Korakrit Poonsuk

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

Abstract 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.

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

ABSTRACT 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 Actinobacillus pleuropneumoniae ApxIV toxin antibody in serum and oral fluid specimens from pigs inoculated under experimental conditions

Abstract 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.

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.