Incomplete autophagy promotes the proliferation of Mycoplasma hyopneumoniae through the JNK and Akt pathways in porcine alveolar macrophages.

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.

Development of an indirect competitive enzyme linked immunosorbent assay for the quantitative detection of Mycoplasma hyopneumoniae during the vaccine production process.

Inactivated Mycoplasma hyopneumoniae vaccine is used extensively to control M. hyopneumoniae infection worldwide. Quantification techniques are essential in the process of standardizing and validating vaccines. In this study, we developed and optimized an indirect competitive enzyme linked immunosorbent assay (ic-ELISA) for the rapid quantification of M. hyopneumoniae antigen during vaccine production. Briefly, whole M. hyopneumoniae antigen was coated onto microtiter plates, and a polyclonal antibody against M. hyopneumoniae recombinant elongation factor thermo unstable (EF-Tu) protein was prepared and added with the samples to be tested. The methods were optimized and showed significant reproducibility, with coefficients of variation of 4.01% and 6.14% for the intra-and inter-assays, respectively. Quantification of M. hyopneumoniae cultures at different growth stages using the ic-ELISA test showed a similar curve to that of the traditional color changing units (CCU) assay, with a delay in the time when the amount reached the peak and started to fall. In the inactivated vaccine production process, the cultures could be harvested later than that for the live vaccine, at about 12 h after the end of the logarithmic growth phase. Different batches of cultures were measured for their relative potency value compared with the in-house reference vaccine, which was used to determine whether the cultures met the antigen amount requirements for vaccine preparation. The curves of the CCU titer and ic-ELISA titer in the logarithmic phase correlated strongly and a linear regression equation was established to calculate the CCU values rapidly using the ic-ELISA results. In conclusion, an ic-ELISA method was established to rapidly assess the amount of antigen in an M. hyopneumoniae culture during the vaccine production process.

Incomplete autophagy promotes the replication of Mycoplasma hyopneumoniae.

Autophagy is an important cellular homeostatic mechanism for recycling of degradative proteins and damaged organelles. Autophagy has been shown to play an important role in cellular responses to bacteria and bacterial replication. However, the role of autophagy in Mycoplasma hyopneumoniae infection and the pathogenic mechanism is not well characterized. In this study, we showed that M. hyopneumoniae infection significantly increases the number of autophagic vacuoles in host cells. Further, we found significantly enhanced expressions of autophagy marker proteins (LC3-II, ATG5, and Beclin 1) in M. hyopneumoniae-infected cells. Moreover, immunofluorescence analysis showed colocalization of P97 protein with LC3 during M. hyopneumoniae infection. Interestingly, autophagic flux marker, p62, accumulated with the induction of infection. Conversely, the levels of p62 and LC3-II were decreased after treatment with 3-MA, inhibiting the formation of autophagosomes, during infection. In addition, accumulation of autophagosomes promoted the expression of P97 protein and the survival of M. hyopneumoniae in PK-15 cells, as the replication of M. hyopneumoniae was down-regulated by adding 3-MA. Collectively, these findings provide strong evidence that M. hyopneumoniae induces incomplete autophagy, which in turn enhances its reproduction in host cells. These findings provide novel insights into the interaction of M. hyopneumoniae and host.

Transfer of Mycoplasma hyopneumoniae-specific cell mediated immunity to neonatal piglets.

Mycoplasma hyopneumoniae is the primary agent of enzootic pneumonia in pigs. Although cell mediated immunity (CMI) may play a role in protection against M. hyopneumoniae, its transfer from sows to their offspring is poorly characterized. Therefore, maternally-derived CMI was studied in piglets from vaccinated and non-vaccinated sows. The potential influence of cross-fostering before colostrum ingestion on the transfer of CMI from dam to piglets was also investigated. Six M. hyopneumoniae vaccinated sows from an endemically infected herd and 47 of their piglets, of which 24 piglets were cross-fostered, were included, as well as three non-vaccinated control sows from an M. hyopneumoniae-free herd and 24 of their piglets. Vaccinated sows received a commercial bacterin intramuscularly at 6 and 3 weeks prior to farrowing. The TNF-α, IFN-γ and IL-17A production by different T-cell subsets in blood of sows, colostrum and blood of piglets was assessed using a recall assay. In blood of sows cytokine producing T-cells were increased upon M. hyopneumoniae vaccination. Similarly, M. hyopneumoniae-specific T-cells were detected in blood of 2-day-old piglets born from these vaccinated sows. In contrast, no M. hyopneumoniae-specific cytokine producing T-cells were found in blood of piglets from control sows. No difference was found in M. hyopneumoniae-specific CMI between cross-fostered and non-cross-fostered piglets. In conclusion, different M. hyopneumoniae-specific T-cell subsets are transferred from the sow to the offspring. Further studies are required to investigate the role of these transferred cells on immune responses in piglets and their potential protective effect against M. hyopneumoniae infections.

Identification of a novel TLR5 agonist derived from the P97 protein of Mycoplasma hyopneumoniae.

By modulating specific immune responses against antigens, adjuvants are used in many vaccine preparations to enhance protective immunity. The C-terminal domain of the protein P97 (P97c) of Mycoplasma hyopneumoniae, which is the etiologic agent of porcine enzootic pneumonia, has been shown to increase the specific humoral response against an antigen when this antigen is merged with P97c and delivered by adenovectors. However, the immunostimulating mechanism of this protein remains unknown. In the present study, recombinantly expressed P97c triggered a concentration-dependent TLR5 activation and stimulates the production of interleukin-8 from HEK-Blue mTLR5 cells. Circular dichroism spectroscopy and prediction of 3-dimensional conformation exposed a relevant secondary and tertiary structural homology between P97c and flagellin, the known potent TLR5 agonist. P97c adjuvanticity was evaluated by fusing the conserved epitope of the ectodomain matrix 2 protein (M2e) of the influenza A virus to the protein. Mice immunized with P97c-3M2e revealed a high antibody titer against the M2e epitope associated with a mixed Th1/Th2 immune response. Overall, this study identifies a novel agonist of the pattern recognition receptor TLR5 and reveals that P97c is a potential adjuvant through the activation of the innate immune system.

Paracellular Pathway-Mediated Mycoplasma hyopneumoniae Migration across Porcine Airway Epithelial Barrier under Air-Liquid Interface Conditions.

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. hyopneumoniaein 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.

Lung consolidation caused by Mycoplasma hyopneumoniae has a negative effect on productive performance and economic revenue in finishing pigs.

This study aimed to measure the impact of productivity and the consequent economic losses related to lung lesions caused by M. hyopneumoniae. Five-hundred 75 days-old pigs were selected and weighed at the beginning and at the end of the finishing phase to assess the average daily gain (ADG). These animals were evaluated at the slaughter, and samples were collected for laboratory analysis to confirm the presence of M. hyopneumoniae DNA. The lungs of each pig were examined and classified into groups based on the extension of macroscopic lung lesions. Four-hundred eighty-six lungs were examined and 68.5% (n = 333) had macroscopic lung lesions. All pigs with lesions were positive for M. hyopneumoniae in qPCR. Linear mixed regression models (proc Glimmix) were performed on SAS to estimate the effect of macroscopic lung lesion scores on the ADG of finishing pigs. All pairwise comparisons among lesion score groups were performed using p < 0.05. For each increase of one percent in the lesion area, there was a decrease of 1.8 g in the daily weight gain. All the groups had a numerically lower ADG when compared to Group 1 (no lesions). The economic analysis was performed by simulation on Excel to estimate and compare the financial performance of the different lung lesion score groups. The negative correlation found between the group with no lung lesions and the group with more than 15.1% of lesions, showed a statistical difference in ADG, which could mean an opportunity to gain up to $ 6.55 per pig at slaughter. The presence of lesions causes the animals to decrease their productive potential, causing financial loss and generating impacts on the production system.

Analysis of the mutant selection window and killing of Mycoplasma hyopneumoniae for doxycycline, tylosin, danofloxacin, tiamulin, and valnemulin.

Mycoplasma hyopneumoniae is the major pathogenic microorganism causing enzootic pneumonia in pigs. With increasing resistance of M. hyopneumoniae to conventional antibiotics, treatment is becoming complicated. Herein, we investigated the mutant selection window (MSW) of doxycycline, tylosin, danofloxacin, tiamulin, and valnemulin for treating the M. hyopneumoniae type strain (ATCC 25934) to determine the likelihood of promoting resistance with continued use of these antibiotics. Minimum inhibitory concentration (MIC) values against M. hyopneumoniae were determined for each antimicrobial agent based on microdilution broth and agar dilution methods (bacterial numbers ranged from 105 colony-forming units (CFU)/mL to 109 CFU/mL). The minimal concentration inhibiting colony formation by 99% (MIC99) and the mutant prevention concentration (MPC) were determined by the agar dilution method with three inoculum sizes. Antimicrobial killing was determined based on MIC99 and MPC values for all five agents. MIC values ranged from 0.001 to 0.25 µg/mL based on the microdilution broth method, and from 0.008 to 1.0 µg/mL based on the agar dilution method. MPC values ranged from 0.0016 to 10.24 µg/mL. MPC/MIC99 values were ordered tylosin > doxycycline > danofloxacin > tiamulin > valnemulin. MPC achieved better bactericidal action than MIC99. Based on pharmacodynamic analyses, danofloxacin, tylosin, and doxycycline are more likely to select resistant mutants than tiamulin and valnemulin.

Mycoplasma hyopneumoniae Inhibits Porcine Beta-Defensin 2 Production by Blocking the Unfolded Protein Response To Facilitate Epithelial Adhesion and Infection.

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.

Survival analysis of two Mycoplasma hyopneumoniae eradication methods.

Mycoplasma hyopneumoniae is an important respiratory pathogen causing significant losses in the swine industry. Eradication of this bacterium from herds results in increased pig performance, productivity, and animal welfare. The objective of this study was to compare the time-to-detection of M. hyopneumoniae in breed-to-wean farms after the application of one of two methods for M. hyopneumoniae eradication. The two methods compared in this study were: 1) Herd closure and medication, and 2) Whole herd medication without extended closure. Fifty-six breed-to-wean farms located in the US Midwest constituted the cohort for this investigation. Herd closure and medication was applied in 45 farms, while whole herd medication was applied in 11 farms. Two mutually exclusive events were recorded for each farm, either detection of M. hyopneumoniae, which was considered the event of interest, or end of follow-up, which was the right-censored event. Farms were monitored until recording the event of interest, or until the end of follow-up, whichever occurred first. Detection of M. hyopneumoniae was assessed by identification of antibodies against the bacterium in sentinel pigs using a commercially available ELISA assay within 6 months post-eradication completion. Moreover, clinical presentation of disease was recorded if observed post-eradication completion. The censored event occurred at the end of the study in November 2016 (administrative censoring). Time-to-detection of M. hyopneumoniae was analyzed with a Cox proportional hazards model. The proportional hazards assumption was assessed using graphical methods. A sensitivity analysis to evaluate the assumption of outcome-independent censoring was also performed. The cumulative incidence of M. hyopneumoniae detection at the end of follow-up was 18.6 % (95% CI: 6.5%, 46.8%) for herd closure and medication, and 36.4% (95% CI: 15.5%, 70.3%) for whole herd medication. An interaction term between the type of eradication method and follow-up time was included in the model to account for the non-proportional hazards. An overall effect of eradication method was present (P = 0.0442). The hazard ratio associated to the time-invariant effect of eradication method was 29.2 (95% CI: 0.95, 894; P = 0.053). The hazard ratio associated with the interaction term was 0.88 (95% CI: 0.65, 1.2; P = 0.405). Under these conditions, eradication using herd closure and medication reduced the likelihood of detecting cases of M. hyopneumoniae in breed-to-wean farms compared to whole herd medication. Detection of M. hyopneumoniae was concentrated during the first 64 months of follow-up in herd closure and medication, and in the first 8 months in whole herd medication.

Influence of pig gut microbiota on Mycoplasma hyopneumoniae susceptibility.

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_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 evades phagocytic uptake by porcine alveolar macrophages in vitro.

Mycoplasma hyopneumoniae, the agent of porcine enzootic pneumonia (EP), is able to persist in the lung tissue and evade destruction by the host for several weeks. To understand the mechanism of pathogen survival, phagocytic uptake of M. hyopneumoniae by primary porcine alveolar macrophages was investigated. Intracellular location and survival of the pathogen were explored using gentamicin survival assays, flow cytometry and confocal microscopy of M. hyopneumoniae 232 labelled with green fluorescent protein (GFP). Following 1 h and 16 h of co-incubation, few viable M. hyopneumoniae were recovered from inside macrophages. Flow cytometric analysis of macrophages incubated with M. hyopneumoniae expressing GFP indicated that the mycoplasmas became associated with macrophages, but were shown to be extracellular when actin-dependent phagocytosis was blocked with cytochalasin D. Confocal microscopy detected GFP-labelled M. hyopneumoniae inside macrophages and the numbers increased modestly with time of incubation. Neither the addition of porcine serum complement or convalescent serum from EP-recovered pigs was able to enhance engulfment of M. hyopneumoniae. This investigation suggests that M. hyopneumoniae evades significant uptake by porcine alveolar macrophages and this may be a mechanism of immune escape by M. hyopneumoniae in the porcine respiratory tract.

Lipid peroxidation impairs growth and viability of nursery pigs reared under commercial conditions1.

The objective of this study was to investigate the impact of lipid peroxidation in a dose-dependent manner on growth, health, and oxidative stress status of nursery pigs. A total of 2,200 weaned pigs (5.95 ± 0.20 kg BW) were housed in 100 pens (22 pigs per pen) in a randomized complete block design based on initial BW and sex. Pigs were randomly assigned within blocks to 5 dietary treatments, consisting of a corn-soybean meal-based diet supplemented with 5% of either control corn oil (iodine value = 118, FFA = 0.06%, anisidine value = 3, peroxide value = 3 mEq/kg oil) or peroxidized corn oil (iodine value = 120, FFA = 0.35%, anisidine value = 30, peroxide value = 163 mEq/kg oil). These 2 diets were blended to obtain 5 levels of peroxidation with final treatments designated as 0 (diet with 5% control oil), 25%, 50%, 75%, and 100% (diet with peroxidized corn oil) peroxidation. Diets were fed ad libitum for 43 d. Blood samples were collected on d 33 from 20 pigs per treatment to determine serum oxidative stress markers and vitamin E concentrations and again on d 43 (14 d after vaccination) to determine immune response to porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae (Mhyo). Gain:feed ratio decreased linearly (P = 0.023) with increasing peroxidation, but pen ADG and ADFI were not affected. Number of pigs removed for medical treatment, total number medically treated, pigs culled for low end weight, and mortality increased, and full-value pigs linearly decreased (P < 0.04) with increasing peroxidation. Consequently, total pen gain (weight of viable pigs that remained in test pens at the end of the study minus weight of pigs placed) decreased linearly (P < 0.01) with increasing peroxidation. Antibody titers to Mhyo and PCV2 increased postvaccination (P < 0.001), but did not differ due to dietary treatment. Serum concentrations of malondialdehyde, 8-hydroxy-2'-deoxyguanosine, and protein carbonyl were not affected by peroxidation. Total antioxidant capacity and serum vitamin E concentrations decreased (P = 0.01) linearly with increasing peroxidation. Data show a dose-dependent negative impact of lipid peroxidation on pig productivity when determined under field population conditions, being primarily manifested by increased mortality, number of pigs medically treated, and number of culled pigs (≤13.6 kg BW). Results underscore the importance of proper assessment of lipid peroxidation as part of quality control to prevent oxidative stress and performance losses in weaned pigs.

Mycoplasma hyopneumoniae-Lawsonia intracellularis dual challenge modulates intestinal integrity and function1.

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.

Differential responses to stress of two Mycoplasma hyopneumoniae strains.

Mycoplasma hyopneumoniae is a respiratory pathogen, causing porcine enzootic pneumonia. To survive in the porcine respiratory tract, M. hyopneumoniae must cope with both oxidative and heat stress imposed by the host. To get insights into M. hyopneumoniae stress responses and pathogenicity mechanisms, the protein profiles of two M. hyopneumoniae strains, pathogenic 7448 strain and non-pathogenic strain J, were surveyed under oxidative (OS) or heat (HS) stress. M. hyopneumoniae strains were submitted to OS (0.5% hydrogen peroxide) or HS (temperature shifts to 42 °C) conditions and protein profiling was carried out by LC-MS/MS and label-free quantitative analyses. Data are available via ProteomeXchange with identifier PXD012742. Qualitative and quantitative differences involving 40-60 M. hyopneumoniae proteins were observed for both strains when comparing bacteria exposed to OS or HS to non-treated controls. However, no differences in abundance were found in proteins classically related to stress responses, as peroxidases and chaperones, suggesting that these proteins would be constitutively present in both strains in the tested conditions. Interestingly, under stress conditions, more virulence-related proteins were detected in M. hyopneumoniae 7448 differentially represented proteins than in M. hyopneumoniae J, suggesting that stress may trigger a differential response of the corresponding genes, shared by both strains.

Extracellular DNA release from the genome-reduced pathogen Mycoplasma hyopneumoniae is essential for biofilm formation on abiotic surfaces.

Mycoplasma hyopneumoniae is an economically devastating, globally disseminated pathogen that can maintain a chronic infectious state within its host, swine. Here, we depict the events underpinning M. hyopneumoniae biofilm formation on an abiotic surface and demonstrate for the first time, biofilms forming on porcine epithelial cell monolayers and in the lungs of pigs, experimentally infected with M. hyopneumoniae. Nuclease treatment prevents biofilms forming on glass but not on porcine epithelial cells indicating that extracellular DNA (eDNA), which localises at the base of biofilms, is critical in the formation of these structures on abiotic surfaces. Subpopulations of M. hyopneumoniae cells, denoted by their ability to take up the dye TOTO-1 and release eDNA, were identified. A visually distinct sub-population of pleomorphic cells, that we refer to here as large cell variants (LCVs), rapidly transition from phase dark to translucent "ghost" cells. The translucent cells accumulate the membrane-impermeable dye TOTO-1, forming readily discernible membrane breaches immediately prior to lysis and the possible release of eDNA and other intracellular content (public goods) into the extracellular environment. Our novel observations expand knowledge of the lifestyles adopted by this wall-less, genome-reduced pathogen and provide further insights to its survival within farm environments and swine.

Hsp90/Sec22b promotes unconventional secretion of mature-IL-1ß through an autophagosomal carrier in porcine alveolar macrophages during Mycoplasma hyopneumoniae infection.

Interleukin-1ß (IL-1ß) is a critical inflammatory regulator in response to Mycoplasma hyopneumoniae infection. However, the mechanism involved in the secretion of IL-1ß during Mycoplasma hyopneumoniae infection is unclear. In this study, we demonstrated that Mycoplasma hyopneumoniae infection increased the secretion of mature-IL-1ß (m-IL-1ß), but not pro-IL-1ß, in porcine alveolar macrophages. Moreover, Mycoplasma hyopneumoniae infection promoted the generation of autophagosomes, which attributed to the unconventional secretion of m-IL-1ß. Further results revealed that Hsp90 was required for the entry of m-IL-1ß into autophagosomes during Mycoplasma hyopneumoniae infection. The fusion of m-IL-1ß-containing autophagosome and plasma membranes was regulated by Sec22b and independent of lysosomal dysfunction. In conclusion, we provide evidence that Hsp90/Sec22b promotes the unconventional secretion of IL-1ß through an autophagosomal carrier during Mycoplasma hyopneumoniae infection. The elucidation of the molecular and cellular machinery in Mycoplasma hyopneumoniae infected mammalian cells in this study suggests avenues for further study and applications and paves the way for novel therapeutic strategies to prevent tissue damage in mycoplasma-associated diseases.

Metabolic adaptation of pigs to a Mycoplasma hyopneumoniae and Lawsonia intracellularis dual challenge.

Respiratory and enteric pathogens such as Mycoplasma hyopneumoniae (Mh) and Lawsonia intracellularis (LI) reduce lean accretion and feed efficiency (FE) in growing pigs. However, the metabolic mechanism by which this occurs is still unknown. Therefore, the primary aim of this study was to examine the metabolic adaptation of pigs presented with a dual Mh and LI challenge (MhLI). A secondary objective was to examine if selection for high FE, modeled by selection for low residual feed intake (RFI), alters molecular response to disease. Using a 2 × 2 factorial design, 6 littermate pairs from a high RFI (HRFI) and 6 littermate pairs from a low RFI (LRFI) line (barrows, 66 ± 2 kg BW) were selected, with 1 pig from each pair assigned to individual pens in either the challenge or the nonchallenge (control) rooms (n = 6 barrows per line/challenge). On days post inoculation (dpi) 0, MhLI pigs were inoculated intragastrically with LI and intratracheally with Mh. Pig and feeder weights were recorded at dpi 0, 7, 14, and 21. On dpi 21, pigs were euthanized and tissues and blood were collected. Markers of oxidative stress, skeletal muscle metabolism and proteolysis, and liver gluconeogenesis were evaluated to determine the effects of MhLI, RFI line, and their interaction. The interaction of line and challenge was not significant (P > 0.05) for any measure. Overall, MhLI pigs had lower ADG (38%, P < 0.001), ADFI (25%, P < 0.001), and G:F (19%, P = 0.012) compared with controls. As expected, LRFI pigs had lower ADFI (P = 0.028) for the same ADG, giving them greater G:F (P = 0.021) than HRFI pigs. Challenged pigs had greater reactive oxygen species (ROS) production in the LM and liver (P < 0.10) but did not have greater skeletal muscle proteolysis. Liver gluconeogenesis was also not upregulated (P > 0.05) due to MhLI. These results provide further evidence that selection for LRFI does not negatively affect response to disease. In addition, these results suggest that postabsorptive metabolic functions are altered due to MhLI challenge. The MhLI challenge induced mitochondrial dysfunction, evident by greater ROS production, and caused pigs to favor glycolytic energy generation. However, skeletal muscle proteolysis and liver gluconeogenesis were not upregulated during MhLI challenge. These data suggest that during mild disease stress, pigs can meet energy demands without reliance on nutrient mobilization and gluconeogenesis.

Assessment of the in vitro growing dynamics and kinetics of the non-pathogenic J and pathogenic 11 and 232 Mycoplasma hyopneumoniae strains.

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.

Cholesterol exacerbates Mycoplasma hyopneumoniae-induced apoptosis via stimulating proliferation and adhesion to porcine alveolar macrophages.

Mycoplasma hyopneumoniae (M. hyo) is the agent of porcine enzootic pneumonia, a disease that causes considerable economic losses in the swine industry. Induction of apoptosis in porcine alveolar macrophages is an important pathogenic mechanism of M. hyo. Cholesterol has been reported to influence cell adherence and cell invasion of Mycoplasma gallisepticum and Mycoplasma fermentans leading to apoptosis, but the role of cholesterol on the apoptotic inducing activity of M. hyo remains unknown. In this study, we found a positive correlation between cholesterol level and M. hyo infection in porcine serum and lung tissue. Cholesterol exacerbated M. hyo-induced apoptosis in porcine alveolar macrophages (PAMs) in a dosage-dependent manner, which was associated with increased hydrogen peroxide (H2O2) and nitric oxide (NO) production, up-regulated TNF-α mRNA expression, and activated caspase-3. The pathogenicity-enhancing effect of cholesterol was related to increased M. hyo proliferation with an up-regulation of M. hyo genes responsible for DNA and protein synthesis, which led to improved M. hyo adherence to PAMs, presumably via increased mRNA expression of adhesin genes. In conclusion, cholesterol promotes the apoptotic effect of M. hyo through stimulating proliferation and enhancing its adherence to PAMs. Hence, the study gives new insights into the role of cholesterol on the PAM - M. hyo interations.