A diagnostic approach to confirm Mycoplasma hyopneumoniae "Day zero" for pathogen eradication.

Breeding herds in the US are trending toward eradication of Mycoplasma hyopneumoniae (M. hyopneumoniae) due to the positive impact on welfare and downstream production. In an eradication program, "Day 0″ is the time point when the last replacement gilts to enter the herd were exposed to M. hyopneumoniae and marks the beginning of a herd closure. However, no specific diagnostic protocols are available for confirmation of successful exposure to define Day 0. Therefore, the objective of this study was to develop diagnostic guidelines, including sample collection approaches, for two common gilt exposure methods to confirm an entire population has been infected with M. hyopneumoniae following purposeful exposure. Forty gilts, age 21-56 days, were ear-tagged for longitudinal sample collection at five commercial gilt developer units (GDUs) and were exposed to M. hyopneumoniae by natural contact or aerosolization. Study gilts originated from sources known to be negative to major swine pathogens, including M. hyopneumoniae, and were sampled prior to exposure to confirm negative status, then every two weeks. Blood samples were collected for antibody detection, while laryngeal and deep tracheal secretions and pen based oral fluids were collected for PCR, and sampling continued until at least 85% of samples were positive by PCR. Detection of M. hyopneumoniae varied greatly based on sample type. Oral fluids showed the lowest detection in groups of gilts detected positive by other sample types. Detection by PCR in deep tracheal secretions was higher than in laryngeal secretions. Seroconversion to and PCR detection of M. hyopneumoniae on oral fluids were delayed compared to PCR detection at the individual level. By two weeks post-exposure, at least 85% of study gilts in three GDUs exposed by aerosolization were PCR positive in deep tracheal secretions. Natural contact exposure resulted in 22.5% of study gilts becoming PCR positive by two weeks post-initial exposure, 61.5% at four weeks, and 100% at six weeks on deep tracheal secretions. Deep tracheal secretions required the lowest number of gilts to sample for the earliest detection compared to all other samples evaluated. As observed in one of the GDUs using aerosolization, demonstration of failure to expose gilts to M. hyopneumoniae allowed for early intervention in the exposure protocol and delay of Day 0, for accurate timing of the eradication protocol. Sampling guidelines proposed in this study can be used for verification of M. hyopneumoniae infection of gilts following exposure to determine Day 0 of herd closure.

Duration of Mycoplasma hyopneumoniae detection in pigs following purposeful aerosol exposure.

Swine disease elimination programs for Mycoplasma hyopneumoniae are commonly applied in the North American swine industry and may include the aerosolization of medium containing lung tissue to achieve population exposure prior to start. Field data has indicated M. hyopneumoniae PCR detection in pigs beyond 240 days post-herd closure (dphc; planned end of an elimination program) and is thought to contribute to disease elimination programs' failure. Here, the duration of M. hyopneumoniae detection in sows and replacement gilts following aerosolized lung homogenate exposure, as part of a dual disease elimination program, was determined. A subset of sows and gilts from a commercial sow herd and off-site gilt development unit were longitudinally sampled to collect deep tracheal catheter secretions at various times post-exposure. Samples were tested for M. hyopneumoniae using a species-specific real-time PCR. A proportion of 58, 51, 52, 19, and 2% females were detected positive at 30, 60, 120, 180 and 240 dphc, respectively. Noteworthy, a greater proportion of gilts exposed at the off-site GDU were detected PCR positive for M. hyopneumoniae at each sampling event, compared to sows. In this study, assaying for genetic material in live female pigs showed extended detection of M. hyopneumoniae until at least 240 dphc. This data suggests persistence of M. hyopneumoniae longer than previously reported and highlights the importance of performing diagnostic testing to confirm negativity to the bacterium, prior to opening sow herds, especially late in the herd closure timeline.

Improving Mycoplasma hyopneumoniae diagnostic capabilities by harnessing the infection dynamics.

Mycoplasma hyopneumoniae remains one of the most problematic bacterial pathogens for pig production. Despite an abundance of observational and laboratory testing capabilities for this organism, diagnostic interpretation of test results can be challenging and ambiguous. This is partly explained by the chronic nature of M. hyopneumoniae infection and its tropism for lower respiratory tract epithelium, which affects diagnostic sensitivities associated with sampling location and stage of infection. A thorough knowledge of the available tools for routine M. hyopneumoniae diagnostic testing, together with a detailed understanding of infection dynamics, are essential for optimizing sampling strategies and providing confidence in the diagnostic process. This study reviewed known information on sampling and diagnostic tools for M. hyopneumoniae and summarized literature reports of the dynamics of key infection outcomes, including clinical signs, lung lesions, pathogen detection, and humoral immune responses. The information gathethered in this manuscript can facilitate better understanding of the performance of different diagnostic approaches at various stages of infection with Mycoplasma hyopneumoniae.

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.

Relationships among Fecal, Air, Oral, and Tracheal Microbial Communities in Pigs in a Respiratory Infection Disease Model.

The association of the lower respiratory tract microbiome in pigs with that of other tissues and environment is still unclear. This study aimed to describe the microbiome of tracheal and oral fluids, air, and feces in the late stage of Mycoplasma hyopneumoniae infection in pigs, and assess the association between the tracheal microbiome and those from air, feces, and oral fluids. Tracheal fluids (n = 73), feces (n = 71), oropharyngeal fluids (n = 8), and air (n = 12) were collected in seeder pigs (inoculated with M. hyopneumoniae) and contact pigs (113 days post exposure to seeder pigs). After DNA extraction, the V4 region from 16S rRNA gene was sequenced and reads were processed using Divisive Amplicon Denoising Algorithm (DADA2). Clostridium and Streptococcus were among the top five genera identified in all sample types. Mycoplasma hyopneumoniae in tracheal fluids was associated with a reduction of diversity and increment of M. hyorhinis, Glaesserella parasuis, and Pasteurella multocida in tracheal fluids, as well as a reduction of Ruminiclostridium, Barnesiella, and Lactobacillus in feces. Air contributed in a greater proportion to bacteria in the trachea compared with feces and oral fluids. In conclusion, evidence suggests the existence of complex interactions between bacterial communities from distant and distinct niches.

Mycoplasma hyopneumoniae genetic variability within swine production flows.

The aim of this study was to assess the genetic variability of Mycoplasma hyopneumoniae within various swine production flows. Four M. hyopneumoniae positive production flows, composed of 4 production stages, were selected for this study. Laryngeal and/or bronchial swabs were collected from each production stage within a flow, for a period of 4 months up to 3 years. A multiple-locus variable-number tandem repeat analysis was performed to assess the genetic variation of M. hyopneumoniae within and across production flows through the identification of variable-number tandem repeat (VNTR) types. A maximum of 6 M. hyopneumoniae VNTR types were identified in a single flow, in which VNTR types appeared to be flow specific. An identical VNTR type was detected across several production stages for up to 3 years. In this study, minimal M. hyopneumoniae genetic variation was evidenced within and across production flows.

L'objectif de cette étude était d'évaluer la variabilité génétique de Mycoplasma hyopneumoniae au sein de différents flux de production porcine. Quatre flux de production positifs pour M. hyopneumoniae, composés de quatre stades de production, furent sélectionnés pour cette étude. Des écouvillons laryngés et/ou bronchiaux furent prélevés de chaque stade de production à l'intérieur d'un flux, pour une période de 4 mois jusqu'à 3 ans. Une analyse multi-locus du polymorphisme des séquences répétées en tandem fut effectuée afin d'évaluer la variation génétique de M. hyopneumoniae au sein et à travers les flux de production par l'identification des types de polymorphismes de séquences répétées en tandem (VNTR). Un maximum de six types de VNTR de M. hyopneumoniae fut identifié dans un flux unique, dans lequel les types de VNTR apparaissaient être spécifiques de flux. Un type de VNTR identique fut détecté à travers plusieurs stades de production et jusqu'à 3 ans. Dans cette étude, une variation génétique minime de M. hyopneumoniae fut notée au sein et à travers des flux de production.(Traduit par Docteur Serge Messier).

Comparison of the sensitivity of laryngeal swabs and deep tracheal catheters for detection of Mycoplasma hyopneumoniae in experimentally and naturally infected pigs early and late after infection.

Detection of Mycoplasma hyopneumoniae infection in live pigs is a critical component to measure the success of disease control or elimination strategies. However, in vivo diagnosis of M. hyopneumoniae is difficult and the imperfect sensitivity of diagnostic tools has been deemed as one of the main challenges. Here, the sensitivity of laryngeal swabs and deep tracheal catheters for detection of M. hyopneumoniae early and late after infection was determined using inoculation status as a gold standard in experimentally infected pigs and a Bayesian approach in naturally infected pigs. Three-hundred and twenty 8-week old seeder pigs were intra-tracheally inoculated with M. hyopneumoniae strain 232 and immediately placed with 1920 contact pigs to achieve a 1:6 seeder-to-contact ratio. A subset of seeders and contacts were longitudinally sampled at 7, 28, 97, and 113 days post-inoculation (dpi) and at 28, 56, 84, and 113 days post-exposure (dpe), respectively, using laryngeal swabs and deep tracheal catheters. Samples were tested for M. hyopneumoniae by a species-specific real-time PCR. The sensitivity of deep tracheal catheters was higher than the one obtained in laryngeal swabs at all samplings (seeders: 36% higher than laryngeal swabs at 7 dpi, 29% higher at 97 dpi, and 44% higher at 113 dpi; contacts: 51% higher at 56 dpe, 42% higher at 84 dpe, and 32% higher at 113 dpe). Our study indicates that deep tracheal catheters were a more sensitive sample than laryngeal swabs. The sensitivity of both sample types varied over time and by exposure method, and these factors should be considered when designing diagnostic strategies.

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.