A comparison of three sampling approaches for detecting PRRSV in suckling piglets.

Determining whether porcine reproductive and respiratory syndrome virus (PRRSV) is circulating within a breeding herd is a longstanding surveillance challenge. Most commonly, piglets in farrowing rooms are sampled to infer the PRRSV status of the sow herd, with sample size based on the expectation of hypergeometric distribution and piglet selection based on simple random sampling (SRS), i.e., randomly selecting individuals from a population in a manner that all individuals have equal chance of being selected. Conceptually straightforward, the assumptions upon which it is based (homogeneous population and independence of individuals) rarely hold in modern swine facilities. Alternative approaches for sample selection include two-stage stratified sampling (2SS), i.e., randomly selecting litters (first stratum) and randomly selecting piglets (second stratum) within selected litters, and risk-based sampling (RBS), i.e., selecting litters with a higher risk of having viremic piglets, and randomly selecting pigs within those litters. The objectives of this study were to 1) characterize the pattern of distribution of PRRSV-viremic piglets in farrowing rooms and 2) compare the efficiency of SRS, 2SS, and RBS for the detection of PRRSV-viremic piglets. In 12 sow farms, serum samples were collected from all 4510 piglets in 422 litters housed in 23 farrowing rooms and tested for PRRSV RNA. At the population level, the distribution of PRRSV-viremic pigs was analyzed for population homogeneity and spatial clustering. At the litter level, litter size and sow parity were evaluated as risk factors. A non-homogeneous distribution of PRRSV-viremic piglets was observed in nearly all farrowing rooms (15/16), and spatial clustering detected on 11 occasions (11/16). Simulated sampling based on farrowing room data determined that 2SS required 1-to-25 fewer samples than SRS to detect ≥ 1 viremic piglet in 13 of 16 rooms and the same number of samples in 3 rooms. RBS required 1-to-7 fewer samples than 2SS to detect ≥ 1 viremic piglet in 7 of 16 rooms, the same number of samples in 6 rooms, and 1 more sample in 3 rooms. Notably, SRS was less efficient than either 2SS or RBS in detecting PRRSV-viremic piglets in farrowing rooms, regardless of the confidence level. It may be concluded that the core assumptions upon which most current surveillance methods are based do not hold in modern farrowing room facilities. Simulation-based sample size tables for SRS and 2SS are provided.

Finding PRRSV in sow herds: Family oral fluids vs. serum samples from due-to-wean pigs.

The aim of this study was to compare the detection of porcine reproductive and respiratory syndrome virus (PRRSV) in due-to-wean litters in commercial swine breeding herds using family oral fluids (FOF) vs. individual piglet serum samples. FOF and piglet serum samples were collected in 199 due-to-wean litters on six farms containing 2177 piglets. All samples were individually tested for PRRSV RNA by RT-rtPCR. A litter was considered PRRSV-positive when PRRSV RNA was detected in ≥ 1 piglet serum sample or the FOF sample. Mixed effect logistic regression with farm as a random effect was used 1) to evaluate the probability of obtaining a PRRSV RNA positive FOF as a function of the proportion of viremic piglets in a litter and 2) the effect of litter size and parity on the probability that a litter would test PRRSV RNA positive in FOF. A Bayesian prevalence estimation under misclassification (BayesPEM) analysis was used to calculate the PRRSV prevalence and 95 % credible interval given the condition that all samples (FOF and serum) tested negative. In total, 34 of 199 litters (17.1 %) contained ≥ 1 viremic piglet(s), and 28 of 199 litters (14.1 %) were FOF positive. When all piglet serum samples within a litter tested negative, 1 of 165 FOF (0.6 %) tested PRRSV RNA positive. The probability of a PCR-positive FOF sample from litters with 10 %, 20 %, 30 %, 40 %, and 50 % within-litter PRRSV prevalence was 3.5 %, 35.1 %, 88.8 %, 99.2 %, and >99.9 %, respectively. The odds of a PCR-positive FOF in a first parity litter were 3.36 times (95 % CI: 2.10-5.38) that of a parity ≥ 2 litter. The odds of a positive FOF result in a litter with ≤ 11 piglets were 9.90 times (95 % CI: 4.62-21.22) that of a litter with > 11 piglets. FOF was shown to be an efficacious sample type for PRRSV detection in farrowing rooms. A risk-based approach for litter selection combined with FOF collection can be used to improve on-farm PRRSV detection with a limited sample size, compared to sampling multiple individual pigs. Finally, the BayesPEM analysis showed that PRRSV may still be present in breeding herds when all samples (serum and FOF) test PRRSV RNA negative, i.e., negative surveillance results should be interpreted with caution.

Collecting oral fluid samples from due-to-wean litters.

Oral fluids are a common diagnostic sample in group-housed nursery, grow-finish, and adult swine. Although oral fluids from due-to-wean litters could be a valuable tool in monitoring pathogens and predicting the health status of pig populations post-weaning, it is generally not done because of inconsistent success in sample collection. The objective of this study was to determine the optimum procedure for collecting oral fluid samples from due-to-wean litters. Successful collection of oral fluids from due-to-wean litters using "Litter Oral Fluid" (LOF) or "Family Oral Fluid" (FOF) sampling techniques were compared in 4 phases involving 920 attempts to collect oral fluids. Phase 1 testing showed that prior exposure to a rope improved the success rates of both LOF (33.4%) and FOF (16.4%) techniques. Phase 2 determined that longer access to the rope (4 h vs 30 min) did not improve the success rate for either LOF or FOF. Phase 3 evaluated the effect of attractants and found that one (Baby Pig Restart®) improved the success rate when used with the FOF technique. Phase 4 compared the success rates of "optimized LOF" (litters previously trained) vs "optimized FOF" (litter previously trained and rope treated with Baby Pig Restart®) vs standard FOF. No difference was found between the FOF-based techniques, but both were superior to the "optimized LOF" technique. Thus, FOF-based procedures provided a significantly higher probability of collecting oral fluids from due-to-wean litters (mean success rate 84.9%, range 70% to 92%) when compared to LOF-based methods (mean success rate 24.1%, range 16.5% to 32.2%).

Assessment of abattoir based monitoring of PRRSV using oral fluids.

Various porcine reproductive and respiratory syndrome virus (PRRSV) regional elimination projects have been implemented in the U.S., but none have yet succeeded. In part, this reflects the need for efficient methods to monitor over time the progress of PRRSV status of participating herds. This study assessed the feasibility of monitoring PRRSV using oral fluids collected at the abattoir. A total of 36 pig lots were included in the study. On-farm oral fluid (n = 10) and serum (n = 10) collected within two days of shipment to the abattoir were used to establish the reference PRRSV status of the population. Oral fluids (n = 3 per lot) were successfully collected from 32 lots (89%) at the lairage. Three veterinary diagnostic laboratories (VDLs) tested the sera (VDL1 and VDL3: n = 316, VDL2: n = 315) and oral fluids (VDL1 and VDL3: n = 319, VDL2: n = 320) for PRRSV antibodies (ELISA) and RNA (rRT-PCR). Environmental samples (n = 64, 32 before and 32 after pigs were placed in lairage) were tested for PRRSV RNA at one VDL. All oral fluids (farm and abattoir) tested positive for PRRSV antibody at all VDLs. PRRSV positivity frequency on serum ranged from 92.4% to 94.6% among VDLs, with an overall agreement of 97.6%. RNA was detected on 1.3% to 1.9%, 8.1% to 17.7%, and 8.3% to 17.7% of sera, on-farm and abattoir oral fluids, respectively. Between-VDLs rRT-PCR agreement on sera and oral fluids (farm and abattoir) ranged from 97.8% to 99.0%, and 79.0% to 81.2%, respectively. Between-locations agreement of oral fluids varied from 31.3% to 50% depending on the VDL. This study reported the application of swine oral fluids collected at the abattoir for monitoring PRRSV, and describes the between-VDL agreement for PRRS testing of serum and oral fluid field samples.

A Four-Biomarker Blood Signature Discriminates Systemic Inflammation Due to Viral Infection Versus Other Etiologies.

The innate immune system of humans and other mammals responds to pathogen-associated molecular patterns (PAMPs) that are conserved across broad classes of infectious agents such as bacteria and viruses. We hypothesized that a blood-based transcriptional signature could be discovered indicating a host systemic response to viral infection. Previous work identified host transcriptional signatures to individual viruses including influenza, respiratory syncytial virus and dengue, but the generality of these signatures across all viral infection types has not been established. Based on 44 publicly available datasets and two clinical studies of our own design, we discovered and validated a four-gene expression signature in whole blood, indicative of a general host systemic response to many types of viral infection. The signature's genes are: Interferon Stimulated Gene 15 (ISG15), Interleukin 16 (IL16), 2',5'-Oligoadenylate Synthetase Like (OASL), and Adhesion G Protein Coupled Receptor E5 (ADGRE5). In each of 13 validation datasets encompassing human, macaque, chimpanzee, pig, mouse, rat and all seven Baltimore virus classification groups, the signature provides statistically significant (p < 0.05) discrimination between viral and non-viral conditions. The signature may have clinical utility for differentiating host systemic inflammation (SI) due to viral versus bacterial or non-infectious causes.

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.

Detection of influenza A virus nucleoprotein antibodies in oral fluid specimens from pigs infected under experimental conditions using a blocking ELISA.

In commercial swine populations, influenza is an important component of the porcine respiratory disease complex (PRDC) and a pathogen with major economic impact. Previously, a commercial blocking ELISA (FlockChek(™) Avian Influenza Virus MultiS-Screen(®) Antibody Test Kit, IDEXX Laboratories, Inc., Westbrook, ME, USA) designed to detect influenza A nucleoprotein (NP) antibodies in avian serum was shown to accurately detect NP antibodies in swine serum. The purpose of this study was to determine whether this assay could detect NP antibodies in swine oral fluid samples. Initially, the procedure for performing the NP-blocking ELISA on oral fluid was modified from the serum testing protocol by changing sample dilution, sample volume, incubation time and incubation temperature. The detection of NP antibody was then evaluated using pen-based oral fluid samples (n = 182) from pigs inoculated with either influenza A virus subtype H1N1 or H3N2 under experimental conditions and followed for 42 days post inoculation (DPI). NP antibodies in oral fluid were detected from DPI 7 to 42 in all inoculated groups, that is, the mean sample-to-negative (S/N) ratio of influenza-inoculated pigs was significantly different (P < 0.0001) from uninoculated controls (unvaccinated or vaccinated-uninoculated groups) through this period. Oral fluid versus serum S/N ratios from the same pen showed a correlation of 0.796 (Pearson's correlation coefficient, P < 0.0001). The results showed that oral fluid samples from influenza virus-infected pigs contained detectable levels of NP antibodies for ≥42 DPI. Future research will be required to determine whether this approach could be used to monitor the circulation of influenza virus in commercial pig populations.

Surveillance of Bungowannah pestivirus in the upper Midwestern USA.

Pestiviruses, a genetically and antigenically highly diverse group, include one of the most historically significant swine pathogens, that is, classical swine fever virus. In Australia, investigations into swine outbreaks characterized by neonatal mortality, stillbirths and mummified foetuses resulted in the discovery of a new pestivirus, Bungowannah virus. This finding raised the possibility that Bungowannah virus, or a variant thereof, was circulating in swine herds elsewhere in the World. If so, it raised the possibility of a pestivirus emerging as a new swine disease with unknown consequences for animal health and food safety. Thus, we developed three specific qRT-PCR assays to evaluate tissue samples from undiagnosed cases of abortion or respiratory disease for evidence of Bungowannah virus. Examination of 64 samples collected between the Fall of 2007 and Spring of 2010 tested negative for all three genes examined. We conclude that Bungowannah-like pestivirus is unlikely to be present in swine in the upper Midwestern USA.

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.

Prolonged detection of PCV2 and anti-PCV2 antibody in oral fluids following experimental inoculation.

The onset, level and duration of PCV2 and anti-PCV2 antibody in oral fluid were evaluated using samples collected from experimentally inoculated pigs for 98 days post-inoculation (DPI). Pigs (n = 24) were obtained at 3 weeks of age and randomly allocated to 4 treatment pens of 6 pigs each: (i) negative control group; (ii) inoculated with PCV2a (strain ISU 40895) on DPI 0; (iii) inoculated with PCV2a (strain ISU-40895) on DPI 0 and re-challenged at DPIs 35 and 70; (iv) inoculated with PCV2a (ISU-40895), PCV2b (PVG4072) and PCV2a (ISU-4838) on DPIs 0, 35 and 70, respectively. Serum was collected from each animal, and one oral fluid sample was collected from each pen (group) every other day from DPI 2 through DPI 14 and weekly through 98 DPI. Oral fluid samples were assayed for the presence of PCV2 by quantitative polymerase chain reaction (qPCR) and anti-PCV2 IgG, IgA and IgM antibody isotypes by enzyme-linked immunosorbant assay (ELISA). Serum was assayed for anti-PCV2 IgG by ELISA. Anti-PCV2 antibodies (IgG, IgM and IgA) were detected in oral fluid from experimentally inoculated pigs from DPI 14 with IgG and IgA clearly present at 98 DPI. PCV2 was detected in oral fluid samples from all pens of inoculated pigs at 2 DPI. Thereafter, PCV2 was detected in oral fluid throughout 98 DPI. Overall, the data indicated that PCV2 infection in swine is prolonged, persists in the face of an active antibody response and can be efficiently monitored using oral fluid specimens.

Using the systematic review methodology to evaluate factors that influence the persistence of influenza virus in environmental matrices.

Understanding factors that influence persistence of influenza virus in an environment without host animals is critical to appropriate decision-making for issues such as quarantine downtimes, setback distances, and eradication programs in livestock production systems. This systematic review identifies literature describing persistence of influenza virus in environmental samples, i.e., air, water, soil, feces, and fomites. An electronic search of PubMed, CAB, AGRICOLA, Biosis, and Compendex was performed, and citation relevance was determined according to the aim of the review. Quality assessment of relevant studies was performed using criteria from experts in virology, disease ecology, and environmental science. A total of 9,760 abstracts were evaluated, and 40 appeared to report the persistence of influenza virus in environmental samples. Evaluation of full texts revealed that 19 of the 40 studies were suitable for review, as they described virus concentration measured at multiple sampling times, with viruses detectable at least twice. Seven studies reported persistence in air (six published before 1970), seven in water (five published after 1990), two in feces, and three on surfaces. All three fomite and five air studies addressed human influenza virus, and all water and feces studies pertained to avian influenza virus. Outcome measurements were transformed to half-lives, and resultant multivariate mixed linear regression models identified influenza virus surviving longer in water than in air. Temperature was a significant predictor of persistence over all matrices. Salinity and pH were significant predictors of persistence in water conditions. An assessment of the methodological quality review of the included studies revealed significant gaps in reporting critical aspects of study design.

Evaluation of the risk of PRRSV transmission via ingestion of muscle from persistently infected pigs.

The objectives of this experiment were to determine how long porcine reproductive and respiratory syndrome virus (PRRSV) could be detected in muscle tissues of experimentally infected pigs and to evaluate the transmissibility of PRRSV to pigs via ingestion of quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR)-positive muscle tissues. Serum, lymphoid tissues, and muscle (M. longissimus dorsi) samples were collected from 135 pigs (89 PRRSV-inoculated pigs and 46 negative control). Between 28 and 202 days post-inoculation, 13 of 89 (14.6%) muscle samples were positive by qRT-PCR. Among these 13, PRRSV was isolated from four of the 13 corresponding serum samples and three of 13 lymphoid tissue samples. In addition, infectious virus was detected in lymphoid tissue homogenates of six of 13 pigs by intramuscular bioassay. Swine transmissibility studies were performed by feeding thirteen 3-week-old PRRSV-naive pigs (recipient pigs) qRT-PCR-positive muscle and then monitoring recipients for evidence of PRRSV viremia by qRT-PCR. No transmission of PRRSV to recipient pigs via consumption of muscle samples was observed. These data suggested that qRT-PCR detected non-infectious PRRSV in pig meat and/or PRRSV is not highly transmissible to susceptible pigs via consumption of PRRSV-contaminated meat.

A method to provide improved dose-response estimates for airborne pathogens in animals: an example using porcine reproductive and respiratory syndrome virus.

This paper describes a method to provide improved probability estimates that exposure to a specific dose of an airborne infectious pathogen will result in animal infection. Individual animals were exposed to a specific dose of airborne pathogen. Following exposure, animals were individually housed and monitored for evidence of infection. The detection of specific antibodies and/or the pathogen in diagnostic specimens was evidence that the exposure dose resulted in infection. If replicated over a range of doses, the results can be used to derive a dose-response curve for a variety of animal species and infectious pathogens. This information is useful in estimating the likelihood of infection associated with exposure to airborne infectious microorganisms. Applications include predicting the risk of transmission associated with exposure to airborne pathogens, modeling the transmission of airborne pathogens, and determining requirements for effective exposure doses for vaccines delivered in aerosols.

Immune response against porcine reproductive and respiratory syndrome virus during acute and chronic infection.

A significant obstacle to the prevention and control of porcine reproductive and respiratory syndrome virus (PRRSV) is the inability of current diagnostic tests to provide information concerning the stage of PRRSV infection. To explore possible prognostic combinations of cell-mediated and humoral immune responses, 3-week-old pigs (n=10) were intramuscularly (IM) inoculated with PRRSV isolate VR-2332 and followed for 193 days post-inoculation (DPI). Negative control pigs (n=10) were IM inoculated with minimum essential medium (MEM). At approximately 2-week intervals, blood samples were collected from all animals and tested for the number of interferon (IFN)-gamma-secreting peripheral blood mononuclear cells (enzyme-linked immunosorbent spot, Elispot), PRRSV viremia (quantitative reverse-transcriptase polymerase chain reaction, qRT-PCR), and serum antibodies using PRRSV protein ELISAs (N, GP5 3', GP5 5', M 5', M 3', GP5-M, and nsp2p) and a commercial PRRSV ELISA (IDEXX Laboratories Inc.). All pigs were viremic by 7 days post-inoculation, with 50% of the pigs resolving viremia by 56 DPI. A PRRSV-specific IFN-gamma response was detected at DPI 28, reached a plateau at 42 DPI, declined slightly, and remained relatively stable from 56 to 193 DPI. On the basis of ROC area under the curve (AUC) analysis, the ELISAs that most reliably differentiated PRRSV-inoculated pigs from negative control pigs were the commercial ELISA (AUC=0.97), the N ELISA (AUC=0.96), and the M 3' ELISA (AUC=0.93). Multivariate analyses were performed to evaluate the relationship between the immune response and the duration and level of viremia. With all antibody assays and Elispot included in the models, the analysis determined that the serum-virus neutralizing antibody response was the best predictor of both level and duration of viremia. It was concluded that humoral antibody responses, particularly the commercial ELISA, N ELISA, and M 3' ELISA were good predictors of prior exposure to PRRSV, but provided little information regarding the ontogeny of the protective immune response. Likewise, cell-mediated immunity based on the number of IFN-gamma-secreting lymphocytes was a poor prognosticator of PRRSV infection status.

Impaired cerebral autoregulation and 6-month outcome in children with severe traumatic brain injury: preliminary findings.

The objective of this study was to describe the incidence of impaired cerebral autoregulation and to describe the relationship between impaired cerebral autoregulation and outcome after severe pediatric traumatic brain injury (TBI). We prospectively examined cerebral autoregulation in 28 children

Optimization of a sampling system for recovery and detection of airborne porcine reproductive and respiratory syndrome virus and swine influenza virus.

The objective of this research was to optimize sampling parameters for increased recovery and detection of airborne porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza virus (SIV). Collection media containing antifoams, activated carbons, protectants, and ethylene glycol were evaluated for direct effects on factors impacting the detection of PRRSV and SIV, including virus infectivity, viability of continuous cell lines used for the isolation of these viruses, and performance of reverse transcriptase PCR assays. The results showed that specific compounds influenced the likelihood of detecting PRRSV and SIV in collection medium. A subsequent study evaluated the effects of collection medium, impinger model, and sampling time on the recovery of aerosolized PRRSV using a method for making direct comparisons of up to six treatments simultaneously. The results demonstrated that various components in air-sampling systems, including collection medium, impinger model, and sampling time, independently influenced the recovery and detection of PRRSV and/or SIV. Interestingly, it was demonstrated that a 20% solution of ethylene glycol collected the greatest quantity of aerosolized PRRSV, which suggests the possibility of sampling at temperatures below freezing. Based on the results of these experiments, it is recommended that air-sampling systems be optimized for the target pathogen(s) and that recovery/detection results should be interpreted in the context of the actual performance of the system.

Probability of porcine reproductive and respiratory syndrome (PRRS) virus infection as a function of exposure route and dose.

At the most elemental level, the design of effective strategies to control and/or eliminate porcine reproductive and respiratory syndrome (PRRS) virus depend on an accurate and comprehensive understanding of virus transmission. As a general rule, transmission is highly dependent on the route of exposure and the dose of virus. The objective of this study was to derive PRRS virus isolate VR-2332 dose-response curves for oral and intranasal routes of exposure, i.e., determine the probability that a specific virus dose would result in infection. Individually housed pigs approximately 21 days of age were exposed to specific doses of PRRS virus isolate VR-2332 by either oral or intranasal routes. Positive controls were intramuscularly inoculated with 10(2.2) 50% tissue culture infective dose (TCID50) of PRRS virus and negative controls were orally administered 100ml of diluent with no virus. Pigs were monitored for evidence of infection for 21 days following exposure, i.e., serum samples were collected on days 0, 7, 14, 21, and tested for virus and PRRS virus-specific antibodies. Dose-response curves and 95% confidence intervals for oral and intranasal routes of exposure were derived using logistic models (logit and probit). The infectious dose50 (ID50) for oral exposure was estimated to be 10(5.3) TCID50 (95% CI, 10(4.6) and 10(5.9)); the ID50 for intranasal exposure was estimated to be 10(4.0) TCID50 (95% CI, 10(3.0) and 10(5.0)). Given these estimates, it is worth noting that intramuscular exposure of animals to 10(2.2) TCID50 (positive controls) resulted in infection in all animals. Thus pigs were the most susceptible to infection via parenteral exposure.

Effect of dietary Echinacea purpurea on viremia and performance in porcine reproductive and respiratory syndrome virus-infected nursery pigs.

The effect of dietary Echinacea purpurea on performance, viremia, and ontogeny of the humoral antibody response against porcine reproductive and respiratory syndrome virus (PRRSV) infection was evaluated in weaned pigs. In three replicates, 120 weaned pigs (25 +/- 1 d of age; 8.46 +/- 0.48 kg of BW) from a PRRSV-naive herd were allotted randomly to one of eight pens (diets) in two separate rooms (four pens/room), with each pen containing five pigs. Pigs began one of four dietary treatments (as-fed basis) 1 wk before inoculation with PRRSV: 1) basal diet composed of corn, soybean meal, whey, and essential vitamins and minerals; 2) basal diet plus carbadox (0.055 g/kg of diet; as-fed basis); 3) basal diet plus Echinacea 2% (2% of the total diet); 4) basal diet plus Echinacea 4% (4% of the total diet). The diets were formulated to be isocaloric and isolysinic. Echinacea purpurea was purchased in powder form and determined by chemical analysis to contain 1.35% cichoric acid (as-fed basis). Seven days after starting the diets, all pigs in one room were intranasally inoculated with PRRSV isolate ATCC VR-2332 at a concentration of 10(4) tissue culture infectious dose50/mL. To monitor the effects of Echinacea and PRRSV challenge, BW and blood samples were obtained from all pigs at 7-d intervals. Serum samples were analyzed for the presence of PRRSV and PRRSV-specific antibodies. All challenged pigs became infected with PRRSV, and all unchallenged pigs remained free of infection. No differences (P > 0.10) in ADG, ADFI, or gain:feed (G:F) were observed in PRRSV-challenged compared with unchallenged animals. For PRRSV-challenged animals receiving diets supplemented with Echinacea at 2 or 4%, no differences (P > 0.10) were observed in ADG, ADFI, or G:F ratio. Among PRRSV-challenged pigs, dietary Echinacea did not affect (P > 0.10) the rate or level of the ELISA-detectable antibody response from d 7 to 42 or the level and duration of PRRSV in serum. For PRRSV-unchallenged animals receiving diets supplemented with Echinacea at 2 or 4%, no differences (P > 0.10) were observed in ADG, ADFI, and G:F ratio. Under the conditions of this study, dietary Echinacea did not enhance growth, exhibit antiviral effects to PRRSV, or show any evidence of immune enhancing properties.

Evolution of porcine reproductive and respiratory syndrome virus during sequential passages in pigs.

Porcine reproductive and respiratory syndrome (PRRS) viruses are recognized as possessing a high degree of genetic and antigenic variability. Viral diversity has led to questions regarding the association of virus mutation and persistent infection in the host and has raised concerns vis-à-vis protective immunity, the ability of diagnostic assays to detect novel variants, and the possible emergence of virulent strains. The purpose of this study was to describe ongoing changes in PRRS virus during replication in pigs under experimental conditions. Animals were inoculated with a plaque-cloned virus derived from VR-2332, the North American PRRS virus prototype. Three independent lines of in vivo replication were maintained for 367 days by pig-to-pig passage of virus at 60-day intervals. A total of 315 plaque-cloned viruses were recovered from 21 pigs over the 367-day observation period and compared to the original plaque-cloned virus by virus neutralization assay, monoclonal antibody analysis, and sequencing of open reading frames (ORFs) 1b (replicase), 5 (major envelope protein), and 7 (nucleocapsid) of the genome. Variants were detected by day 7 postinoculation, and multiple variants were present concurrently in every pig sampled over the observation period. Sequence analysis showed ORFs 1b and 7 to be highly conserved. In contrast, sequencing of ORF 5 disclosed 48 nucleotide variants which corresponded to 22 amino acid variants. Although no epitopic changes were detected under the conditions of this experiment, PRRS virus was shown to evolve continuously in infected pigs, with different genes of the viral genome undergoing various degrees of change.