Apisit Kittawornrat

Porcine reproductive and respiratory syndrome virus (PRRSV) in serum and oral fluid samples from individual boars: will oral fluid replace serum for PRRSV surveillance?

The purpose of this study was to determine whether oral fluid samples could be used to monitor individually-housed adult boars for porcine reproductive and respiratory syndrome virus (PRRSV) infection. In 3 trials, 24 boars were intramuscularly (IM) inoculated with a modified-live PRRSV (MLV) vaccine (Trial 1), a Type 1 PRRSV isolate (Trial 2), or a Type 2 isolate (Trial 3). Oral fluid samples were collected daily and serum samples were collected twice weekly. Following the completion of the study, samples were randomized and blind-tested for PRRSV by real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR). PRRSV was detected in oral fluids at DPI 1 and all oral fluid specimens were PRRSV qRT-PCR positive at DPI 4. Although PRRSV was detected in both serum and oral fluid specimens through DPI 21, a comparison of matched samples from individual boars showed that oral fluid was equal to serum for the detection of PRRSV at DPI 7 and more likely to be positive than serum on DPI 14 and 21. Overall, oral fluid was superior to serum for the detection of PRRSV using PCR over the 21-day observation period in this study. The results of this experiment suggest that individually-penned oral fluid sampling could be an efficient, cost-effective approach to PRRSV surveillance in boar studs and other swine 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.

Toward a better understanding of pig behavior and pig welfare

Abstract Pork production began to flourish in the USA after the practice of finishing pigs on corn was popularized in the late 1600s. By the 1840s, there were 35 million pigs and 20 million people in the USA and Cincinnati was the worlds largest pork market. Between 1890 and the present, the total number of pigs in the USA has remained at 50–60 million, but dramatic changes in swine husbandry over the course of the 20th century have metamorphosed pig production from small, extensive (outdoor), labor-dependent enterprises into large, intensive (indoor), capital-dependent, production systems. This development has led to debate concerning the impact of swine production on animal/human health, the environment, and the welfare of the animals under our care. In a very tangible way, the future of pork production depends on effectively addressing the publics concerns regarding animal welfare and health. Here, we review basic sensory and behavioral aspects of swine with the objective of reaching a better understanding of pig behavior and pig welfare. The premise of this discussion is that safeguarding animal welfare and health is good for pigs, pork producers and the animal-conscious public.

Development of a fluorescent microsphere immunoassay for detection of antibodies against porcine reproductive and respiratory syndrome virus using oral fluid samples as an alternative to serum-based assays.

ABSTRACT For effective disease surveillance, rapid and sensitive assays are needed to detect antibodies developed in response to porcine reproductive and respiratory syndrome virus (PRRSV) infection. In this study, we developed a multiplexed fluorescent microsphere immunoassay (FMIA) for detection of PRRSV-specific antibodies in oral fluid and serum samples. Recombinant nucleocapsid protein (N) and nonstructural protein 7 (nsp7) from both PRRSV genotypes (type I and type II) were used as antigens and covalently coupled to Luminex fluorescent microspheres. Based on an evaluation of 488 oral fluid samples with known serostatus, the oral fluid-based FMIAs achieved >92% sensitivity and 91% specificity. For serum samples (n = 1,639), the FMIAs reached >98% sensitivity and 95% specificity. The assay was further employed to investigate the kinetics of the antibody response in infected pigs. In oral fluid, the N protein was more sensitive for the detection of early infection (7 and 14 days postinfection), but nsp7 detected a higher level and longer duration of antibody response (28 days postinfection). In serum, the antibodies specific to nsp7 and N proteins were detected as early as 7 days postinfection, and the responses lasted more than 202 days. This study provides a framework from which a more robust assay could be developed to profile the immune response to multiple PRRSV antigens in a single test. The development of oral fluid-based diagnostic tests will change the way we survey diseases in swine herds and improve our ability to cheaply and efficiently track PRRSV infections in both populations and individual animals.

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

BackgroundThe 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.ResultsStatistically 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.ConclusionsThis 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 influenza A virus subtypes H1N1 and H3N2 in individual pig nasal swabs and pen-based oral fluid specimens over time.

The probability of detecting influenza A virus (IAV) by virus isolation (VI), point-of-care (POC) antigen detection, and real-time reverse-transcription polymerase chain reaction (rRT-PCR) was estimated for pen-based oral fluid (OF) and individual pig nasal swab (NS) specimens. Piglets (n=82) were isolated for 30 days and confirmed negative for porcine reproductive and respiratory syndrome virus, Mycoplasma hyopneumoniae, and IAV infections. A subset (n=28) was vaccinated on day post inoculation (DPI) -42 and -21 with a commercial multivalent vaccine. On DPI 0, pigs were intratracheally inoculated with contemporary isolates of H1N1 (n=35) or H3N2 (n=35) or served as negative controls (n=12). OF (n=370) was collected DPI 0-16 and NS (n=924) DPI 0-6, 8, 10, 12, 14, 16. The association between IAV detection and variables of interest (specimen, virus subtype, assay, vaccination status, and DPI) was analyzed by mixed-effect repeated measures logistic regression and the results used to calculate the probability (pˆ) of detecting IAV in OF and NS over DPI by assay. Vaccination (p-value<0.0001), DPI (p-value<0.0001), and specimen-assay interaction (p-value<0.0001) were significant to IAV detection, but virus subtype was not (p-value=0.89). Vaccination and/or increasing DPI reduced pˆ for all assays. VI was more successful using NS than OF, but both VI and POC were generally unsuccessful after DPI 6. Overall, rRT-PCR on OF specimens provided the highest pˆ for the most DPIs, yet significantly different results were observed between the two laboratories independently performing rRT-PCR testing.

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.

Kinetics of influenza A virus nucleoprotein antibody (IgM, IgA, and IgG) in serum and oral fluid specimens from pigs infected under experimental conditions.

Indirect influenza A virus (IAV) nucleoprotein (NP) antibody ELISAs were used to compare the kinetics of the NP IgM, IgA, and IgG responses in serum and pen-based oral fluid samples collected from 82 pigs followed for 42 days post inoculation (DPI). Treatment categories included vaccination (0, 1) and inoculation (0, 1) with contemporary H1N1 or H3N2 isolates. Antibody ontogeny was markedly affected by vaccination status, but no significant differences were detected between H1N1 and H3N2 inoculated groups of the same vaccination status (0, 1) in IgM, IgA, or IgG responses. Therefore, these data were combined in subsequent analyses. The correlation between serum and oral fluid responses was evaluated using the pen-based oral fluid sample-to-positive (S/P) ratios versus the mean serum S/P ratios of pigs within the pen. 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, IgM responses were not correlated in vaccinated groups and the correlation between serum and oral fluid IgA was weak (r∼0.3), regardless of vaccination status. In general, vaccinated animals exhibited a suppressed IgM response and accelerated IgG response. 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.

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.

Evaluation of Screening Assays for the Detection of Influenza A Virus Serum Antibodies in Swine

Increased surveillance of influenza A virus (IAV) infections in human and swine populations is mandated by public health and animal health concerns. Antibody assays have proven useful in previous surveillance programmes because antibodies provide a record of prior exposure and the technology is inexpensive. The objective of this research was to compare the performance of influenza serum antibody assays using samples collected from pigs (vaccinated or unvaccinated) inoculated with either A/Swine/OH/511445/2007 γ H1N1 virus or A/Swine/Illinois/02907/2009 Cluster IV H3N2 virus and followed for 42 days. Weekly serum samples were tested for anti-IAV antibodies using homologous and heterologous haemagglutination-inhibition (HI) assays, commercial swine influenza H1N1 and H3N2 indirect ELISAs, and a commercial influenza nucleoprotein (NP)-blocking ELISA. The homologous HIs showed 100% diagnostic sensitivity, but largely failed to detect infection with the heterologous virus. With diagnostic sensitivities of 1.4% and 4.9%, respectively, the H1N1 and H3N2 indirect ELISAs were ineffective at detecting IAV antibodies in swine infected with the contemporary influenza viruses used in the study. At a cut-off of S/N ≤ 0.60, the sensitivity and specificity of the NP-blocking ELISA were estimated at 95.5% and 99.6%, respectively. Statistically significant factors which affected S/N results include vaccination status, inoculum (virus subtype), day post-inoculation and the interactions between those factors (P < 0.0001). Serum antibodies against NP provide an ideal universal diagnostic screening target and could provide a cost-effective approach for the detection and surveillance of IAV infections in swine populations.