Considerations for improved surveillance of Influenzavirus A in swine populations

This thesis consists of 4 chapters. Chapter 1 is a general introduction and road map for contemporary swine surveillance programs, Protecting our future - A road map for practical, real-time, on-farm infectious disease surveillance . Chapter 2, Ring test evaluation of the detection of influenza A virus in swine oral fluids by real-time, reverse transcription polymerase chain reaction (rRT-PCR) and virus isolation has been submitted for publication in the Journal of Veterinary Diagnostic Investigation. Chapter 3, Probability of the detection of influenza A virus subtypes H1N1 and H3N2 in individual nasal swab and pen-based oral fluid specimens from pigs over time has been submitted for publication to Veterinary Microbiology. Chapter 4, Evaluation of screening assays for the detection of Influenza A virus serum antibodies in swine has been submitted for publication to the Journal of Veterinary Diagnostic Investigation. References, tables, and figures for each manuscript follow each discussion section respectively. The last chapter contains general conclusions of this dissertation

Detection of African Swine Fever Virus Antibodies in Serum and Oral Fluid Specimens Using a Recombinant Protein 30 (p30) Dual Matrix Indirect ELISA

In the absence of effective vaccine(s), control of African swine fever caused by African swine fever virus (ASFV) must be based on early, efficient, cost-effective detection and strict control and elimination strategies. For this purpose, we developed an indirect ELISA capable of detecting ASFV antibodies in either serum or oral fluid specimens. The recombinant protein used in the ELISA was selected by comparing the early serum antibody response of ASFV-infected pigs (NHV-p68 isolate) to three major recombinant polypeptides (p30, p54, p72) using a multiplex fluorescent microbead-based immunoassay (FMIA). Non-hazardous (non-infectious) antibody-positive serum for use as plate positive controls and for the calculation of sample-to-positive (S:P) ratios was produced by inoculating pigs with a replicon particle (RP) vaccine expressing the ASFV p30 gene. The optimized ELISA detected anti-p30 antibodies in serum and/or oral fluid samples from pigs inoculated with ASFV under experimental conditions beginning 8 to 12 days post inoculation. Tests on serum (n = 200) and oral fluid (n = 200) field samples from an ASFV-free population demonstrated that the assay was highly diagnostically specific. The convenience and diagnostic utility of oral fluid sampling combined with the flexibility to test either serum or oral fluid on the same platform suggests that this assay will be highly useful under the conditions for which OIE recommends ASFV antibody surveillance, i.e., in ASFV-endemic areas and for the detection of infections with ASFV isolates of low virulence

Effects of Local and Landscape Factors on Population Dynamics of a Cotton Pest

BACKGROUND: Many polyphagous pests sequentially use crops and uncultivated habitats in landscapes dominated by annual crops. As these habitats may contribute in increasing or decreasing pest density in fields of a specific crop, understanding the scale and temporal variability of source and sink effects is critical for managing landscapes to enhance pest control. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated how local and landscape characteristics affect population density of the western tarnished plant bug, Lygus hesperus (Knight), in cotton fields of the San Joaquin Valley in California. During two periods covering the main window of cotton vulnerability to Lygus attack over three years, we examined the associations between abundance of six common Lygus crops, uncultivated habitats and Lygus population density in these cotton fields. We also investigated impacts of insecticide applications in cotton fields and cotton flowering date. Consistent associations observed across periods and years involved abundances of cotton and uncultivated habitats that were negatively associated with Lygus density, and abundance of seed alfalfa and cotton flowering date that were positively associated with Lygus density. Safflower and forage alfalfa had variable effects, possibly reflecting among-year variation in crop management practices, and tomato, sugar beet and insecticide applications were rarely associated with Lygus density. Using data from the first two years, a multiple regression model including the four consistent factors successfully predicted Lygus density across cotton fields in the last year of the study. CONCLUSIONS/SIGNIFICANCE: Our results show that the approach developed here is appropriate to characterize and test the source and sink effects of various habitats on pest dynamics and improve the design of landscape-level pest management strategies

Ring test evaluation of the detection of influenza A virus in swine oral fluids by real-time reverse-transcription polymerase chain reaction and virus isolation

The probability of detecting influenza A virus (IAV) in oral fluid (OF) specimens was calculated for each of 13 assays based on real-time reverse-transcription polymerase chain reaction (rRT-PCR) and 7 assays based on virus isolation (VI). The OF specimens were inoculated with H1N1 or H3N2 IAV and serially diluted 10-fold (10(-1) to 10(-8)). Eight participating laboratories received 180 randomized OF samples (10 replicates × 8 dilutions × 2 IAV subtypes plus 20 IAV-negative samples) and performed the rRT-PCR and VI procedure(s) of their choice. Analysis of the results with a mixed-effect logistic-regression model identified dilution and assay as variables significant (P \u3c 0.0001) for IAV detection in OF by rRT-PCR or VI. Virus subtype was not significant for IAV detection by either rRT-PCR (P = 0.457) or VI (P = 0.101). For rRT-PCR the cycle threshold (Ct) values increased consistently with dilution but varied widely. Therefore, it was not possible to predict VI success on the basis of Ct values. The success of VI was inversely related to the dilution of the sample; the assay was generally unsuccessful at lower virus concentrations. Successful swine health monitoring and disease surveillance require assays with consistent performance, but significant differences in reproducibility were observed among the assays evaluated

Considerations for improved surveillance of Influenzavirus A in swine populations

This thesis consists of 4 chapters. Chapter 1 is a general introduction and road map for contemporary swine surveillance programs, "Protecting our future - A road map for practical, real-time, on-farm infectious disease surveillance". Chapter 2, "Ring test evaluation of the detection of influenza A virus in swine oral fluids by real-time, reverse transcription polymerase chain reaction (rRT-PCR) and virus isolation" has been submitted for publication in the Journal of Veterinary Diagnostic Investigation. Chapter 3, "Probability of the detection of influenza A virus subtypes H1N1 and H3N2 in individual nasal swab and pen-based oral fluid specimens from pigs over time" has been submitted for publication to Veterinary Microbiology. Chapter 4, "Evaluation of screening assays for the detection of Influenza A virus serum antibodies in swine" has been submitted for publication to the Journal of Veterinary Diagnostic Investigation. References, tables, and figures for each manuscript follow each discussion section respectively. The last chapter contains general conclusions of this dissertation.</p

Shedding and transmission of a live attenuated influenza A virus vaccine in pre-weaned pigs under field conditions.

Influenza A virus (IAV) is one of the most important respiratory viruses affecting pig health and vaccination is the most common strategy to control influenza infections. In this field study we assessed the onset and duration of shedding of a live attenuated influenza virus (LAIV) vaccine, its ability to transmit to non-vaccinated pigs and whether the LAIV could be aerosolized and detected in the environment. Thirty-three litters (n = 33) of a farm using the LAIV vaccine were selected for the study, a subset of them (n = 12) were left unvaccinated and a subset of piglets (n = 3) in vaccinated litters were also left unvaccinated to serve as sentinels. Selected piglets from the litters were sampled multiple days post vaccination (DPV) by collecting nasal swabs and blood, and were tested using a LAIV vaccine specific RT-PCR assay and hemagglutination inhibition assay against the LAIV strains respectively. Environmental specimens consisting of air and surface wipes were also collected. One hundred percent (21/21) of the vaccinated litters tested LAIV positive 1 DPV and until 6 DPV. In contrast, only five (5/33) of the thirty-three non-vaccinated pigs tested positive during the course of the study. Viable LAIV was confirmed in vaccinated pigs by cell culture and whole genome sequencing. In addition, low levels of LAIV RNA (RT-PCR Ct values ranging between 33 and 38) were detected in all air specimens collected on the day of vaccination and until 6 DPV (3/10). Pigs had maternally derived antibodies reactive against the LAIV strains which may have influenced the degree of shedding observed. Under the conditions of this study, shedding of the LAIV from vaccinated pigs was limited in time, resulted in minimal transmission to non-vaccinated pigs and was detected in low levels in aerosols collected in the vaccinated rooms likely influenced by the presence of maternally derived antibodies against the LAIV strains

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.This article is published as González, Wendy, Luis G. Giménez-Lirola, Ashley Holmes, Sergio Lizano, Christa Goodell, Korakrit Poonsuk, Panchan Sitthicharoenchai, Yaxuan Sun, and Jeffrey Zimmerman. "Detection of Actinobacillus pleuropneumoniae ApxIV toxin antibody in serum and oral fluid specimens from pigs inoculated under experimental conditions." Journal of Veterinary Research 61, no. 2 (2017): 163. DOI: 10.1515/jvetres-2017-0021. Copyright 2017 W. González et al. Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0). Posted with permission

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

Characteristics of cotton fields sampled for western tarnished plant bug, <i>Lygus hesperus.</i>

<p>Variables shown are average field area (ha), average closest distance between pairs of sampled cotton fields (km), date of initiation of first and second sampling periods, average date of initiation of flowering, percentage of sampled fields planted to Pima cotton, average number of insecticide sprays and average <i>Lygus</i> density (calculated per 100 sweeps to facilitate comparison with thresholds) for combined sampling periods in each year.</p>*<p>Number of fields sampled: 41 in 2007; 39 in 2008; 56 in 2009.</p>†<p>Standard error in parentheses.</p>‡<p>Date is for onset of sampling period; number of weeks sampled per period is in parentheses.</p>¶<p>Range associated with average flowering date was 22 Jun–7 Jul in 2007, 7 Jul–12 Jul in 2008, and 17 Jun–2 Jul in 2009.</p>§<p>Suggested thresholds for <i>Lygus</i> spraying depend on cotton phenology <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039862#pone.0039862-UC1" target="_blank">[48]</a>. Number of individuals per 100 sweeps that would trigger spraying is: >4–8 adults (early squaring); >14–20 individuals with at least two nymphs (bloom); and >20 individuals with nymphs present (boll filling).</p