C or 25°C). All combinations of the 5 sample-handling steps (i.e., 32 unique treatments) were tested in a completely randomized factorial design with 4 replicates and 1 negative control for each treatment. The entire experiment was repeated on 5 separate days to produce a total of 800 PRRSV RT-rtPCR results. Binary (positive or negative) data were analyzed by logistic regression and results (Ct) were analyzed using a generalized linear model. Overall, 1 false-positive result was observed among 160 negative controls (99.4% specificity), and 85 false-negative results were observed among the 640 known-positive samples (86.7% sensitivity). The most significant factor affecting test outcome was thaw temperature (4°C or 25°C); samples thawed at 4°C had higher positivity rate (94% vs. 80%, p < 0.0001) and lower Ct (36.2 vs. 37.5, p < 0.0001).

The effect of chemical clarification of oral fluids on porcine epidemic diarrhea virus antibody responses.

Routine testing of breeding herd oral fluid (OF) samples for porcine epidemic diarrhea virus (PEDV) IgG and/or IgA is used to track levels of PEDV immunity over time. However, OFs contain particles of feed, feces, and inorganic material that detract from the quality of the sample. We clarified swine OF samples using lyophilized chitosan-based formulas (A-C) tested by PEDV IgG and IgA ELISAs. To evaluate both the immediate and residual effects of treatment on antibody detection, samples were tested immediately post-treatment, then stored at 4°C and retested at 2, 4, and 6 days post-treatment (DPT). Formulations were shown to effectively clarify samples. Statistical analysis comparing treated to untreated OF samples at 0 DPT found that neither chitosan nor Tween 20 affected the OF ELISA IgA and IgG sample-to-positive (S/P) ratio results ( p > 0.05). Furthermore, pairwise comparisons of 0 DPT to 2, 4, and 6 DPT results detected no significant differences ( p > 0.05) in IgA and IgG S/P ratios (i.e., treated OF samples were stable over time). Therefore, chitosan efficiently clarified OF specimens without affecting the results of the PEDV IgG and IgA antibody ELISAs.

Effect of chemical clarification of oral fluids on the detection of porcine reproductive and respiratory syndrome virus IgG.

Routine collection and testing of oral fluid (OF) samples facilitates porcine reproductive and respiratory syndrome virus (PRRSV) surveillance in commercial swine herds in a cost-effective, welfare-friendly fashion. However, OFs often contain environmental contaminants that may affect liquid handling and test performance. Traditional processing methods (e.g., filtration or centrifugation) are not compatible with high-throughput testing because of the burden of additional processing costs and time. OF "clarification" using chemical flocculants is an alternative approach not widely explored. Therefore, we evaluated the effect of chitosan-based clarification treatment on a commercial PRRSV OF ELISA. Serum and individual OFs were collected from vaccinated pigs ( n = 17) at -7 to 42 d post-vaccination and subdivided into 4 aliquots. Each aliquot was clarified (treatment A, B, C), with the 4th aliquot serving as untreated control. All samples were tested by PRRSV OF ELISA immediately after treatment and then were held at 4°C to be re-tested at 2, 4, 6, and 14 d post-treatment. Quantitative and qualitative treatment effects were evaluated. A Kruskal-Wallis test found no significant difference in ELISA S/P responses among treatments by days post-treatment. No difference was detected in the proportion of positive PRRSV antibody samples among treatments (Cochran Q, p > 0.05). Treatment of swine OFs using chitosan-based formulations did not affect the performance of a commercial PRRSV OF ELISA. Chitosan (or other flocculants) could improve OF characteristics and could be adapted for use in the field or in high-throughput laboratories.

Detection of Actinobacillus Pleuropneumoniae ApxIV Toxin Antibody in Serum and Oral Fluid Specimens from Pigs Inoculated Under Experimental Conditions.

INTRODUCTION: The prevention and control of Actinobacillus pleuropneumoniae in commercial production settings is based on serological monitoring. Enzyme-linked immunosorbent assays (ELISAs) have been developed to detect specific antibodies against a variety of A. pleuropneumoniae antigens, including long-chain lipopolysaccharides (LPS) and the ApxIV toxin, a repeats-in-toxin (RTX) exotoxin unique to A. pleuropneumoniae and produced by all serovars. The objective of this study was to describe ApxIV antibody responses in serum and oral fluid of pigs. MATERIAL AND METHODS: Four groups of pigs (six pigs per group) were inoculated with A. pleuropneumoniae serovars 1, 5, 7, or 12. Weekly serum samples and daily oral fluid samples were collected from individual pigs for 56 days post inoculation (DPI) and tested by LPS and ApxIV ELISAs. The ApxIV ELISA was run in three formats to detect immunlgobulins M, G, and A (IgM, IgG and IgA) while the LPS ELISA detected only IgG. RESULTS: All pigs inoculated with A. pleuropneumoniae serovars 1 and 7 were LPS ELISA serum antibody positive from DPI 14 to 56. A transient and weak LPS ELISA antibody response was observed in pigs inoculated with serovar 5 and a single antibody positive pig was observed in serovar 12 at ≥35 DPI. Notably, ApxIV serum and oral fluid antibody responses in pig inoculated with serovars 1 and 7 reflected the patterns observed for LPS antibody, albeit with a 14 to 21 day delay. CONCLUSION: This work suggests that ELISAs based on ApxIV antibody detection in oral fluid samples could be effective in population monitoring for A. pleuropneumoniae.

Quantifying the effect of lactogenic antibody on porcine epidemic diarrhea virus infection in neonatal piglets.

The contribution of lactogenic antibody to the protection of piglets against porcine epidemic diarrhea virus (PEDV) was evaluated. Pregnant multiparous sows and their litters were allocated to one of 3 treatment groups: Group 1-6 serum antibody-negative sows and a subset (n=11) of their piglets. Group 2-8 serum antibody-positive sows and their 91 piglets. Piglets were orally inoculated with PEDV at 4 (Group 1) or 2 (Group 2) days of age. Group 3-2 PEDV serum antibody-negative sows and 22 piglets, provided a baseline for piglet survivability and growth rate. Piglets were monitored daily for clinical signs, body weight, and body temperature through day post-inoculation (DPI) 12 (Groups 2 and 3) or 14 (Group 1). Serum and mammary secretions were tested for PEDV IgG, IgA, and virus-neutralizing antibody. Feces were tested by PEDV real-time, reverse transcriptase PCR (rRT-PCR). Piglets on sows without (Group 1) or with (Group 2) anti-PEDV antibody showed significantly different responses to PEDV infection in virus shedding (p<0.05), thermoregulation (p<0.05), growth rate (p<0.05), and survivability (p<0.0001). Specifically, Group 1 piglets shed more virus on DPIs 1 to 5, were hypothermic at all sampling points except DPIs 9, 11, and 12, gained weight more slowly, and exhibited lower survivability than Group 2 piglets. Within Group 2 litters, significant differences were found in virus shedding (p<0.05), and body temperature (p<0.05), but not in piglet survival rate. The number of sows and litters in Group 2 was insufficient to derive the relationship between specific levels of lactogenic antibody (FFN, IgA, and IgG) and the amelioration of clinical effects. However, when combined with previous PEDV literature, it can be concluded that the optimal protection to piglets will be provided by dams able to deliver sufficient lactogenic immunity, both humoral and cellular, to their offspring.