Cough associated with the detection of Mycoplasma hyopneumoniae DNA in clinical and environmental specimens under controlled conditions.

BACKGROUND: The association of cough with Mycoplasma hyopneumoniae (MHP) DNA detection in specimens was evaluated under conditions in which the MHP status of inoculated and contact-infected pen mates was closely monitored for 59 days post-inoculation (DPI). METHODS: Seven-week-old pigs (n = 39) were allocated to five rooms (with one pen). Rooms contained 9 pigs each, with 1, 3, 6, or 9 MHP-inoculated pigs, respectively, except Room 5 (three sham-inoculated pigs). Cough data (2 × week) and specimens, tracheal swabs (2 × week), oral fluids (daily), drinker wipes (~ 1 × week), and air samples (3 × week) were collected. At 59 DPI, pigs were euthanized, and lung and trachea were evaluated for gross and microscopic lesions. Predictive cough value to MHP DNA detection in drinker and oral fluid samples were estimated using mixed logistic regression. RESULTS: Following inoculation, MHP DNA was first detected in tracheal swabs from inoculated pigs (DPI 3), then oral fluids (DPI 8), air samples (DPI 10), and drinker wipes (21 DPI). MHP DNA was detected in oral fluids in 17 of 59 (Room 1) to 43 of 59 (Room 3) samples, drinker wipes in 4 of 8 (Rooms 2 and 3) to 5 of 8 (Rooms 1 and 4) samples, and air samples in 5 of 26 (Room 2) or 3 of 26 (Room 4) samples. Logistic regression showed that the frequency of coughing pigs in a pen was associated with the probability of MHP DNA detection in oral fluids (P < 0.01) and nearly associated with drinker wipes (P = 0.08). Pathology data revealed an association between the period when infection was first detected and the severity of gross lung lesions. CONCLUSIONS: Dry, non-productive coughs suggest the presence of MHP, but laboratory testing and MHP DNA detection is required for confirmation. Based on the data from this study, oral fluids and drinker wipes may provide a convenient alternative for MHP DNA detection at the pen level when cough is present. This information may help practitioners in specimen selection for MHP surveillance.

Pooled-sample testing for detection of Mycoplasma hyopneumoniae during late experimental infection as a diagnostic tool for a herd eradication program.

Early and accurate detection of Mycoplasma hyopneumoniae infection in live pigs is a critical component to measure the success of disease eradication strategies. However, the imperfect sensitivity of in vivo diagnostic tools, change in sensitivity over the course of infection, and expected low prevalence level at the end of an eradication program create a challenging diagnostic scenario. Here, the individual and pool sensitivities for detection of M. hyopneumoniae during the chronic phase of infection was determined using deep tracheal catheter samples, the in vivo sample type with the highest reported diagnostic sensitivity. Fifty samples from known infected pigs collected at 113 days post-M. hyopneumoniae intra-tracheal inoculation, were diluted in known negative samples to form pools of 1:3 and 1:5. Samples were tested for M. hyopneumoniae by a species-specific PCR. Ninety-eight percent (49/50) of individual samples, 84 % (42/50) of pools of 1:3, and 82 % (41/50) of 1:5 were detected positive for M. hyopneumoniae. To apply the sensitivity estimates for detection of M. hyopneumoniae in a low prevalence scenario, sample sizes with associated sample collection costs were calculated for individual and pooled testing using algorithms within the program EpiTools One-Stage Freedom Analyses. Assumptions included a ≥95 % population sensitivity, infinite population size, prevalence levels of ≥0.5 %, ≥1 %, ≥2 %, ≥3 %, ≥4 %, or ≥5 %, 100 % specificity, along with the mean and lower confidence limit of the individual or pool sensitivity for each pool size, when appropriate. For instance, following completion of a herd eradication program, if a low risk approach is targeted, sample size estimates for ≥2 % prevalence using the lower limit of the diagnostic or pool sensitivity 95 %CI may be followed. If samples were to be tested individually, 167 individuals would be sampled at a cost of 6,012 USD. If pooled by 3, 213 would be sampled (testing cost 3,266 USD), and for pools of 5, 220 individuals would be sampled (testing cost 2,464 USD). Population sensitivity was also calculated for a range of testing scenarios. Our study indicated that pooling samples by 3 or 5 was a cost-effective method for M. hyopneumoniae detection in low prevalence scenarios. Cost-effective detection was evidenced despite the increased sample collection costs associated with large sample sizes in order to offset decreased testing sensitivity attributable to pooling. The post-eradication sample collection scheme, combined with pooling, suggested lower cost options than individual sampling for testing to be applied at the end of an eradication program, without significantly compromising the likelihood of detection.

Mycoplasma hyopneumoniae Surveillance in Pig Populations: Establishing Sampling Guidelines for Detection in Growing Pigs.

Antemortem detection of Mycoplasma hyopneumoniae infection in swine production systems has relied on antibody testing, but the availability of tests based on DNA detection and novel diagnostic specimens, e.g., tracheal swabs and oral fluids, has the potential to improve M. hyopneumoniae surveillance. A field study was performed over a 14-week period during which 10 pigs in one pen at the center of a room with 1,250 6-week-old pigs housed in 46 pens were intratracheally inoculated with M. hyopneumoniae Thereafter, one tracheal sample, four serum samples, and one oral fluid sample were collected from every pen at 2-week intervals. Tracheal and oral fluid samples were tested for M. hyopneumoniae DNA and serum samples for M. hyopneumoniae antibody. Test results were modeled using a hierarchical Bayesian model, based on a latent spatial piecewise exponential survival model, to estimate the probability of detection by within-pen prevalence, number of positive pens in the barn, sample allocation, sample size, and sample type over time. Analysis showed that tracheal samples provided the earliest detection, especially at large sample sizes. While serum samples are more commonly collected and are less expensive to test, high probability of detection estimates were only obtained 30 days postexposure at large sample sizes. In all scenarios, probability of detection estimates for oral fluids within 30 days were significantly lower than those for tracheal and serum samples. Ultimately, the choice of specimen type, sample number, and assay will depend on testing objectives and economics, but the estimates provided here will assist in the design of M. hyopneumoniae surveillance and monitoring programs for different situations.

Survival of swine pathogens in compost formed from preprocessed carcasses.

An introduction of a Foreign Animal Disease (FAD) like African Swine Fever Virus (ASF) would be financially devastating. For example, ASF, a highly contagious pathogen with high mortality rates, is a World Health Organization reportable disease that has recently been spreading across Asia and Europe. Control of ASF would likely require mass euthanasia of infected and exposed animals similar to the United Kingdom's elimination of Foot and Mouth Disease (FMD). Subsequent disposal of infectious carcasses must adequately eliminate the virus and prevent further transmission of the disease. Although composting swine carcasses is widely used throughout the industry, limited data is available describing pathogen survival or elimination during this process. While current methods have evaluated the composting of swine carcasses under temperature-controlled settings, they have not considered the effects of adverse weather conditions (e.g., cold winter conditions) where composting is routinely performed. This study utilized preprocessing (grinding) of swine carcasses prior to composting, which decreases the amount of required carbon material and land space. The ability of composting to reduce the level of viral nucleic acid during cold weather conditions and the risk of environmental contamination that may occur during preprocessing was evaluated. In this study, pigs challenged with Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Porcine Epidemic Diarrhea Virus (PEDV), common domestic diseases, before euthanasia provided infectious carcasses containing pathogen surrogates. Composting of preprocessed carcasses achieved adequate temperatures necessary to eliminate FAD and common swine pathogens during cold weather conditions (monitored by compost temperature over time, virus diagnostic testing, and swine bioassay for PRRSV and PEDV). Under the conditions of this study, composting preprocessed carcasses presents minimal risk to air and groundwater contamination. In conclusion, composting preprocessed euthanized swine under adverse weather conditions is a safe and feasible option for mass disposal of infected carcasses.

Effect of litter aggregation and pooling on detection of porcine reproductive and respiratory virus in piglet processing fluids.

A sampling technique has been validated to monitor porcine reproductive and respiratory syndrome virus 2 (PRRSV-2) using the serosanguinous exudate known as processing fluids (PFs) that accumulate from tissues obtained during tail docking and castration. PFs are an aggregate sample of large numbers of piglets and litters. However, little is known about the effect of litter aggregation on the ability of PCR to correctly classify an aggregated PF sample as positive. We evaluated both the effect of litter aggregation and of PF pooling on PCR detection. We estimated that aggregation of at least 50 litters was possible when a pig with a Ct value of ~22 was present in the sample, and aggregation of up to 40 litters was possible when there was a sample with a Ct value of ~33. Pooling did not affect PCR detection when initial Ct values of 20 and 25 were assessed. However, in litters with initial Ct values of ≥30, the amount of pooling should be reduced. Our results provide producers and practitioners with a general framework to interpret more accurately the results of their PRRSV-2 surveillance programs using PF.

Modeling the spatio-temporal dynamics of porcine reproductive & respiratory syndrome cases at farm level using geographical distance and pig trade network matrices.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating infectious diseases for the swine industry. A better understanding of the disease dynamics and the transmission pathways under diverse epidemiological scenarios is a key for the successful PRRS control and elimination in endemic settings. In this paper we used a two step parameter-driven (PD) Bayesian approach to model the spatio-temporal dynamics of PRRS and predict the PRRS status on farm in subsequent time periods in an endemic setting in the US. For such purpose we used information from a production system with 124 pig sites that reported 237 PRRS cases from 2012 to 2015 and from which the pig trade network and geographical location of farms (i.e., distance was used as a proxy of airborne transmission) was available. We estimated five PD models with different weights namely: (i) geographical distance weight which contains the inverse distance between each pair of farms in kilometers, (ii) pig trade weight (PT ji ) which contains the absolute number of pig movements between each pair of farms, (iii) the product between the distance weight and the standardized relative pig trade weight, (iv) the product between the standardized distance weight and the standardized relative pig trade weight, and (v) the product of the distance weight and the pig trade weight. RESULTS: The model that included the pig trade weight matrix provided the best fit to model the dynamics of PRRS cases on a 6-month basis from 2012 to 2015 and was able to predict PRRS outbreaks in the subsequent time period with an area under the ROC curve (AUC) of 0.88 and the accuracy of 85% (105/124). CONCLUSION: The result of this study reinforces the importance of pig trade in PRRS transmission in the US. Methods and results of this study may be easily adapted to any production system to characterize the PRRS dynamics under diverse epidemic settings to more timely support decision-making.

Unraveling the contact patterns and network structure of pig shipments in the United States and its association with porcine reproductive and respiratory syndrome virus (PRRSV) outbreaks.

The analysis of the pork value chain is becoming key to understanding the risk of infectious disease dissemination in the swine industry. In this study, we used social network analysis to characterize the swine shipment network structure and properties in a typical multisite swine production system in the US. We also aimed to evaluate the association between network properties and porcine respiratory and reproductive syndrome virus (PRRSV) transmission between production sites. We analyzed the 109,868 swine shipments transporting over 93 million swine between more than 500 production sites from 2012 to 2014. A total of 248 PRRSV positive occurrences were reported from 79 production sites during those 3 years. The temporal dynamics of swine shipments was evaluated by computing network properties in one-month and three-month networks. The association of PRRS occurrence in sow farms with centrality properties from one-month and three-month networks was assessed by using the multilevel logistic regression. All monthly networks showed a scale-free network topology with positive degree assortativity. The regression model revealed that out-degree centrality had a negative association with PRRS occurrence in sow farms in both one-month and three-month networks [OR=0.79 (95% CI, 0.63-0.99) in one-month network and 0.56 (95% CI, 0.36, 0.88) in three-month network] and in-closeness centrality model was positively associated with PRRS occurrence in sow farms in the three-month network [OR=2.45 (95% CI, 1.14-5.26)]. We also describe how the occurrence of porcine epidemic diarrheac (PED) outbreaks severely affected the network structure as well as the PRRS occurrence reports and its association with centrality measures in sow farms. The structure of the swine shipment network and the connectivity between production sites influenced on the PRRSV transmission. The use of network topology and characteristics combining with spatial analysis based on fine scale geographical location of production sites will be useful to inform the design of more cost-efficient, risk-based surveillance and control measures for PRRSV as well as other diseases in the US swine industry.

Stochastic model of porcine reproductive and respiratory syndrome virus control strategies on a swine farm in the United States.

OBJECTIVE: To use mathematical modeling to assess the effectiveness of control strategies for porcine reproductive and respiratory syndrome (PRRS) virus on a swine farm. SAMPLE: A hypothetical small, medium, or large farrow-to-weaning swine farm in the Midwestern United States. PROCEDURES: Stochastic models were formulated to simulate an outbreak of PRRS on a farm. Control strategies assessed in those models included none (baseline) and various combinations of mass immunization, herd closure, and gilt acclimatization. Nine different models resulting from the combination of low, moderate, or high PRRS virus virulence and small, medium, or large herd size were simulated. A stabilized status, the outcome of interest, was defined as the absence of positive PCR assay results for PRRS virus in 3-week-old piglets. For each scenario, the percentage of simulations with a stabilized status was used as a proxy for the probability of disease control. RESULTS: Increasing PRRS virus virulence and herd size were negatively associated with the probability of achieving a stabilized status. Repeated mass immunization with herd closure or gilt acclimitization was a better alternative than was single mass immunization for disease control within a farm. CONCLUSIONS AND CLINICAL RELEVANCE: Repeated mass immunization with a PRRS modified-live virus vaccine with herd closure or gilt acclimitization was the scenario most likely to achieve a stabilized status. Estimation of the cost of various PRRS control strategies is necessary.

Financial implications of installing air filtration systems to prevent PRRSV infection in large sow herds.

Air filtration systems implemented in large sow herds have been demonstrated to decrease the probability of having a porcine reproductive and respiratory syndrome virus (PRRSV) outbreak. However, implementation of air filtration represents a considerable capital investment, and does not eliminate the risk of new virus introductions. The specific objectives of the study were: 1) to determine productivity differences between a cohort of filtered and non-filtered sow farms; and 2) to employ those productivity differences to model the profitability of filtration system investments in a hypothetical 3000 sow farm. Variables included in the study were production variables (quarterly) from respective herds; air filtration status; number of pig sites within 4.7 km of the farm; occurrence of a PRRSV outbreak in a quarter, and season. For the investment analyses, three Scenarios were compared in a deterministic spreadsheet model of weaned pig cost: (1) control, (2) filtered conventional attic, and (3) filtered tunnel ventilation. Model outputs indicated that a filtered farm produced 5927 more pigs than unfiltered farms. The payback periods for the investments, were estimated to be 5.35 years for Scenario 2 and 7.13 years for Scenario 3 based solely on sow herd productivity. Payback period sensitivity analyses were performed for both biological and financial inputs. The payback period was most influenced by the premium for weaned pig sales price for PRRSV-negative pigs, and the relative proportions of time that filtered vs. unfiltered farms produced PRRSV-negative pigs. A premium of $5 per pig for PRRS-negative weaned pigs reduced the estimated payback periods to 2.1 years for Scenario 2 and 2.8 years for Scenario 3.

Evaluation of the long-term effect of air filtration on the occurrence of new PRRSV infections in large breeding herds in swine-dense regions.

Airborne transmission of porcine reproductive and respiratory syndrome virus (PRRSV) is a risk factor for the infection of susceptible populations. Therefore, a long­term sustainability study of air filtration as a means to reduce this risk was conducted. Participating herds (n = 38) were organized into 4 independent cohorts and the effect of air filtration on the occurrence of new PRRSV infections was analyzed at 3 different levels from September 2008 to January 2012 including the likelihood of infection in contemporary filtered and non-filtered herds, the likelihood of infection before and after implementation of filtration and the time to failure in filtered and non-filtered herds. Results indicated that new PRRSV infections in filtered breeding herds were significantly lower than in contemporary non-filtered control herds (P < 0.01), the odds for a new PRRSV infection in breeding herds before filtration was 7.97 times higher than the odds after filtration was initiated (P < 0.01) and the median time to new PRRSV infections in filtered breeding herds of 30 months was significantly longer than the 11 months observed in non-filtered herds (P < 0.01). In conclusion, across all 3 levels of analysis, the long-term effect of air filtration on reducing the occurrence of new PRRSV infections in the study population was demonstrated.

A prospective study evaluating duration of swine breeding herd PRRS virus-free status and its relationship with measured risk.

A variety of methods for eliminating the PRRS virus from pig production sites have been successfully applied. However, success in maintaining a PRRS virus-free status for extended periods of time following elimination has been inconsistent and unpredictable. The objective of this study was to evaluate whether risks measured using version 1 of the American Association of Swine Veterinarians (AASV) PRRS Risk Assessment for the Breeding Herd, season of year and method by which swine breeding herd sites were established PRRS virus-free were associated with how long they retained their virus-free status. Thirty-three swine farrow-to-wean breeding herd sites that were established as PRRS virus-free by either populating a new site with virus-free breeding animals or by completely depopulating the site and repopulating with PRRS virus-free breeding animals were enrolled in this study. Survival analysis, using the Cox proportional hazards model and Kaplan-Meier survival curves, was performed where the outcome was the duration of time PRRS virus-free breeding herd sites remained virus-free ("survived"). Covariates evaluated included the internal and external risk scores measured by the PRRS Risk Assessment for the Breeding Herd as well as the season and the method by which the site was established free of the PRRS virus. All but 5 (15%) of the 33 sites became positive to the PRRS virus during the course of the study and approximately 40% became positive within 1 year from when they were established free of the PRRS virus. A higher external risk score was associated with a greater risk of becoming positive to the PRRS virus and shorter survival times. The internal risk score was not significantly associated with survival. Establishing breeding herd sites free of the PRRS virus in winter months (November through February) was associated with a greater risk of becoming positive to the PRRS virus and shorter survival times compared to those established in non-winter months. The association between the risk of becoming positive to the PRRS virus and the external risk score was confounded by the method the site was established PRRS virus-free.

Simultaneous detection of North American and European porcine reproductive and respiratory syndrome virus using real-time quantitative reverse transcriptase-PCR.

Porcine reproductive and respiratory syndrome (PRRS) is 1 of the most economically important diseases of swine. Detection of the etiologic agent, PRRS virus (PRRSV), represents a diagnostic challenge due to the heterogeneity of field isolates as well as the propensity for swine to develop persistent infection in which virus is difficult to detect. Recently European (EU) lineage PRRSV isolates, which are genetically divergent from North American (NA) isolates, have been introduced into NA swine further complicating efforts to diagnose this disease. In this study, real-time (TaqMan) reverse transcriptase (RT)-PCR assays were developed for multiplex detection, differentiation, and quantification of NA and EU PRRSV field isolates. Oligonucleotide primers and dual-labeled probes were selected from conserved regions of open-reading frame 7 and the 3'-untranslated region. The real-time RT-PCR assays described for the NA or EU genotype of PRRSV detected viral RNA from 83/83 strains (74 NA; 9 EU) previously isolated by cell culture between 1992 and 2003. The analytical sensitivity of both assays was consistently found to be less than a single TCID50, which corresponded to 5-10 RNA molecules, and was not significantly reduced when the reactions were performed in a multiplex format. When performing multiplex reactions, sensitive detection was possible even when 1 viral RNA concentration was up to 5,000-fold higher than the second. The diagnostic sensitivity and specificity of the multiplex reaction was found to be at a minimum equivalent to that of both nested RT-PCR and virus isolation.

Validation of a blocking enzyme-linked immunosorbent assay for detection of antibodies against porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) continues to be one of the most significant diseases of swine. IDEXX HerdChek PRRS, a commercially available enzyme-linked immunosorbent assay (ELISA), has become the industry standard for the detection of antibodies against PRRS virus (PRRSV). The need to accurately determine the PRRSV serostatus of herds and individual animals has prompted the development of several follow-up assay methods. A highly specific and repeatable blocking ELISA (bELISA) was developed on the basis of the use of an expressed PRRSV nucleocapsid (N) protein as the antigen and a biotinylated monoclonal antibody. Validation of the bELISA used sera from 316 animals experimentally and naturally infected with North American PRRSV and sera from 370 uninfected animals. Receiver operating characteristic analysis of the data calculated a diagnostic sensitivity of 97.8% and a diagnostic specificity of 100%. The between-run coefficient of variation of an internal quality control serum was 4.24%. The bELISA was able to detect seroconversion as well as the IDEXX ELISA and the indirect fluorescent antibody (IFA) assay; kappa values were 0.94 and 0.96, respectively. A collection of 133 serum samples with unexpected positive IDEXX ELISA results was obtained from 4,038 diagnostic samples submitted from farms from which PRRS-negative results were expected. The bELISA identified 97% of the samples as PRRS seronegative, while the IFA identified 100% as seronegative. The anticipated use of the bELISA is as a follow-up test to the IDEXX ELISA for determining the PRRSV serostatus of individual animals with unexpected positive test results from swine herds from which negative results are expected.