Detection of Mycoplasma hyopneumoniae viability using a PCR-based assay.

Mycoplasma hyopneumoniae detection in clinical specimens is accomplished by PCR targeting bacterial DNA. However, the high stability of DNA and the lack of relationship between bacterial viability and DNA detection by PCR can lead to diagnostic interpretation issues. Bacterial messenger RNA is rapidly degraded after cell death, and consequently, assays targeting mRNA detection can be used for the exclusive detection of viable bacterial cells. Therefore, this study aimed at developing a PCR-based assay for the detection of M. hyopneumoniae mRNA and at validating its applicability to differentiate viable from inert bacteria. Development of the RNA-based PCR encompassed studies to determine its analytical sensitivity, specificity, and repeatability, as well as its diagnostic accuracy. Comparisons between DNA and mRNA detection for the same target gene were performed to evaluate the ability of the RNA-based PCR to detect exclusively viable M. hyopneumoniae after bacterial inactivation using various methods. The RNA-based PCR was also compared to the DNA-based PCR as a tool to monitor the growth of M. hyopneumoniae in vitro. Under the conditions of this study, the developed RNA-based PCR assay detected only viable or very recently inactivated M. hyopneumoniae, while the DNA-based PCR consistently detected cells irrespective of their viability status. Changes in growth activity over time were only observable via RNA-based PCR. This viability PCR assay could be directly applied to evaluate the clearance of M. hyopneumoniae or to determine the viability of the bacterium at late stages of eradication programs.

Characterizing the detection of inactivated Mycoplasma hyopneumoniae DNA in the respiratory tract of pigs.

A positive Mycoplasma hyopneumoniae PCR result in a clinical specimen may eventually represent the mere detection of non-viable bacteria, complicating the diagnostic interpretation. Thus, the objective of this study was to evaluate the PCR detection of non-viable M. hyopneumoniae and its residual cell-free DNA in live pigs. Pigs were inoculated with either active or inactivated M. hyopneumoniae and were sampled for up to 14 days. Mycoplasma hyopneumoniae was not detected by PCR at any timepoint in pigs inoculated with the inactivated bacterium, suggesting that in healthy pigs, the non-viable M. hyopneumoniae DNA was rapidly sensed and cleared.

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.

Evaluation of Immune Nanoparticles for Rapid and Non-Specific Activation of Antiviral and Antibacterial Immune Responses in Cattle, Swine, and Poultry.

Given the rapid potential spread of agricultural pathogens, and the lack of vaccines for many, there is an important unmet need for strategies to induce rapid and non-specific immunity against these viral and bacterial threats. One approach to the problem is to generate non-specific immune responses at mucosal surfaces to rapidly protect from entry and replication of both viral and bacterial pathogens. Using complexes of charged nanoparticle liposomes with both antiviral and antibacterial toll-like receptor (TLR) nucleic acid ligands (termed liposome-TLR complexes or LTC), we have previously demonstrated considerable induction of innate immune responses in nasal and oropharyngeal tissues and protection from viral and bacterial pathogens in mixed challenge studies in rodents, cattle, and companion animals. Therefore, in the present study, we used in vitro assays to evaluate the ability of the LTC immune stimulant to activate key innate immune pathways, particularly interferon pathways, in cattle, swine, and poultry. We found that LTC complexes induced strong production of type I interferons (IFNα and IFNß) in both macrophages and leukocyte cultures from all three species. In addition, the LTC complexes induced the production of additional key protective cytokines (IL-6, IFNγ, and TNFα) in macrophages and leukocytes in cattle and poultry. These findings indicate that the LTC mucosal immunotherapeutic has the capability to activate key innate immune defenses in three major agricultural species and potentially induce broad protective immunity against both viral and bacterial pathogens. Additional animal challenge studies are warranted to evaluate the protective potential of LTC immunotherapy in cattle, swine, and poultry.

Review on the methodology to assess respiratory tract lesions in pigs and their production impact.

Porcine respiratory disease is one of the most important health problems in pig production worldwide. Cranioventral pulmonary consolidation (CVPC) and pleurisy are the two most common lesions in the respiratory tract of slaughtered pigs. The present review paper discusses pathogens involved in the lesions, lesion prevalence, scoring systems, advantages and disadvantages of slaughterhouse examination, and the impact of CVPC and pleurisy on performance, carcass, and meat quality. Cranioventral pulmonary consolidation and pleurisy in slaughter pigs are characteristic for infections with Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae, respectively, although other pathogens may cause similar lesions and/or be involved in their development. The overall prevalence of CVPC and pleurisy in slaughter pigs are still high, being the prevalence of CVPC generally higher than that of chronic pleurisy. The advantages and disadvantages of slaughterhouse examination are discussed in relation to practical aspects, the assessment of lesions, the number and representativeness of the examined animals and the interpretation and value of the results for the stakeholders. The main scoring methods for CVPC and pleurisy are shortly reviewed. In general, scoring methods can be applied rapidly and easily, although significant variation due to abattoir and observer remains. Artificial intelligence-based technologies that automatically score lesions and facilitate processing of data may aid solving these problems. Cranioventral pulmonary consolidation and pleurisy have a major negative impact on pig performance, and the effects increase the extension of the lesions and/or presence of multiple lesions. The performance losses caused by these lesions, however, vary significantly between studies and farms, possibly due to differences in study population and used methodology. Both lesions also have a negative impact on different carcass and meat quality parameters, leading to increased risk for poor processing and storage of the carcasses. Monitoring lung lesions of slaughter pigs should be optimized and implemented routinely; however, it is recommended to complement this information with farm data and laboratory results for specific pathogens.

The effect of gilt flow management during acclimation on Mycoplasma hyopneumoniae detection.

The objective of this study was to characterize the Mycoplasma hyopneumoniae (M. hyopneumoniae) detection and seroconversion patterns in recently acclimated gilts to be introduced to endemically infected farms using different types of replacement management. Three gilt developing units (GDUs) belonging to sow farms were included in this investigation: two farms managed gilts in continuous flow, and one farm managed gilts all-in/all-out. Two replicates of 35 gilts each were selected per GDU and sampled approximately every 60 days for a total of four or five samplings, per replicate and per GDU. Detection of M. hyopneumoniae was evaluated by PCR, while antibodies were measured using a commercial ELISA assay. Also, M. hyopneumoniae genetic variability was evaluated using Multiple-Locus Variable number tandem repeat Analysis. Detection of M. hyopneumoniae was similar across GDUs. Although a significant proportion of gilts was detected positive for M. hyopneumoniae after acclimation, an average of 30.3 % of gilts was negative at any point during the study. Detection of M. hyopneumoniae antibodies was similar among GDUs regardless of flow type or vaccination protocol. The genetic variability analysis revealed a limited number of M. hyopneumoniae types within each GDU. Results of this study showed a similar pattern of M. hyopneumoniae detection by PCR and seroconversion by ELISA among GDUs, regardless of the type of flow management strategies applied to gilts.

Longitudinal Changes in Campylobacter and the Litter Microbiome throughout the Broiler Production Cycle.

Broiler chickens are an important source of Campylobacter to humans and become colonized on the farm, but the role of the litter in the ecology of Campylobacter is still not clear. The aim of this study was to examine the relationship between Campylobacter and the changes in the litter microbiome throughout the broiler production cycle. Twenty-six commercial broiler flocks representing two production types (small and big broilers) were followed from 1 to 2 weeks after placement to the end of the production cycle. Composite litter samples from the broiler chicken house were collected weekly. Litter DNA was extracted and used for Campylobacter jejuni and Campylobacter coli qPCR as well as for 16S rRNA gene V4 region sequencing. Campylobacter jejuni concentration in litter significantly differed by production type and flock age. Campylobacter jejuni concentration in litter from big broilers was 2.4 log10 units higher, on average, than that of small broilers at 3 weeks of age. Sixteen amplicon sequence variants (ASVs) differentially abundant over time were detected in both production types. A negative correlation of Campylobacter with Bogoriella and Pseudogracilibacillus was observed in the litter microbiome network at 6 weeks of flock age. Dynamic Bayesian networks provided evidence of negative associations between Campylobacter and two bacterial genera, Ornithinibacillus and Oceanobacillus, at 2 and 4 weeks of flock age, respectively. In conclusion, dynamic associations between Campylobacter and the litter microbiome were observed during grow-out, suggesting a potential role of the litter microbiome in the ecology of Campylobacter colonization and persistence on farm. IMPORTANCE This study interrogated the longitudinal association between Campylobacter and broiler litter microbiome in commercial broiler flocks. The results of this investigation highlighted differences in Campylobacter dynamics in the litter throughout the broiler production cycle and between small and big broilers. Besides documenting the changing nature of the microbial networks in broiler litter during grow-out, we detected bacterial genera (Oceanobacillus and Ornithinibacillus) negatively associated with Campylobacter abundance and concentration in litter via the Bayesian network framework. These bacteria should be investigated as possible antagonists to Campylobacter colonization of the broiler environment.

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.

The role of co-infections in M. hyopneumoniae outbreaks among heavy fattening pigs: a field study.

Little is known about how co-infections and genotype dynamics affect Mycoplasma hyopneumoniae infection in fattening pigs. This study was aimed at assessing the role of co-infections in M. hyopneumoniae outbreaks, their influence on the presence of M. hyopneumoniae genotypes and their impact on consequent lung lesions. Tracheobronchial swabs (TBS) from 300 finishers were collected from 10 farms at the onset of enzootic pneumonia outbreaks and 1 month later, sampling of 3 groups per farm: Group A showed clinical signs first, Group B was housed near Group A, and Group C was located in a different building. Pigs' lungs were scored at the slaughterhouse. TBS were tested for the main pathogens involved in respiratory diseases, and samples positive for M. hyopneumoniae were genotyped by multiple-locus variable-number tandem repeat analysis (MLVA). Pigs in Group A showed the highest prevalence and load of M. hyopneumoniae. A positive association was detected between M. hyopneumoniae and Mycoplasma hyorhinis, whereas Actinobacillus pleuropneumoniae was more frequent when the M. hyopneumoniae load was higher. Nevertheless, co-infection had no effect on lung lesion scores. The presence of multiple MLVA types (mixed infections) increased in time only in pigs from Group C and was positively associated with porcine reproductive and respiratory syndrome virus infection. Lung lesions were more severe in pigs with at least one TBS positive for M. hyopneumoniae and in pigs with a history of mixed infections. The central role of M. hyopneumoniae and relevance of mixed infections suggest that increased biosecurity might be beneficial for lung lesion sequelae.

Mycoplasma hyopneumoniae detection by PCR in naturally infected finishing pigs.

The aim of this study was to compare the sensitivity of different in vivo and post-mortem samples collected from finishing pigs under field conditions on Mycoplasma hyopneumoniae detection by PCR. Results suggested that tracheobronchial secretions and bronchial swabs conferred the highest sensitivity in vivo and post-mortem, respectively.

Environmental detection of Mycoplasma hyopneumoniae in breed-to-wean farms.

There is a need to develop cost-effective and non-invasive approaches to sample large populations to evaluate the disease status of breeding herds. In this study we assessed the detection of the M. hyopneumoniae genetic material in environmental surfaces and air of farrowing rooms, and skin (udder, snout and vagina) of lactating sows at weaning, in farms having different M. hyopneumoniae infection status (negative, positive sub-clinically infected and positive clinically affected). Mycoplasma hyopneumoniae was detected in air, air deposition particles, dam and stall surfaces of the positive clinically affected herd. Mycoplasma hyopneumoniae could only be detected in dam and stall surfaces in sub-clinically infected herds. Mycoplasma hyopneumoniae was not detected in all samples collected in the negative herd. The cycle threshold of the positive PCR samples were not statistically different between sample types or farms. However, a significant difference (p < 0.05) was observed in the percentage of positive samples between the positive clinically affected farm and the rest. Likewise, M. hyopneumoniae was detected in the environment and surfaces at weaning in positive breeding herds. Further testing and validation is recommended for environmental and surface samples before they can be employed as part of the M. hyopneumoniae diagnostic process. In addition, results from this study highlight potential sources of M. hyopneumoniae infection for piglets in breeding herds, especially during an outbreak.

Effect of antibiotic treatment on Mycoplasma hyopneumoniae detection and infectious potential.

Mycoplasma hyopneumoniae (M. hyopneumoniae) causes significant economic losses in the swine industry. Antibiotics with activity against Mycoplasma spp. are employed for disease mitigation and pathogen elimination. However, veterinarians are often challenged with the detection of M. hyopneumoniae by PCR after antibiotic treatment, thus raising the question whether the bacterium is still infectious. The objective of this study was to evaluate the effect of tulathromycin treatment on M. hyopneumoniae detection and infectious potential during the acute and chronic phases of infection. For each infection phase, one age-matched naïve gilt was placed in contact with one M. hyopneumoniae infected gilt that was either treated with tulathromycin, treated and vaccinated, or non-treated, for 14 days. Four replicates per treatment group were performed for each infection phase. A numerical reduction in relative bacterial load was observed in acutely treated gilts compared to non-treated gilts. The rate at which naïve gilts became infected with M. hyopneumoniae was numerically reduced when co-housed with treated, acutely infected gilts compared to those housed with non-treated, infected gilts. During the chronic infection phase, M. hyopneumoniae was detected by PCR in more than 50 % of treated infected gilts and persisted for up to three months post-treatment. Transmission was not detected in all treatment groups however, the possibility that the pathogen was infectious could not be completely ruled out. Further research focused on assessing M. hyopneumoniae detection and viability post-treatment is necessary to guide control and elimination efforts.

Association of Broiler Litter Microbiome Composition and Campylobacter Isolation.

Infection with Campylobacter species is one of the leading causes of bacterial diarrhea in humans in the US. Chickens, which become colonized on the farm, are important reservoirs of this bacterium. Campylobacter can establish itself in the broiler house via a variety of sources, can survive in the litter of the house, and possibly persist over successive flock cycles. However, the role of the broiler litter microbiome on Campylobacter persistence is not clear. A matched case-control study was conducted to determine whether the broiler litter microbiome composition was associated with Campylobacter isolation within the broiler house. Flocks were classified as cases when either Campylobacter jejuni or Campylobacter coli was isolated in boot sock samples, or as controls otherwise. Case and control flocks were matched at the broiler house level. Composite broiler litter samples were collected and used for DNA extraction and 16S rRNA gene V4 region sequencing. Reads were processed using the DADA2 pipeline to obtain a table of amplicon sequence variants. Alpha diversity and differential bacterial relative abundance were used as predictors of Campylobacter isolation status in conditional logistic regression models adjusting for flock age and sampling season. Beta diversity distances were used as regressors in stratified PERMANOVA with Campylobacter isolation status as predictor, and broiler house as stratum. When Campylobacter was isolated in boot socks, broiler litter microbiome richness and evenness were lower and higher, respectively, without reaching statistical significance. Campylobacter isolation status significantly explained a small proportion of the beta diversity (genus-level Aitchison dissimilarity distance). Clostridium and Anaerostipes were positively associated with Campylobacter isolation status, whereas Bifidobacterium, Anaerosporobacter, and Stenotrophomonas were negatively associated. Our results suggest the presence of bacterial interactions between Campylobacter and the broiler litter microbiome. The negative association of Campylobacter with Bifidobacterium, Anaerosporobacter, and Stenotrophomonas in litter could be potentially exploited as a pre-harvest control strategy.

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.

Genomic variability of Mycoplasma hyopneumoniae within pig lung lobes.

BACKGROUND: Genotypic variability in M. hyopneumoniae has been reported within and among herds. However, information regarding VNTR types within single lung lobes is lacking. The objective of his study was to analyse M. hyopneumoniae infections and their association with VNTR types and lung lesions at the lobe level. Lungs from 300 pigs from 10 farms experiencing an enzootic pneumonia outbreak were collected and scored. M. hyopneumoniae was detected by real-time PCR and genotyped by MLVA assay in all samples. RESULTS: The results showed genotypic variability within single pigs and among lung lobes. At the lobe level, infection with one VNTR type (SN infection) was dominant. Lobes with lesion scores > 0 were associated with positive results for real-time PCR. At the lobe level, no relationship was observed between infections with more than one genotype (MX infections) and the proportion of Mycoplasma-like lesions. Lesion-free lobes presented a higher proportion of MX infections than lobes scored > 0. M. hyopneumoniae was detected more frequently in the right lobe of the lung (p < 0.05), with a similar distribution within lobes for SN and MX infections. The anatomic conformation of swine lungs led to a higher prevalence of infections in the right lobe. However, this study showed that this condition did not affect the distribution of infections with multiple VNTR types. Nevertheless, careful consideration of sample selection should be practised for M. hyopneumoniae genotype analyses, including lung lobes with no visible lesions. CONCLUSION: The results did not show a significant association between the number of detected genotypes and the severity of the lesions at the lung lobe level, but revealed the unexpected detection of M. hyopneumoniae genotypes in lesion-free lobes. These results imply that a representative sampling of all lobes may lead to an accurate identification of the VNTR-type distribution. Further studies including factors that can affect pathogenetic evolution of this bacterium could shed light on the complexity of the relationship between genotypes and the lung lesions magnitude.

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.

Effect of multiple vaccinations on transmission and degree of Mycoplasma hyopneumoniae infection in gilts.

Mycoplasma hyopneumoniae (M. hyopneumoniae) infections continue to result in significant respiratory challenges in the swine industry worldwide. Vaccination for M. hyopneumoniae is commonly utilized, as reduction in bacterial loads and clinical severity in vaccinated pigs have been shown. However, the effect of M. hyopneumoniae vaccination on transmission across different pig populations has been minimally investigated. The aim of this pilot study was to evaluate the effect of multiple vaccinations on M. hyopneumoniae infection, transmission, and genetic variability in infected and susceptible gilt populations. Thirty-two naïve gilts were allocated to four treatment groups: (1) Vaccinated seeder (VS); (2) Non-vaccinated seeder (NVS); (3) Vaccinated contact (VC); and (4) Non-vaccinated contact (NVC). At 5, 7, and 9 weeks of age, all gilts selected to be vaccinated received a commercial M. hyopneumoniae bacterin for a total of 3 doses. At 11 weeks of age, VS and NVS gilts were inoculated with M. hyopneumoniae to become seeders. At 28 days post-inoculation (dpi), VS and NVS gilts were individually relocated to clean experimental rooms, where they were placed in contact with one age-matched VC or NVC gilt (1:1 ratio) for 14 days. Blood and tracheal samples, bronchial swabs, and lung lesions were collected and/or evaluated for M. hyopneumoniae infection. In this study, a three-dose vaccination strategy against M. hyopneumoniae significantly reduced bacterial load in seeder gilts. Furthermore, a numerical reduction in M. hyopneumoniae lung lesions at 28 dpi was observed in VS gilts. All VC gilts in the VS:VC treatment group pairing remained M. hyopneumoniae negative, compared to other groups in which 1-2 transmission events occurred per treatment group. Results from this investigation provide insight on the potential impact of multiple vaccinations on reducing M. hyopneumoniae transmission and infection. Further research encompassing vaccinations of gilt groups in field settings is necessary to validate findings.

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).