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Tracheal pooling for Mycoplasma hyopneumoniae (M. hyopneumoniae) DNA detection allows for decreased diagnostic cost, one of the main constraints in surveillance programs. The objectives of this study were to estimate the sensitivity of pooled-sample testing for the detection of M. hyopneumoniae in tracheal samples and to develop probability of M. hyopneumoniae detection estimates for tracheal samples pooled by 3, 5, and 10. A total of 48 M. hyopneumoniae PCR-positive field samples were pooled 3-, 5-, and 10-times using field M. hyopneumoniae DNA-negative samples and tested in triplicate. The sensitivity was estimated at 0.96 (95% credible interval [Cred. Int.]: 0.93, 0.98) for pools of 3, 0.95 (95% Cred. Int: 0.92, 0.98) for pools of 5, and 0.93 (95% Cred. Int.: 0.89, 0.96) for pools of 10. All pool sizes resulted in PCR-positive if the individual tracheal sample Ct value was < 33. Additionally, there was no significant decrease in the probability of detecting at least one M. hyopneumoniae-infected pig given any pool size (3, 5, or 10) of tracheal swabs. Furthermore, this manuscript applies the probability of detection estimates to various real-life diagnostic testing scenarios. Combining increased total animals sampled with pooling can be a cost-effective tool to maximize the performance of M. hyopneumoniae surveillance programs., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

Mycoplasma hyopneumoniae is a difficult-to-control bacterium since commercial vaccines do not prevent colonization and excretion. The present study aimed to evaluate the performance of an orally administered vaccine composed of antigens extracted from Mycoplasma hyopneumoniae and incorporated into mesoporous silica (SBA-15), which has an adjuvant-carrier function, aiming to potentiate the action of the commercial intramuscular vaccine. A total of 60 piglets were divided into four groups (n = 15) submitted to different vaccination protocols as follows, Group 1: oral SBA15 + commercial vaccine at 24 days after weaning, G2: oral vaccine on the third day of life + vaccine commercial vaccine at 24 days, G3: commercial vaccine at 24 days, and G4: commercial vaccine + oral vaccine at 24 days. On the first day, the piglets were weighed and, from the third day onwards, submitted to blood collections for the detection and quantification of anti- Mycoplasma hyopneumoniae IgG. Nasal swabs were collected to monitor IgA by ELISA, and oropharyngeal swabs were used to assess the bacterial load by qPCR. Biological samples were collected periodically from the third day of life until the 73rd day. At 41 days of life, 15 individuals of the same age, experimentally challenged with an inoculum containing M. hyopneumoniae , were co-housed with the animals from groups (1 to 4) in a single pen to increase the infection pressure during the nursery period. At 73 days, all piglets were euthanized, and lungs were evaluated by collecting samples for estimation of bacterial load by qPCR. Quantitative data obtained from physical parameters and laboratory investigation were analyzed by performing parametric or non-parametric statistical tests. Results indicate that animals from G2 showed smaller affected lung areas compared to G3. Animals from G2 and G4 had a low prevalence of animals shedding M. hyopneumoniae at 61 days of age. Additionally, no correlation was observed between lung lesions and M. hyopneumoniae load in lung and BALF samples in animals that received the oral vaccine, while a strong correlation was observed in other groups. In the present study, evidence points to the effectiveness of the oral vaccine developed for controlling M. hyopneumoniae in pig production under field conditions.

Autophagy is an important conserved homeostatic process related to nutrient and energy deficiency and organelle damage in diverse eukaryotic cells and has been reported to play an important role in cellular responses to pathogens and bacterial replication. The respiratory bacterium Mycoplasma hyopneumoniae has been identified to enter porcine alveolar macrophages, which are considered important immune cells. However, little is known about the role of autophagy in the pathogenesis of M. hyopneumoniae infection of porcine alveolar macrophages. Our experiments demonstrated that M. hyopneumoniae infection enhanced the formation of autophagosomes in porcine alveolar macrophages but prevented the fusion of autophagosomes with lysosomes, thereby blocking autophagic flux and preventing the acidification and destruction of M. hyopneumoniae in low-pH surroundings. In addition, using different autophagy regulators to intervene in the autophagy process, we found that incomplete autophagy promoted the intracellular proliferation of M. hyopneumoniae. We also found that blocking the phosphorylation of JNK and Akt downregulated the autophagy induced by M. hyopneumoniae, but pathways related to two mitogen-activated protein kinases (Erk1/2 and p38) did not affect the process. Collectively, M. hyopneumoniae induced incomplete autophagy in porcine alveolar macrophages through the JNK and Akt signalling pathways; conversely, incomplete autophagy prevented M. hyopneumoniae from entering and degrading lysosomes to realize the proliferation of M. hyopneumoniae in porcine alveolar macrophages. These findings raise the possibility that targeting the autophagic pathway may be effective for the prevention or treatment of M. hyopneumoniae infection., (© 2022. The Author(s).)

Respiratory diseases constitute a major health challenge for the worldwide pork industry. Porcine enzootic pneumonia (PES) is caused by Mycoplasma hyopneumoniae (Mhyo). Mycoplasmas have the ability to produce extracellular vesicles (EVs), which can be useful for pathogenicity studies and as delivery systems for vaccines. The aim of this study was to demonstrate and compare, under laboratory conditions, EVs produced by Mhyo strain J and wild isolate in stressed and non-stressed in vitro conditions. Using differential centrifugation, density gradient ultracentrifugation, and transmission electron microscopy, the ability of Mhyo strains to produce EVs was demonstrated under favorable and unfavorable conditions., (© 2022. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Mycoplasma (M.) hyopneumoniae interacts with the respiratory microbiota and facilitates colonization of other pathogens. The present study investigated the pulmonary and nasal microbiota of M. hyopneumoniae-infected and M. hyopneumoniae-free pigs. Sixty-six pigs from three commercial herds were selected at the end of the finishing phase: 44 originated from two M. hyopneumoniae-positive herds and 22 from a M. hyopneumoniae-negative farm. At the slaughterhouse, samples of nasal turbinate (NT) and bronchus-alveolar lavage fluid (BALF) were collected. DNA was extracted with a commercial kit and the infection status was confirmed by qPCR. All samples from the same herd were pooled, and next-generation sequencing based on the hypervariable region V3-V4 of the 16 s bacterial rDNA was performed. Data analysis included the taxonomic analysis, Alpha diversity indexes, and Principal coordinates analysis (Pcoa) using Jaccard, Bray-Curtis, Weighted Unifrac, and Unweighted Unifrac distances. All pigs from the infected herds tested PCR positive for M. hyopneumoniae, whereas all pigs from the negative farm were negative. There was a greater diversity of microorganisms in BALF when compared to NT samples in all the farms. BALF samples from infected animals showed higher abundance of M. hyopneumoniae than NT samples and a predominance of Pasteurella multocida among the main species identified, which was also abundant in the M. hyopneumoniae-free herd. PCoa diagrams indicated that for most of the samples, dissimilarity on bacterial composition was observed, regardless of infection status and sample type. Therefore, the lung microbiota was modulated by M. hyopneumoniae infection, which could play a role in the pathogenesis of M. hyopneumoniae-disease., (© 2022. The Author(s).)

Mycoplasma (M.) hyopneumoniae is the main pathogen of porcine enzootic pneumonia (PEP). Its controlling is challenging, and requires alternative strategies. This study aimed to develop an oral vaccine against M. hyopneumoniae using a nanostructured mesoporous silica (SBA-15) as an adjuvant, and compare its effect with an intramuscular (IM) commercial vaccine (CV). Fifty 24 day-old M. hyopneumoniae-free piglets composed five equal groups for different immunization protocols, consisting of a CV and/or oral immunization (OI). Control piglets did not receive any form of immunization. All piglets were challenged with M. hyopneumoniae strain 232 on D49 by tracheal route. IgA antibody response in the respiratory tract, bacterial shedding and serum IgG were evaluated. The piglets were euthanized on 28 (D77) and 56 (D105) days post-infection. Lung lesions were macroscopically evaluated; lung fragments and bronchoalveolar fluid (BALF) were collected for estimation of bacterial loads by qPCR and/or histopathology examination. All immunization protocols induced reduction on Mycoplasma-like macroscopic lung lesions. IgA Ab responses anti-M. hyopneumoniae, the expression of IL-4 cytokine and a lower expression of IL-8 were induced by CV and OI vaccines, while IgG was induced only by CV. Oral immunization using silica as a carrier-adjuvant can be viable in controlling M. hyopneumoniae infection., (© 2021. The Author(s).)

Background: Porcine respiratory diseases remain the biggest challenge in pig-based food production and are a public health concern. Despite control measures, persistent outbreaks have been reported worldwide., Objective: To establish an early detection mechanism for pig farm disease outbreaks based on slaughterhouse risk and environmental assessment., Methods: We investigated the prevalence and risk factors of porcine respiratory disease-causing pathogens including Mycoplasma hyopneumoniae (MHP), porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV) and Haemophilus parasuis (HPS). Polymerase chain reaction (PCR) was used to analyse the lungs of 491 pigs from 19 slaughterhouses across 11 cities in Shanxi Province, China., Results: PCR detected MHP, PCV2, PPRSV and HPS in 76.99%, 67.00%, 11.82% and 19.55% of the samples, respectively; 10.12% were negative for all four pathogens. Co-positivity rates for two and three pathogens were identified. The results confirmed significant correlations between PCV2 and MHP (p = .001, p < .05), HPS and PCV2 (p = .01, p < .05) and MHP and PRRSV (p = .01, p < .05). No significant correlation was observed between HPS and MHP (p = .067, p > .05). Positive MHP and PCV2 rates were low in areas with high vegetation coverage. The overall pathogen positivity rate was higher in both lower and higher temperature environments., Conclusions: Interactions among pathogens may increase disease severity. Furthermore, environmental assessment and pathogen surveillance within pig slaughterhouses can be an effective approach for early detection and mitigation of new disease threats before broad dissemination occurs among a herd., (© 2021 The Authors. Veterinary Medicine and Science Published by John Wiley & Sons Ltd.)

Mycoplasma hyopneumoniae (M. hyopneumoniae) is one of the primary agents involved in the porcine respiratory disease complex, economically one of the most important diseases in pigs worldwide. The pathogen adheres to the ciliated epithelium of the trachea, bronchi, and bronchioles, causes damage to the mucosal clearance system, modulates the immune system and renders the animal more susceptible to other respiratory infections. The pathogenesis is very complex and not yet fully understood. Cell-mediated and likely also mucosal humoral responses are considered important for protection, although infected animals are not able to rapidly clear the pathogen from the respiratory tract. Vaccination is frequently practiced worldwide to control M. hyopneumoniae infections and the associated performance losses, animal welfare issues, and treatment costs. Commercial vaccines are mostly bacterins that are administered intramuscularly. However, the commercial vaccines provide only partial protection, they do not prevent infection and have a limited effect on transmission. Therefore, there is a need for novel vaccines that confer a better protection. The present paper gives a short overview of the pathogenesis and immune responses following M. hyopneumoniae infection, outlines the major limitations of the commercial vaccines and reviews the different experimental M. hyopneumoniae vaccines that have been developed and tested in mice and pigs. Most experimental subunit, DNA and vector vaccines are based on the P97 adhesin or other factors that are important for pathogen survival and pathogenesis. Other studies focused on bacterins combined with novel adjuvants. Very few efforts have been directed towards the development of attenuated vaccines, although such vaccines may have great potential. As cell-mediated and likely also humoral mucosal responses are important for protection, new vaccines should aim to target these arms of the immune response. The selection of proper antigens, administration route and type of adjuvant and carrier molecule is essential for success. Also practical aspects, such as cost of the vaccine, ease of production, transport and administration, and possible combination with vaccines against other porcine pathogens, are important. Possible avenues for further research to develop better vaccines and to achieve a more sustainable control of M. hyopneumoniae infections are discussed.

Background: The field efficacy of a bivalent vaccine containing porcine circovirus type 2b (PCV2b) and Mycoplasma hyopneumoniae was evaluated on three pig farms., Methods: Three pig farms were used, two of which had a history of subclinical PCV2 and clinical M. hyopneumoniae infections between 84 and 126 days of age while concurrent porcine circovirus-associated disease and clinical M. hyopneumoniae infection between 70 and 105 days of age. Each farm vaccinated pigs with a single dose of a bivalent vaccine at 10 days of age while unvaccinated pigs were administered a single dose of phosphate buffered-saline at the same age., Results: Vaccination improved growth performance and reduced clinical scores significantly (p < .05) when compared with unvaccinated animals. The amount of PCV2d loads in blood and M. hyopneumoniae loads in nasal swabs of vaccinated animals were also significantly lower (p < .05) when compared with unvaccinated animals. Immunologically, vaccinated groups elicited a significantly higher (p < .05) level of protective immunity against PCV2d such as neutralizing antibodies and interferon-γ secreting cells (IFN-γ-SC), as well as protective immunity against M. hyopneumoniae such as IFN-γ-SC when compared with unvaccinated animals. Pathologically, vaccination significantly lowered (p < .05) the scores of M. hyopneumoniae-induced pneumonia and PCV2-associated lymphoid lesions when compared with unvaccinated animals., Conclusions: The evaluated bivalent vaccine provided good protection against PCV2d and M. hyopneumoniae infection under field conditions., (© 2020 The Authors Veterinary Medicine and Science Published by John Wiley & Sons Ltd.)

Mycoplasma hyopneumoniae : is the etiological agent of porcine enzootic pneumonia (EP), a disease that impacts the swine industry worldwide. Pathogen-induced damage, as well as the elicited host-response, contribute to disease. Here, we provide an overview of EP epidemiology, control and prevention, and a more in-depth review of M. hyopneumoniae pathogenicity determinants, highlighting some molecular mechanisms of pathogen-host interactions relevant for pathogenesis. Based on recent functional, immunological, and comparative "omics" results, we discuss the roles of many known or putative M. hyopneumoniae virulence factors, along with host molecules involved in EP. Moreover, the known molecular bases of pathogenicity mechanisms, including M. hyopneumoniae adhesion to host respiratory epithelium, protein secretion, cell damage, host microbicidal response and its modulation, and maintenance of M. hyopneumoniae homeostasis during infection are described. Recent findings regarding M. hyopneumoniae pathogenicity determinants also contribute to the development of novel diagnostic tests, vaccines, and treatments for EP.

Background: Mycoplasma hyopneumoniae (M. hyopneumoniae), a representative pathogen causing swine enzootic pneumonia, generally infects piglets vertically. However, it is difficult to ascertain the M. hyopneumoniae infection state of sows due to limited detection methods. This report investigated sow herd stability by applying nested PCR to laryngeal swabs of suckling pigs, which is reportedly the most sensitive method., Results: M. hyopneumoniae was detected in 14 farms (63.6%) and 127 piglets (6.5%). The prevalence of sows likely to transmit M. hyopneumoniae in herds (11.1%) was calculated. In addition, there was a significant difference in detection rates among farms depending on herd size, gilt replacement rate, acclimation method, and antibiotic usage, suggesting various parameters that influence sow stability., Conclusions: The results demonstrated that laryngeal swabs from suckling pigs have provided useful information regarding vertical transmission from sows in South Korean farm conditions. This result demonstrated that farms with larger herd sizes, higher gilt replacement rates, and a practice of naturally exposing gilts for acclimation had higher detection rates in weaning piglets, indicating an unstable sow infection state.

Mycoplasma hyopneumoniae is an important respiratory pathogen of pigs that causes persistent and secondary infections. However, the mechanisms by which this occurs are unclear. In this study, we established air-liquid interface culture systems for pig bronchial epithelial cells (ALI-PBECs) that were comparable to the conditions in the native bronchus in vivo We used this ALI-PBECs model to study the infection and migration characteristics of M. hyopneumoniae in vitro Based on the results, we confirmed that M. hyopneumoniae was able to adhere to ALI-PBECs and disrupt mucociliary function. Importantly, M. hyopneumoniae could migrate to the basolateral chamber through the paracellular route but not the transcellular pathway, and this was achieved by reversibly disrupting tight junctions (TJs) and increasing the permeability and damaging the integrity of the epithelial barrier. We examined the migration ability of M. hyopneumoniae using an ALI-PBECs model for the first time. The disruption of the epithelial barrier allowed M. hyopneumoniae to migrate to the basolateral chamber through the paracellular route, which may be related to immune evasion, extrapulmonary dissemination, and persistent infection of M. hyopneumoniae ., (Copyright © 2020 American Society for Microbiology.)

Mycoplasma hyopneumoniae is the most costly pathogen for swine production. Although several studies have focused on the host-bacterium association, little is known about the changes in gene expression of swine cells upon infection. To improve our understanding of this interaction, we infected swine epithelial NPTr cells with M. hyopneumoniae strain J to identify differentially expressed mRNAs and miRNAs. The levels of 1,268 genes and 170 miRNAs were significantly modified post-infection. Up-regulated mRNAs were enriched in genes related to redox homeostasis and antioxidant defense, known to be regulated by the transcription factor NRF2 in related species. Down-regulated mRNAs were enriched in genes associated with cytoskeleton and ciliary functions. Bioinformatic analyses suggested a correlation between changes in miRNA and mRNA levels, since we detected down-regulation of miRNAs predicted to target antioxidant genes and up-regulation of miRNAs targeting ciliary and cytoskeleton genes. Interestingly, most down-regulated miRNAs were detected in exosome-like vesicles suggesting that M. hyopneumoniae infection induced a modification of the composition of NPTr-released vesicles. Taken together, our data indicate that M. hyopneumoniae elicits an antioxidant response induced by NRF2 in infected cells. In addition, we propose that ciliostasis caused by this pathogen is partially explained by the down-regulation of ciliary genes.

Mycoplasma hyopneumoniae causes the disease porcine enzootic pneumonia, a highly contagious and chronic disease affecting pigs. Understanding the molecular mechanisms of its pathogenicity is critical for developing effective interventions to control this swine respiratory disease. Here, we describe a novel virulence mechanism by which M. hyopneumoniae interferes with the host unfolded protein response (UPR) and eventually facilitates bacterial adhesion and infection. We observed that M. hyopneumoniae infection suppressed the UPR target molecules GRP78 and CHOP by reducing PKR-like endoplasmic reticulum kinase/eukaryotic initiation factor 2 alpha (PERK/eIF2α) phosphorylation, ATF6 cleavage, and X-box binding protein 1 (XBP1) splicing. Interestingly, further analyses revealed that host UPR inhibition subsequently suppressed the NF-κB pathway, leading to the reduced production of porcine beta-defensin 2 (PBD-2), thus facilitating M. hyopneumoniae adherence and infection. This study provides new insights into the molecular pathogenesis of M. hyopneumoniae and sheds light upon its interactions with the host., (Copyright © 2020 American Society for Microbiology.)

Mycoplasma hyopneumoniae is the etiologic agent of porcine enzootic pneumonia, responsible for major production losses worldwide. The bacteria have a limited metabolism and need to obtain molecules from the growth environment, which causes multiple difficulties for in vitro culture. These limitations have a negative influence on the ability to carry out research for the development of the rational use of antimicrobials and vaccines. The objective of this investigation was to evaluate the genetic profile and in vitro susceptibility of field isolates of M. hyopneumoniae to different antimicrobials. All 16 isolates obtained from the samples presented 100% of identity in the partial sequence of 16S rRNA gene when compared to M. hyopneumoniae. A dendrogram was created using the PCR results of the genes related to pathogenicity, and the isolates were distributed into four clusters, suggesting genetic variability among four different isolates circulating on the same farm. The minimum inhibitory concentration of the isolates was higher for the antimicrobials tylosin (< 0.001-16 mg/L) and spiramycin (< 0.001-16 mg/L) than for enrofloxacin (< 0.001-0.125 mg/L) and tiamulin (< 0.001-0.125 mg/L). Our results demonstrate the genetic variability among M. hyopneumoniae isolates from pigs of the same farm, with differences in their susceptibility to antimicrobial agents.

Mycoplasma hyopneumoniae causes highly contagious swine enzootic pneumonia worldwide. It has been reported that highly diversified M. hyopneumoniae strains exist in different parts of the world. We found p146 gene sequencing analysis, an affordable and simple-to-perform typing method, to be specific and highly sensitive when applied to the molecular typing of 113 M. hyopneumoniae -positive clinical samples directly without culture. The samples were submitted to the Animal Health Laboratory at the University of Guelph (Ontario, Canada) during 2009-2017 from 40 different geographic areas in Ontario. Using a previously described criterion of grouping strains with < 4-bp differences into the same molecular type ( p146 type), the 113 clinical samples clustered into 19 p146 genotypes. Dominant types were found in 2016 and 2017 only, indicating that highly diversified M. hyopneumoniae strains existed in Ontario. Some strains from the same geographic location but different years had the same sequence types, indicating that the same strain types circulate persistently in the field. Different p146 genotypes were also identified from similar geographic locations, indicating that either M. hyopneumoniae strains are prone to mutation or that multiple strains can infect the same or nearby swine production units.

The objective of this study was to determine the influence of a dual respiratory and enteric pathogen challenge on growth performance, carcass composition, and pork quality of high and low feed efficient pigs. Pigs divergently selected for low and high residual feed intake (RFI, ~68 kg) from the 11th generation of Iowa State University RFI project were used to represent high and low feed efficiency. To elicit a dual pathogen challenge, half of the pigs (n = 12/line) were inoculated with Mycoplasma hyopneumoniae (Mh) and Lawsonia intracellularis (MhLI) on days post-inoculation (dpi) 0. Pigs in a separate room of the barn were not inoculated and used as controls (n = 12/RFI line). Pigs were weighed and feed intake was recorded to calculate ADG, ADFI, and G:F for the acclimation period (period 1: dpi -21 to 0), during peak infection (period 2: dpi 0 to 42), and during the remaining growth period to reach market weight (period 3: dpi 42 to harvest). At ~125 kg, pigs were harvested using standard commercial procedures. Carcasses were evaluated for composition (weight, fat free lean, loin eye area, 10th rib fat depth) and meat quality (pH decline, temperature decline, Hunter L, a, and b, subjective color and marbling, star probe, drip loss, cook loss, proximate composition, and desmin degradation). Challenged pigs had lesser ADFI than controls during period 2 (P < 0.05), but had greater ADG and G:F during period 3 (P < 0.05). Selection for feed efficiency did not result in a differential response to MhLI (P > 0.05). Loin chops from the less feed efficient, high RFI pigs, had greater drip loss, greater cook loss, lesser moisture content, greater Hunter L values, and greater Hunter b values (P < 0.05) than loin chops from low RFI pigs. Infection status did not significantly affect carcass composition or pork quality traits (P > 0.05). These results indicate that a MhLI challenge early in growth did not significantly affect ultimate carcass composition or meat quality traits. Selection for greater feed efficiency in pigs did not affect their response to pathogenic challenge., (© The Author(s) 2019. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Mycoplasma hyopneumoniae, the primary pathogenic bacterium causing enzootic pneumonia, significantly affects worldwide swine production. The infection is usually persistent and bacterial identification and isolation of M. hyopneumoniae in clinical samples are challenging due to the fastidious requirements for its growth. Hence, new practical surveillance tools that improve or complement existing diagnostics on M. hyopneumoniae are desirable, especially in early infection. The objective of this study was to identify potential metabolite markers of early M. hyopneumoniae infection in pigs through metabolomics analysis. Samples obtained from pigs in a previous M. hyopneumoniae experimental infection were used in this study. Briefly, two pigs served as mock inoculated controls and ten pigs were intra-tracheally inoculated with M. hyopneumoniae. Sera, laryngeal swabs (LS), and tracheo-bronchial lavage fluid (TBLF) were collected from all pigs at 0, 2, 5, 9, 14, 21 and 28 days post-inoculation (dpi). Bronchial swabs (BS) were collected post-mortem at 28 dpi. Mycoplasma hyopneumoniae infection was confirmed by PCR in LS, TBLF and BS. Serum metabolites were profiled using high-resolution liquid chromatography-mass spectrometry (LC-MS) analysis. Metabolite markers were identified by structural analysis following multivariate analysis of LC-MS data. The results showed that M. hyopneumoniae infection time-dependently altered the serum levels of selective amino acids and fatty acids. α-Aminobutyric acid and long-chain fatty acids were markedly increased at 14 and 21 dpi in inoculated pigs (p < 0.05). These results indicated that M. hyopneumoniae infection caused systemic changes in host metabolism, warranting further studies to determine underlying biochemical and physiological mechanisms responsible for the observed changes.

New vaccine formulations that include novel strains of Mycoplasma hyopneumoniae and innovative adjuvants designed to induce cellular immunity could improve vaccine efficacy against this pathogen. The aim of this experimental study was to assess the efficacy of three experimental bacterin formulations based on M. hyopneumoniae field strain F7.2C which were able to induce cellular immunity. The formulations included a cationic liposome formulation with the Mincle receptor ligand trehalose 6,6-dibehenate (Lipo&#95;DDA:TDB), a squalene-in-water emulsion with Toll-like receptor (TLR) ligands targeting TLR1/2, TLR7/8 and TLR9 (SWE&#95;TLR), and a poly(lactic-co-glycolic acid) micro-particle formulation with the same TLR ligands (PLGA&#95;TLR). Four groups of 12 M. hyopneumoniae-free piglets were primo- (day (D) 0; 39 days of age) and booster vaccinated (D14) intramuscularly with either one of the three experimental bacterin formulations or PBS. The pigs were endotracheally inoculated with a highly and low virulent M. hyopneumoniae strain on D28 and D29, respectively, and euthanized on D56. The main efficacy parameters were: respiratory disease score (RDS; daily), macroscopic lung lesion score (D56) and log copies M. hyopneumoniae DNA determined with qPCR on bronchoalveolar lavage (BAL) fluid (D42, D56). All formulations were able to reduce clinical symptoms, lung lesions and the M. hyopneumoniae DNA load in the lung, with formulation SWE&#95;TLR being the most effective (RDS D28-D56 -61.90%, macroscopic lung lesions -88.38%, M. hyopneumoniae DNA load in BAL fluid (D42) -67.28%). Further experiments raised under field conditions are needed to confirm these results and to assess the effect of the vaccines on performance parameters.

Mycoplasma hyopneumoniae (M. hyopneumoniae) is the pathogen of swine enzootic pneumonia, a chronic respiratory disease affecting pigs of all ages. The ciliated epithelial cells of the respiratory tract are the main target invaded and colonized by M. hyopneumoniae. Therefore, the ideal vaccine would be mucosally administered and able to stimulate suitable mucosal immunity and prevent the adherence of pathogens to mucosal cell surfaces. Currently, Bacillus subtilis as a recombinant vaccine carrier has been used for antigen delivery and proved to be effectively enhancing the innate immunity of nasal mucosa. Here, our study attempts to construct recombinant Bacillus subtilis (B.S-P97R1, B.S-P46), which can express the P97R1 or P46 antigen of M. hyopneumoniae, and to evaluate the immune responses in BALB/c mice. Initially, we respectively successfully constructed recombinant B.S-P97R1, B.S-P46 and validated the expression of antigen proteins by Western analysis. Then, recombinant B.S-P97R1 or B.S-P46 were respectively intranasally (i.n.) immunized in mice. Both strong P97R1-specific and P46-specific immunoglobulin G (IgG), secretory immunoglobulin A (SIgA) antibodies were induced in sera, bronchoalveolar lavage fluids (BALs) by ELISA analysis. Moreover, the levels of specific IL-4, IFN-γ in the immunized mice were elevated, and the proliferation of lymphocytes was also enhanced. In general, intranasal inoculation of recombinant B.S-P97R1 or B.S-P46 resulted in strong mucosal immunity, cell-mediated and humoral immunity, which was a mixed Th1/Th2-type response. In addition, our results provided a potential novel strategy that may be applied to the development of vaccines against M. hyopneumoniae., (© 2019 The Author(s).)

Background: Porcine enzootic pneumonia is a worldwide problem in swine production. The infected host demonstrates a respiratory disease whose etiologic agent is Mycoplasma hyopneumoniae (Mhp). A total of 266 lung samples with Mycoplasma-like lesions were collected from two slaughterhouses. We analyzed the genetic profile of Mhp field samples using 16 genes that encode proteins involved in the mechanisms of bacterial pathogenesis and/or the immune responses of the host. Bioinformatic analyses were performed to classify the Mhp field samples based on their similarity according to the presence of the studied genes., Results: Our results showed variations in the frequency of the 16 studied genes among different Mhp field samples. It was also noted that samples from the same farm were genetically different from each other and samples from different regions could be genetically similar, which is evidence of the presence of different genetic profiles among the Mhp field strains that circulate in Brazilian swine herds., Conclusion: This work demonstrated the genetic diversity of several Mhp field strains based on 16 selected genes related to virulence and/or immune response in Brazil. Our findings demonstrate the difference between Mhp field strains could influence the virulence, and we hypothesize that the most frequent genes in Mhp field strains could possibly be used as vaccine candidates. Based on our results, we suspect that Mhp genetic variability may be associated with the frequency of genes among the field strains and we have demonstrated that some Mhp field samples could not have many important genes described in the literature.

This study investigated the influence of gut microbiome composition in modulating susceptibility to Mycoplasma hyopneumoniae in pigs. Thirty-two conventional M. hyopneumoniae free piglets were randomly selected from six different litters at 3 weeks of age and were experimentally inoculated with M. hyopneumoniae at 8 weeks of age. Lung lesion scores (LS) were recorded 4 weeks post-inoculation (12 weeks of age) from piglet lungs at necropsy. Fecal bacterial community composition of piglets at 3, 8 and 12 weeks of age were targeted by amplifying the V3-V4 region of the 16S rRNA gene. The LS ranged from 0.3 to 43% with an evident clustering of the scores observed in piglets within litters. There were significant differences in species richness and alpha diversity in fecal microbiomes among piglets within litters at different time points (p < 0.05). The dissimilarity matrices indicated that at 3 weeks of age, the fecal microbiota of piglets was more dissimilar compared to those from 8 to 12 weeks of age. Specific groups of bacteria in the gut that might predict the decreased severity of M. hyopneumoniae associated lesions were identified. The microbial shift at 3 weeks of age was observed to be driven by the increase in abundance of the indicator family, Ruminococcaceae in piglets with low LS (p < 0.05). The taxa, Ruminococcus&#95;2 having the highest richness scores, correlated significantly between litters showing stronger associations with the lowest LS (r = -0.49, p = 0.005). These findings suggest that early life gut microbiota can be a potential determinant for M. hyopneumoniae susceptibility in pigs.

Mycoplasma hyopneumoniae and Mycoplasma hyorhinis are two phylogenetically related species colonizing the respiratory tract of pigs but differing in pathogenicity, the basis of which is not well resolved. We hypothesize that genes belonging to the species-specific portion of the genome and being non-essential during ideal laboratory growth conditions encode possible virulent determinants and are the driver of interspecies differences. To investigate this, transposon mutant libraries were generated for both species and a transposon sequencing (Tn-seq) method for mycoplasmas was established to identify non-essential genes. Tn-seq datasets combined with bidirectional Blastp analysis revealed that 101 out of a total 678 coding sequences (CDS) are species-specific and non-essential CDS of M. hyopneumoniae strain F7.2C, while 96 out of a total 751 CDS are species-specific and non-essential CDS in the M. hyorhinis strain JF5820. Among these species-specific and non-essential CDS were genes involved in metabolic pathways. In particular, the myo-inositol and the sialic acid pathways were found to be non-essential and therefore could be considered important to the specific pathogenicity of M. hyopneumoniae and M. hyorhinis, respectively. Such pathways could enable the use of an alternative energy source providing an advantage in their specific niche and might be interesting targets to knock out in order to generate attenuated live vaccines.

In Argentina, enzootic pneumonia (EP) is highly prevalent and different genetic types of Mycoplasma hyopneumoniae have been identified. However, there is a lack of information about prevalence and other epidemiological aspects of EP in Mendoza province. A multiple Locus variable-number tandem repeat analysis (MLVA) targeting P97 R1, P97 R1A and P146 R3 loci was used to assess the genetic diversity of M. hyopneumoniae from clinical specimens recovered from pigs from five herds located in different districts of Mendoza province. M. hyopneumoniae could be typed from 27 bronchoalveolar lavages (BAL) specimens, and eight different MLVA types were identified. This is the first report about diversity of M. hyopneumoniae in Mendoza. Results obtained in this work allow drawing a better picture of the genetic diversity of this pathogen in Argentina., (Copyright © 2018 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

Enolase is an evolutionarily conserved enzyme involved in the processes of glycolysis and gluconeogenesis. Mycoplasma hyopneumoniae belongs to Mycoplasma , whose species are wall-less and among the smallest self-replicating bacteria, and is an important colonizing respiratory pathogen in the pig industry worldwide. Mycoplasma hyopneumoniae enolase ( Mhp Eno) expression is significantly increased after infection and was previously found to be a virulence factor candidate. Our studies show that Mhp Eno is a cell surface-localized protein that can adhere to swine tracheal epithelial cells (STECs). Adhesion to STECs can be specifically inhibited by an Mhp Eno antibody. Mhp Eno can recognize and interact with plasminogen with high affinity. Here, the first crystal structure of the mycoplasmal enolase from Mycoplasma hyopneumoniae was determined. The structure showed unique features of Mhp Eno in the S3/H1, H6/S6, H7/H8, and H13 regions. All of these regions were longer than those of other enolases and were exposed on the Mhp Eno surface, making them accessible to host molecules. These results show that Mhp Eno has specific structural characteristics and acts as a multifunctional adhesin on the Mycoplasma hyopneumoniae cell surface.

Lawsonia intracellularis (LI) and Mycoplasma hyopneumoniae (Mh) are 2 globally distributed pathogens that cause significant morbidity and mortality in grow-finish pigs. However, mechanisms that reduce growth and feed efficiency during LI and Mh infection are poorly defined. We hypothesized that reductions in performance are partially due to declines in intestinal function and integrity; thus, this study aimed to evaluate intestinal function and integrity of pigs during a 21-d Mh and LI dual challenge (MhLI). Littermate pairs of barrows (48.1 ± 6.7 kg BW) were selected; 1 pig from each pair was assigned to either MhLI challenge or nonchallenge treatments (n = 12). Pigs were individually housed, fed a corn-soybean diet, and allowed to acclimate for 21 d prior to inoculation. On days postinoculation (dpi) 0, MhLI pigs were dual inoculated with LI and Mh. On dpi 21, all pigs were euthanized for ileal and colon tissue collection. Formalin-fixed tissues were clinically scored and morphology analyzed, frozen tissues assayed for digestive enzyme activities, and fresh tissues mounted into modified Ussing Chambers to assess active nutrient transport, barrier integrity, and bacterial translocation. Data were analyzed using the Mixed Procedure of SAS with treatment as a fixed effect, age and start BW as covariates, and litter as a random effect. Compared with controls, MhLI pigs had decreased ADG (38%, P < 0.001), ADFI (25%, P < 0.001), and G:F (19%, P = 0.012). The MhLI dual challenge did not alter ileum morphology or transepithelial resistance (P > 0.10); however, ex vivo mucosal to serosal translocation of S. Typhimurium in the colon was increased (60%, P = 0.003) in MhLI pigs compared with controls. Additionally, MhLI pigs had increased ileal glucose transport (30%, P = 0.05) and decreased sucrase activity (30%, P = 0.049) compared with controls. This MhLI challenge antagonized intestinal function and integrity, and this may be a contributing factor to reduced pig performance., (© The Author(s) 2019. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Enzootic pneumonia incurs major economic losses to pork production globally. The primary pathogen and causative agent, Mycoplasma hyopneumoniae, colonises ciliated epithelium and disrupts mucociliary function predisposing the upper respiratory tract to secondary pathogens. Alleviation of disease is reliant on antibiotics, vaccination, and sound animal husbandry, but none are effective at eliminating M. hyopneumoniae from large production systems. Sustainable pork production systems strive to lower reliance on antibiotics but lack of a detailed understanding of the pathobiology of M. hyopneumoniae has curtailed efforts to develop effective mitigation strategies. M. hyopneumoniae is considered an extracellular pathogen. Here we show that M. hyopneumoniae associates with integrin β1 on the surface of epithelial cells via interactions with surface-bound fibronectin and initiates signalling events that stimulate pathogen uptake into clathrin-coated vesicles (CCVs) and caveosomes. These early events allow M. hyopneumoniae to exploit an intracellular lifestyle by commandeering the endosomal pathway. Specifically, we show: (i) using a modified gentamicin protection assay that approximately 8% of M. hyopneumoniae cells reside intracellularly; (ii) integrin β1 expression specifically co-localises with the deposition of fibronectin precisely where M. hyopneumoniae cells assemble extracellularly; (iii) anti-integrin β1 antibodies block entry of M. hyopneumoniae into porcine cells; and (iv) M. hyopneumoniae survives phagolysosomal fusion, and resides within recycling endosomes that are trafficked to the cell membrane. Our data creates a paradigm shift by challenging the long-held view that M. hyopneumoniae is a strict extracellular pathogen and calls for in vivo studies to determine if M. hyopneumoniae can traffic to extrapulmonary sites in commercially-reared pigs.

Mycoplasma hyopneumoniae is an important respiratory pathogen that causes great economic losses to the pig industry worldwide. Although some putative virulence factors have been reported, pathogenesis remains poorly understood. Herein, we evaluated the relative abundance of proteins in virulent 168 (F107) and attenuated 168L (F380) M. hyopneumoniae strains to identify virulence-associated factors by two-dimensional electrophoresis (2-DE). Seven proteins were found to be ≥ 1.5-fold more abundant in 168, and protein-protein interaction network analysis revealed that all seven interact with putative virulence factors. Unexpectedly, six of these virulence-associated proteins are encoded by core rather than accessory genomic elements. The most differentially abundant of the seven, fructose-1,6-bisphosphate aldolase (FBA), was successfully cloned, expressed and purified. Flow cytometry demonstrated the surface localisation of FBA, recombinant FBA (rFBA) mediated adhesion to swine tracheal epithelial cells (STEC), and anti-rFBA sera decreased adherence to STEC. Surface plasmon resonance showed that rFBA bound to fibronectin with a moderately strong K D of 469 nM. The results demonstrate that core gene expression contributes to adhesion and virulence in M. hyopneumoniae, and FBA moonlights as an important adhesin, mediating binding to host cells via fibronectin.

Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia. In our previous work, we reconstructed the metabolic models of this species along with two other mycoplasmas from the respiratory tract of swine: Mycoplasma hyorhinis, considered less pathogenic but which nonetheless causes disease and Mycoplasma flocculare, a commensal bacterium. We identified metabolic differences that partially explained their different levels of pathogenicity. One important trait was the production of hydrogen peroxide from the glycerol metabolism only in the pathogenic species. Another important feature was a pathway for the metabolism of myo-inositol in M. hyopneumoniae. Here, we tested these traits to understand their relation to the different levels of pathogenicity, comparing not only the species but also pathogenic and attenuated strains of M. hyopneumoniae. Regarding the myo-inositol metabolism, we show that only M. hyopneumoniae assimilated this carbohydrate and remained viable when myo-inositol was the primary energy source. Strikingly, only the two pathogenic strains of M. hyopneumoniae produced hydrogen peroxide in complex medium. We also show that this production was dependent on the presence of glycerol. Although further functional tests are needed, we present in this work two interesting metabolic traits of M. hyopneumoniae that might be directly related to its enhanced virulence., (© 2018 John Wiley & Sons Ltd.)

Information on the in vitro growth of pathogenic and non-pathogenic Mycoplasma hyopneumoniae (M. hyopneumoniae) strains is scarce and controversial. Despite its limitations, the colour changing units (CCU) assay is still considered the golden standard titration technique for M. hyopneumoniae culture. Thus, the aims of the present study were: (1) to describe the growth dynamics and kinetics of pathogenic and non-pathogenic M. hyopneumoniae strains, and (2) to monitor the strains' daily growth by ATP luminometry, CCU, colony forming units (CFU), and DNA quantification by real time quantitative PCR (qPCR) and by fluorescent double-stranded DNA (dsDNA) staining, to evaluate them as putative titration methodologies. The growth of the non-pathogenic J (ATCC ® 25934™) type strain and the pathogenic 11 (ATCC ® 25095™) reference strain and 232 strain was modelled by the Gompertz model. Globally, all three-strain cultures showed the same growing phases as well as similar maximal titres within a particular technique, but for CFU. However, the J strain displayed the fastest growing. During the logarithmic phase of growing, CCU, ATP and M. hyopneumoniae copy titres were strongly and linearly associated, and correlation between techniques could be reliably established. In conclusion, real-time culture titration by means of ATP or molecular assays was useful to describe the in vitro growth of the tested strains. Knowledge about the in vitro growth behaviour of a specific strain in a specific medium may provide several advantages, including information about the time required to reach maximal titres by the culture. Noteworthy, the obtained results refers to the three strains used, so extrapolation to other M. hyopneumoniae strains or culture conditions should be made cautiously.

The objective of this study was to compare clinical, microbiologic, immunologic, and pathologic parameters in pigs each concurrently administered porcine reproductive and respiratory syndrome virus (PRRSV), Mycoplasma hyopneumoniae, and porcine circovirus type 2 (PCV2) vaccine from 1 of 2 commercial sources at 21 days of age and challenged with field strains of each of the 3 pathogens. Pigs were challenged with PRRSV and M. hyopneumoniae at 42 days of age (-14 days post-challenge, dpc) followed by a challenge with PCV2 at 56 days of age (0 dpc). Significant differences were observed between vaccinated challenged and unvaccinated challenged groups in clinical (average daily gain and clinical signs), microbiologic (viremia and nasal shedding), immunologic (antibodies and interferon-γ secreting cells), and pathologic (lesions) outcomes. Significant differences were observed among the 3 vaccinated challenged groups in microbiologic (nasal shedding of M. hyopneumoniae and viremia of PCV2) and immunologic ( M. hyopneumoniae - and PCV2-specific interferon-γ secreting cells) outcomes. The vaccination regimen for PRRSV vaccine, M. hyopneumoniae vaccine, and PCV2 vaccine is efficacious for controlling triple challenge with PRRSV, M. hyopneumoniae, and PCV2 from weaning to finishing period.

Mycoplasma hyopneumoniae and Mycoplasma flocculare are genetically similar bacteria, which coinhabit the porcine respiratory tract. These mycoplasmas share most of the known virulence factors, but, while M. hyopneumoniae causes porcine enzootic pneumonia (PEP), M. flocculare is a commensal species. To identify potential PEP determinants and provide novel insights on mycoplasma-host interactions, the whole cell proteomes of two M. hyopneumoniae strains, one pathogenic (7448) and other non-pathogenic (J), and M. flocculare were compared. A cell fractioning approach combined with mass spectrometry (LC-MS/MS) proteomics was used to analyze cytoplasmic and surface-enriched protein fractions. Average detection of ~ 50% of the predicted proteomes of M. hyopneumoniae 7448 and J, and M. flocculare was achieved. Many of the identified proteins were differentially represented in M. hyopneumoniae 7448 in comparison to M. hyopneumoniae J and M. flocculare, including potential PEP determinants, such as adhesins, proteases, and redox-balancing proteins, among others. The LC-MS/MS data also provided experimental validation for several genes previously regarded as hypothetical for all analyzed mycoplasmas, including some coding for proteins bearing virulence-related functional domains. The comprehensive proteome profiling of two M. hyopneumoniae strains and M. flocculare provided tens of novel candidates to PEP determinants or virulence factors, beyond those classically described.

The study was aimed to investigate biofilm forming ability of Mycoplasma hyopneumoniae and to determine the minimum biofilm eradication concentrations of antibiotics. Biofilm forming ability of six strains of M. hyopneumoniae was examined using crystal violet staining on coverslips. The results demonstrated an apparent line of biofilm growth in 3 of the strains isolated from swine with confirmed cases of enzootic pneumonia. BacLight bacterial viability assay revealed that the majority of the cells were viable after 336 hr of incubation. Moreover, M. hyopneumoniae persists in the biofilm after being exposed to 10 fold higher concentration of antibiotics than the minimum inhibitory concentrations in planktonic cells. To the best of our knowledge, this is the first report of biofilm formation in M. hyopneumoniae. However, comprehensive studies on the mechanisms of biofilm formation are needed to combat swine enzootic pneumonia caused by resistant M. hyopneumoniae.

Pneumocystis carinii f. sp. suis (PCS) nucleic acid and antibody profiles on two Austrian-farrow-to-finish farms were investigated. Furthermore, associations with other respiratory pathogens were evaluated. Respiratory specimen and sera from pigs of five age classes between the 1st week and the 3rd month of life as well as samples from sows were analyzed. On Farm A, PCS infection occurred early in life. The suckling piglets were already infected in the 1st week of life and the pigs remained positive until the 3rd month of life. On Farm B, pigs were infected later, between 3 and 4 months of age. The maximum PCS nucleic acid load on Farm A was 8.3 log10 genome copies/mL BALF, whereas on Farm B the PCS burden was significantly lower, with 4.0 log10 genome copies/mL BALF. Anti-PCS antibodies were detected in sows, as maternal antibodies in suckling piglets and as an immunological reaction to infection. On both farms, PCS infection was accompanied by several co-infections. On Farm A, there were concurrent infections with PRRSV, a virulent strain of Haemophilus parasuis, and Mycoplasma hyopneumoniae. On Farm B, PCS was accompanied by infections with swine influenza virus, Mycoplasma hyopneumoniae, and a non-virulent strain of Haemophilus parasuis. The results clearly show that the PCS profiles can vary between farms. Younger pigs may be more susceptible as they had higher PCS burdens. It is possible that PCS may contribute to a respiratory disease in pigs and further investigation of its potential role is warranted.

Background: Mycoplasma hyopneumoniae (Mhp) causes porcine enzootic pneumonia, a disease that cause major economic losses in the pig industry. Dendritic cells (DCs), the most effective antigen-presenting cells, are widely distributed beneath respiratory epithelium, DCs uptake and present antigens to T cells, to initiate protective immune responses in different infections. In this study, we investigated the role of porcine DCs in vaccine Mhp-168 exposure., Results: The antigen presenting ability of DCs were improved by vaccine Mhp-168 exposure. DCs could activate T-cell proliferation by up-regulating the antigen presenting molecule MHCII expression and co-stimulatory molecule CD80/86. However, the up-regulation of IL-10 and accompany with down-regulation of IFN-γ gene level may account for the limitation of attenuated Mhp-168 strain use as vaccine alone., Conclusion: These findings are benefit for exploring the protection mechanisms and the possible limitations of this attenuated Mhp-168 vaccine.

One of the main Mycoplasma hyopneumoniae (M. hyopneumoniae) swine experimental model objectives is to reproduce mycoplasmal pneumonia (MP). Unfortunately, experimental validated protocols to maximize the chance to successfully achieve lung lesions induced by M. hyopneumoniae are not available at the moment. Thus, the objective of this work was to identify those factors that might have a major influence on the effective development of MP, measured as macroscopic lung lesions, under experimental conditions. Data from 85 studies describing M. hyopneumoniae inoculation experiments were compiled by means of a systematic review and analyzed thereafter. Several variables were considered in the analyses such as the number of pigs in the experiment, serological status against M. hyopneumoniae, source of the animals, age at inoculation, type of inoculum, strain of M. hyopneumoniae, route, dose and times of inoculation, study duration and co-infection with other swine pathogens. Descriptive statistics were used to depict M. hyopneumoniae experimental model main characteristics whereas a recursive partitioning approach, using regression trees, assessed the importance of the abovementioned experimental variables as MP triggering factors. A strong link between the time period between challenge and necropsies and lung lesion severity was observed. Results indicated that the most important factors to explain the observed lung lesion score variability were: (1) study duration, (2) M. hyopneumoniae strain, (3) age at inoculation, (4) co-infection with other swine pathogens and (5) animal source. All other studied variables were not relevant to explain the variability on M. hyopneumoniae lung lesions. The results provided in the present work may serve as a basis for debate in the search for a universally accepted M. hyopneumoniae challenge model.

Transcriptional regulation, a multiple-step process, is still poorly understood in the important pig pathogen Mycoplasma hyopneumoniae. Basic motifs like promoters and terminators have already been described, but no other cis-regulatory elements have been found. DNA repeat sequences have been shown to be an interesting potential source of cis-regulatory elements. In this work, a genome-wide search for tandem and palindromic repetitive elements was performed in the intergenic regions of all coding sequences from M. hyopneumoniae strain 7448. Computational analysis demonstrated the presence of 144 tandem repeats and 1,171 palindromic elements. The DNA repeat sequences were distributed within the 5' upstream regions of 86% of transcriptional units of M. hyopneumoniae strain 7448. Comparative analysis between distinct repetitive sequences found in related mycoplasma genomes demonstrated different percentages of conservation among pathogenic and nonpathogenic strains. qPCR assays revealed differential expression among genes showing variable numbers of repetitive elements. In addition, repeats found in 206 genes already described to be differentially regulated under different culture conditions of M. hyopneumoniae strain 232 showed almost 80% conservation in relation to M. hyopneumoniae strain 7448 repeats. Altogether, these findings suggest a potential regulatory role of tandem and palindromic DNA repeats in the M. hyopneumoniae transcriptional profile., Competing Interests: The authors have declared that no competing interests exist.

In pigs, influenza A viruses and Mycoplasma hyopneumoniae (Mhp) are major contributors to the porcine respiratory disease complex. Pre-infection with Mhp was previously shown experimentally to exacerbate the clinical outcomes of H1N1 infection during the first week after virus inoculation. In order to better understand the interactions between these pathogens, we aimed to assess very early responses (at 5, 24 and 48 h) after H1N1 infection in pigs pre-infected or not with Mhp. Clinical signs and macroscopic lung lesions were similar in both infected groups at early times post-H1N1 infection; and Mhp pre-infection affected neither the influenza virus replication nor the IFN-induced antiviral responses in the lung. However, it predisposed the animals to a higher inflammatory response to H1N1 infection, as revealed by the massive infiltration of neutrophils and macrophages into the lungs and the increased production of pro-inflammatory cytokines (IL-6, IL-1β and TNF-α). Thus, it seems it is this marked inflammatory state that would play a role in exacerbating the clinical signs subsequent to H1N1 infection.

Mycoplasma hyopneumoniae causes porcine enzootic pneumonia, an economically important disease of swine. A more sensitive and reliable method for detection of serum antibodies is needed for epidemiological investigations and to evaluate the effect of immunization. We expressed the M. hyopneumoniae protein P65 in Escherichia coli and produced a monoclonal antibody (mAb) that bound specifically to recombinant P65. Using this mAb, a blocking enzyme linked immunosorbent assay (ELISA) was developed. The blocking ELISA had similar specificity to and sensitivity with the commercial ELISA produced by IDEXX. Thus, this blocking ELISA is a useful test for serological confirmation of M. hyopneumoniae infection.

Background: The respiratory tract of swine is colonized by several bacteria among which are three Mycoplasma species: Mycoplasma flocculare, Mycoplasma hyopneumoniae and Mycoplasma hyorhinis. While colonization by M. flocculare is virtually asymptomatic, M. hyopneumoniae is the causative agent of enzootic pneumonia and M. hyorhinis is present in cases of pneumonia, polyserositis and arthritis. The genomic resemblance among these three Mycoplasma species combined with their different levels of pathogenicity is an indication that they have unknown mechanisms of virulence and differential expression, as for most mycoplasmas., Methods: In this work, we performed whole-genome metabolic network reconstructions for these three mycoplasmas. Cultivation tests and metabolomic experiments through nuclear magnetic resonance spectroscopy (NMR) were also performed to acquire experimental data and further refine the models reconstructed in silico., Results: Even though the refined models have similar metabolic capabilities, interesting differences include a wider range of carbohydrate uptake in M. hyorhinis, which in turn may also explain why this species is a widely contaminant in cell cultures. In addition, the myo-inositol catabolism is exclusive to M. hyopneumoniae and may be an important trait for virulence. However, the most important difference seems to be related to glycerol conversion to dihydroxyacetone-phosphate, which produces toxic hydrogen peroxide. This activity, missing only in M. flocculare, may be directly involved in cytotoxicity, as already described for two lung pathogenic mycoplasmas, namely Mycoplasma pneumoniae in human and Mycoplasma mycoides subsp. mycoides in ruminants. Metabolomic data suggest that even though these mycoplasmas are extremely similar in terms of genome and metabolism, distinct products and reaction rates may be the result of differential expression throughout the species., Conclusions: We were able to infer from the reconstructed networks that the lack of pathogenicity of M. flocculare if compared to the highly pathogenic M. hyopneumoniae may be related to its incapacity to produce cytotoxic hydrogen peroxide. Moreover, the ability of M. hyorhinis to grow in diverse sites and even in different hosts may be a reflection of its enhanced and wider carbohydrate uptake. Altogether, the metabolic differences highlighted in silico and in vitro provide important insights to the different levels of pathogenicity observed in each of the studied species.

The purpose of this study was to assess the effect of three different inoculation routes into mycoplasmal pneumonia (MP) in pigs challenged with Mycoplasma hyopneumoniae (M. hyopneumoniae). Thirty six-week-old M. hyopneumoniae seronegative piglets were randomly assigned to four groups: three challenged groups with experimentally inoculated pigs by either the endotracheal (ET; n = 8), intranasal (IN; n = 8) or aerosol (AE; n = 8) routes and one uninfected group (Control; n = 6). Blood samples were collected 1 day before challenge and at necropsy, 28 days post-inoculation (dpi), to assess seroconversion. Laryngeal swabs were collected at -1, 7, 14, 21 and 28 dpi in order to evaluate colonization. At necropsy, lung lesions were scored and lung tissue was collected for histopathological studies and M. hyopneumoniae DNA detection. Broncho-alveolar lavage fluid (BALF) was also obtained to detect M. hyopneumoniae DNA, specific IgA antibodies and cytokines. MP was observed in all inoculated groups, but the ET group displayed a significantly higher number of animals affected by MP as well as a higher mean lung lesion score. These results were paralleled with an earlier seroconversion and upper respiratory tract colonization of M. hyopneumoniae. Additionally, in the ET group, higher levels of pro-inflammatory cytokines and specific IgA antibodies in BALF were found. Under the conditions of the present study, MP was reproduced by the three evaluated inoculation routes. Obtained results suggest that the ET route is the most effective in order to induce MP in pigs experimentally challenged with M. hyopneumoniae.

The objective of our study was to characterize the Mycoplasma hyopneumoniae genetic diversity within a swine operation comingling weaned pigs. Bronchial swabs and tracheal aspirates were collected from 3 nursery-to-finish farms. During the finishing production stages, samples were obtained from mortalities and from live coughing pigs in rooms where mortality was not observed. A total of 105 samples were examined by a M. hyopneumoniae real-time polymerase chain reaction and subjected to genetic typing using a multilocus variable number tandem repeat analysis (MLVA) assay. The MLVA was used to identify genetic variants based on the number of repeats in 2 variable number tandem repeats loci, namely P97 and P146, thought to mediate adherence of M. hyopneumoniae to swine cilia. Four distinguishable M. hyopneumoniae variants were identified: MVLA variants 9-15, 11-21, 9-21, and 7-15. Variant 9-15 was the most prevalent, observed in 79% of rooms, and detected on all 3 farms. Variant 11-21 was present in 37% of the rooms on 2 of the 3 farms. Only one 9-21 variant was identified in 1 farm, and all samples of variant 7-15 were recovered from another farm. Based on the low prevalence and limited geographic distribution of the last 2 variants, it is hypothesized that they might be the result of in-situ recombination. All variants detected in this investigation appeared to belong to 3 clusters. Overall, a limited number of variants and clusters were identified in a system that comingles pigs from different sources, suggesting limited M. hyopneumoniae genetic variation within commercial swine production environments., (© 2016 The Author(s).)

Background: The objective of this study was to elucidate the pathogenic mechanisms of how Mycoplasma hyopneumoniae enhances secondary Pasteurella multocida type A infection which leads to porcine enzootic pneumonia in infected pigs. Sixteen pigs were experimentally infected with M. hyopneumoniae and then euthanized at 7, 14, 21 and 28 days post inoculation. In situ hybridization for M. hyopneumoniae DNA and Ulex europaeus agglutinin-I (UEA-I) lectin histochemistry for fucosyl glycoconjugate, was performed in serial lung sections to determine alteration of fucosyl glycoconjugate in M. hyopneumoniae-infected bronchial and bronchiolar epithelium. Bacterial overlay assay was performed to determine the affinity of P. multocida type A with L-fucose., Results: The luminal surface of bronchial and bronchiolar epithelial cells that were stained with UEA-I always showed hybridization signals for M. hyopneumoniae but it was negative in the unaffected parts of the lung from M. hyopneumoniae-infected pigs and in lung from negative control pigs. Colocalization of M. hyopneumoniae and UEA-I was especially prominent in the luminal surface of bronchial and bronchiolar epithelial cells in serial section of lung. The mean number of M. hyopneumoniae-positive cells correlated with the mean number of UEA-I-positive cells in lungs from infected pigs throughout the experiment. All eight P. multocida type A isolates from naturally occurring enzootic pneumonia, bound strongly at levels of 2 μg and 5 μg of L-fucose., Conclusions: The results of the present study demonstrate that M. hyopneumoniae increases the L-fucose composition to enhance adherence of P. multocida type A to the bronchial and bronchiolar epithelial cells.

The swine industry currently lacks validated antemortem methods of detecting baseline herd prevalence of Mycoplasma hyopneumoniae. The focus of our study was to evaluate alternative antemortem detection techniques and to determine baseline litter prevalence in preweaned pig populations utilizing the selected technique and a real-time polymerase chain reaction (qPCR) assay. Preliminary data was analyzed on weaned piglets with evidence of respiratory disease (n = 32). Five sample types (antemortem nasal swab, tracheobronchial mucus, postmortem deep airway swab, bronchoalveolar lavage, and lung tissue) were collected from each pig. Individual samples were tested for M. hyopneumoniae using qPCR. Compared to nasal swabs, tracheobronchial mucus demonstrated higher test sensitivity (P < 0.0001). Tracheobronchial mucus was collected from apparently healthy preweaned piglets (n = 1,759; sow farms 1-3) and preweaned piglets exhibiting signs of respiratory disease (n = 32; sow farm 4), ranging in age from 12 to 25 days. Samples from sow farms 1-3 were pooled into 2 groups of 5 per litter (n = 360 pools from 180 litters), and qPCR was utilized to analyze each pool. A qPCR-positive result, threshold cycle <37, from either pool designated the litter positive for M. hyopneumoniae. Two out of 180 litters revealed a positive result (1.1%). Individual qPCR assays were run on the samples collected from sow farm 4. Five out of 30 samples revealed a positive result (16.7%). Tracheobronchial mucus collection in combination with qPCR is a sensitive antemortem sampling technique that can be used to estimate the prevalence of M. hyopneumoniae in preweaned pigs, thus providing insight into the infection dynamics across the entire farrow-to-finish process., (© 2015 The Author(s).)

The Jiangquhai porcine lean strain (JQHPL) is a new pork meat-type strain that has been developed in recent years from the parent lines Duroc, Fengjing, and Jiangquhai pigs (DurocxFengjing pigxJiangquhai pig). Enzootic pneumonia (EP) in pigs induced by Mycoplasma hyopneumoniae (M. hyopneumoniae) is a chronic respiratory disease of pigs, generating high economic losses in the swine industry. Here, we investigated the degree of resistance to M. hyopneumoniae for the Jiangquhai porcine lean strain and the Duroc x Landrace x Yorkshire (DLY) pigs, which are Western commercial pigs that have been introduced in China. A total of 209 DLY piglets and 221 JQHPL piglets from 19 Landrace x Yorkshire and 22 JQHPL M. hyopneumoniae positive gestating sows with different expected dates of confinement were selected and raised in the same M. hyopneumoniae positive farrowing barn. When the oldest suckling piglets were 37 days old, nasal swabs were collected from all the piglets (ranging from 4 to 37 days old) to detect the M. hyopneumoniae pathogen using n-PCR and M. hyopneumoniae specific SIgA using ELISA. Positive M. hyopneumoniae infection rates in both the strains increased with age; however, positive rates for JQHPL were lower compared to DLY at 14 to 35 days old. The level of the specific SIgA rose rapidly in JQHPL respiratory tracts, particularly in piglets 21 to 35 days in age compared to DLY piglets of the same age; however, the level of the specific SIgA in DLY also marginally increased. In conclusion, JQHPL pigs exhibits higher resistance to M. hyopneumoniae compared to DLY. It is possible that this characteristic is caused by the faster and stronger mucosal immunity phenotype of the JQHPL strain.

Mycoplasma hyopneumoniae is the cause of enzootic pneumonia in pigs, a chronic respiratory disease associated with significant economic losses to swine producers worldwide. The molecular pathogenesis of infection is poorly understood due to the lack of genetic tools to allow manipulation of the organism and more generally for the Mycoplasma genus. The objective of this study was to develop a system for generating random transposon insertion mutants in M. hyopneumoniae that could prove a powerful tool in enabling the pathogenesis of infection to be unraveled. A novel delivery vector was constructed containing a hyperactive C9 mutant of the Himar1 transposase along with a mini transposon containing the tetracycline resistance cassette, tetM. M. hyopneumoniae strain 232 was electroporated with the construct and tetM-expressing transformants selected on agar containing tetracycline. Individual transformants contained single transposon insertions that were stable upon serial passages in broth medium. The insertion sites of 44 individual transformants were determined and confirmed disruption of several M. hyopneumoniae genes. A large pool of over 10 000 mutants was generated that should allow saturation of the M. hyopneumoniae strain 232 genome. This is the first time that transposon mutagenesis has been demonstrated in this important pathogen and could be generally applied for other Mycoplasma species that are intractable to genetic manipulation. The ability to generate random mutant libraries is a powerful tool in the further study of the pathogenesis of this important swine pathogen.

Mycoplasma hyopneumoniae has a primary role in the porcine respiratory disease complex (PRDC). The objective of this study was to determine whether fumonisin mycotoxins influence the character and/or the severity of pathological processes induced in the lungs of pigs by Mycoplasma hyopneumoniae. Four groups of pigs (n = 7/group) were used, one fed 20 ppm fumonisin B1 (FB1) from 16 days of age (group F), one only infected with M. hyopneumoniae on study day 30 (group M), and a group fed FB1 and infected with M. hyopneumoniae (group MF), along with an untreated control group (group C). Computed tomography (CT) scans of infected pigs (M and MF) on study day 44 demonstrated lesions extending to the cranial and middle or in the cranial third of the caudal lobe of the lungs. The CT images obtained on study day 58 showed similar but milder lesions in 5 animals from group M, whereas lungs from 2 pigs in group MF appeared progressively worse. The evolution of average pulmonary density calculated from combined pixel frequency values, as measured by quantitative CT, was significantly influenced by the treatment and the age of the animals. The most characteristic histopathologic lesion in FB1-treated pigs was pulmonary edema, whereas the pathomorphological changes in Mycoplasma-infected pigs were consistent with catarrhal bronchointerstitial pneumonia. FB1 aggravated the progression of infection, as demonstrated by severe illness requiring euthanasia observed in 1 pig and evidence of progressive pathology in 2 pigs (group MF) between study days 44 and 58.

Mycoplasma hyopneumoniae is a prevalent swine respiratory pathogen that is a major cause of economic loss to pig producers. Control is achieved by a combination of antimicrobials, vaccination and management practices, but current vaccines offer only partial control and there is a need for improved preventative strategies. A major barrier to advances in understanding the pathogenesis of M. hyopneumoniae and in developing new vaccines is the lack of tools to genetically manipulate the organism. We describe the development and optimisation of the first successful plasmid-based system for the genetic manipulation of M. hyopneumoniae. Our artificial plasmids contain the origin of replication (oriC) of M. hyopneumoniae along with tetM, conferring resistance to tetracycline. With these plasmids, we have successfully transformed M. hyopneumoniae strain 232 by electroporation, generating tetracycline resistant organisms. The persistence of extrachromosomal plasmid and maintenance of plasmid DNA over serial passages shows that these artificial plasmids are capable of self-replication in M. hyopneumoniae. In addition to demonstrating the amenability of M. hyopneumoniae to genetic manipulation and in optimising the conditions necessary for successful transformation, we have used this system to determine the minimum functional oriC of M. hyopneumoniae. In doing so, we have developed a plasmid with a small oriC that is stably maintained over multiple passages that may be useful in generating targeted gene disruptions. In conclusion, we have generated a set of plasmids that will be valuable in studies of M. hyopneumoniae pathogenesis and provide a major step forward in the study of this important swine pathogen.

Enzootic pneumonia, resulting from infection with Mycoplasma hyopneumoniae, is of considerable economic importance to the pig industry and normally is controlled through active vaccination of piglets. We have demonstrated that administration of an inactivated Mycoplasma hyopneumoniae vaccine to piglets less than 1 week old is efficacious under field conditions and reduces the level of lung lesions observed in comparison to that in control pigs. Here, the results of two separate studies, one in piglets with and the second one in piglets without maternal antibodies, conducted to satisfy the requirements of the European Pharmacopoeia (monograph no. 07/2009:2448), are reported. Piglets received either minimal titer Suvaxyn MH-One or saline at less than 1 week of age and were challenged with Mycoplasma hyopneumoniae 2 weeks later. The number of lung lesions was recorded 4 weeks after challenge, and bronchial swab and lung tissue specimens were analyzed for quantification of Mycoplasma hyopneumoniae DNA. In the presence and absence of maternal antibodies, vaccination of piglets at less than 1 week of age was efficacious, with vaccinated piglets having significantly lower percentages of lung with lesions and lower Mycoplasma hyopneumoniae counts detected in bronchial swab and lung tissue specimens at necropsy. In conclusion, the vaccination of piglets at 1 week of age with Suvaxyn MH-One is efficacious in the presence of high levels of maternal antibodies.