The influence of regulatory elements on Mycoplasma hyopneumoniae 7448 transcriptional response during oxidative stress and heat shock.

BACKGROUND: The comprehension of genome organization and gene modulation is essential for understanding pathogens' infection mechanisms. Mycoplasma hyopneumoniae 7448 genome is organized in transcriptional units (TUs), which are flanked by regulatory elements such as putative promoters, terminators and repetitive sequences. Yet the relationship between the presence of these elements and bacterial responses during stress conditions remains unclear. Thus, in this study, in silico and RT-qPCR analyses were associated to determine the effect of regulatory elements in gene expression regulation upon heat shock and oxidative stress conditions. METHODS AND RESULTS: Thirteen TU's organizational profiles were found based on promoters and terminators distribution. Differential expression in genes sharing the same TUs was observed, suggesting the activity of internal regulatory elements. Moreover, 88.8% of tested genes were differentially expressed under oxidative stress in comparison to the control condition, being 81.3% of them surrounded by their own regulatory elements. Similarly, under heat shock, 44.4% of the genes showed regulation when compared to control condition, being 75.0% of them surrounded by their own regulatory elements. CONCLUSIONS: Altogether, this data suggests the activity of internal regulatory elements in gene modulation of M. hyopneumoniae 7448 transcription.

Mycoplasma hyopneumoniae J elicits an antioxidant response and decreases the expression of ciliary genes in infected swine epithelial cells.

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.

Quantitative Proteomic Analyses of a Pathogenic Strain and Its Highly Passaged Attenuated Strain of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia, a chronic respiratory disease in swine resulting in enormous economic losses. To identify the components that contribute to virulence and unveil those biological processes potentially related to attenuation, we used isobaric tags for relative and absolute quantification technology (iTRAQ) to compare the protein profiles of the virulent M. hyopneumoniae strain 168 and its attenuated highly passaged strain 168L. We identified 489 proteins in total, 70 of which showing significant differences in level of expression between the two strains. Remarkably, proteins participating in inositol phosphate metabolism were significantly downregulated in the virulent strain, while some proteins involved in nucleoside metabolism were upregulated. We also mined a series of novel promising virulence-associated factors in our study compared with those in previous reports, such as some moonlighting adhesins, transporters, lipoate-protein ligase, and ribonuclease and several hypothetical proteins with conserved functional domains, deserving further research. Our survey constitutes an iTRAQ-based comparative proteomic analysis of a virulent M. hyopneumoniae strain and its attenuated strain originating from a single parent with a well-characterized genetic background and lays the groundwork for future work to mine for potential virulence factors and identify candidate vaccine proteins.

Evaluation of growth and gene expression of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis in defined medium.

Mycoplasmas belong to the Mollicutes class and possess low GC content and lack a cell wall, and also simplified metabolic pathways. Due to its reduced metabolic ability mycoplasmas are fastidious organisms growing with difficult under laboratory conditions. Its complex nutritional requirements render mycoplasmas to depend on external supplies of biosynthetic precursors. Aiming to develop and test defined media that could be used as a tool for Mycoplasma research, Mycoplasma hyopneumoniae and Mycoplasma hyorhinis were cultivated in a complex medium supplemented with serum (Friis broth) and in four different defined media (YUS, YUSm, CMRL and CMRL+, that was developed in the present study). The cell concentration of both Mycoplasma species was assessed, by flow cytometry. Cellular viability was also analyzed in all defined media, indicating the presence of viable mycoplasma cells. All the defined media tested were able to maintain cell concentrations and viability and, amongst them, CMRL+ was the most suitable. For both Mycoplasma species, only the CMRL+ media showed similar cell density when compared to the complex medium. The transcriptional response of M. hyopneumoniae in CMRL+ broth was assessed by RT-qPCR, and the transcriptional profile of 18 genes in three cultures conditions (standard, heat shock and oxidative stress) was analyzed demonstrating gene expression regulation in response to the medium composition and to the culture conditions tested. The medium developed enables the definition of mycoplasmal nutritional requirements and metabolic pathways as well as genetic analysis.

Clinical impact of deoxynivalenol, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol on the severity of an experimental Mycoplasma hyopneumoniae infection in pigs.

BACKGROUND: The mycotoxin deoxynivalenol (DON) is highly prevalent in cereals in moderate climates and therefore pigs are often exposed to a DON-contaminated diet. Pigs are highly susceptible to DON and intake of DON-contaminated feed may lead to an altered immune response and may influence the pathogenesis of specific bacterial diseases. Therefore, the maximum guidance level in feed is lowest in this species and has been set at 900 µg/kg feed by the European Commission. This study aimed to determine the effect of in-feed administration of a moderately high DON concentration (1514 µg/kg) on the severity of an experimental Mycoplasma hyopneumoniae (M. hyopneumoniae) infection in weaned piglets. Fifty M. hyopneumoniae-free piglets were assigned at 30 days of age [study day (D)0] to four different groups: 1) negative control group (NCG; n = 5), 2) DON-contaminated group (DON; n = 15), 3) DON-contaminated and M. hyopneumoniae-inoculated group (DONMHYO; n = 15), 4) M. hyopneumoniae-inoculated group (MHYO; n = 15). The piglets were fed the experimental diets ad libitum for five weeks and were monitored during this period and euthanized at day 35 [27 days post infection (DPI)] or 36 (28 DPI). The main parameters under investigation were macroscopic lung lesions (MLL) at euthanasia, respiratory disease score (RDS) from day 8 until day 35, histopathologic lesions and log copies of M. hyopneumoniae DNA detected by qPCR, determined at the day of euthanasia. RESULTS: No significant difference was obtained for MLL at euthanasia, RDS (8-35), histopathologic lung lesions and log copies of M. hyopneumoniae DNA in the DONMHYO and MHYO group and consequently, no enhancement of the severity of the M. hyopneumoniae infection could be detected in the DONMHYO compared to the MHYO group. CONCLUSIONS: Under present conditions, the findings imply that feed contaminated with DON (1514 µg/kg) provided to weaned pigs for five weeks did not increase the severity of an experimental M. hyopneumoniae infection. Further research is needed to investigate the impact of DON on M. hyopneumoniae infections in a multi-mycotoxin and multi-pathogen environment.

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.

Determinants for swine mycoplasmal pneumonia reproduction under experimental conditions: A systematic review and recursive partitioning analysis.

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.

Low temperature and dust favour in vitro survival of Mycoplasma hyopneumoniae: time to revisit indirect transmission in pig housing.

Porcine enzootic pneumonia (EP) caused by Mycoplasma hyopneumoniae adversely affects pig welfare and is associated with major economic losses in the pig industry worldwide. Transmission is predominantly by direct contact, but the role of indirect transmission remains poorly understood. This study examined survival of six M. hyopneumoniae isolates dried onto five different surfaces encountered in pig units and exposed to temperatures of 4, 25 and 37°C for up to 12 days. Survival of the organisms was determined by recovering the organism from the surface material and culturing in Friis broth. Data were analysed by logistic regression to identify factors influencing survival of M. hyopneumoniae. Maximum survival was 8 days for all isolates on at least one surface (except stainless steel) at 4°C and was limited to 2 days at 25 and 37°C. Overall, dust and polypropylene copolymer supported M. hyopneumoniae survival the longest when compared with other surface materials. In conclusion, we have demonstrated that M. hyopneumoniae can survive outside the host for at least 8 days. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the transmission of Mycoplasma hyopneumoniae and optimizing biosecurity practices are keys to reducing the use of antimicrobial agents to control this pathogen. Direct transmission of the pathogen between pigs is the main route of spread and its lack of cell wall may compromise its resilience outside the host. The results from our study show that M. hyopneumoniae can survive for up to several days on dry surfaces and therefore may have the potential to infect pigs by indirect transmission. Factors influencing the survival of M. hyopneumoniae outside the host are further elucidated.

Optimization of culture media of pathogenic Mycoplasma hyopneumoniae by a response surface methodology.

Composition of culture medium for mass production of Mycoplasma hyopneumoniae was optimized using a response surface methodology (RSM). Initially, the influence of glucose, thallium acetate, fresh yeast extract, horse serum, and porcine serum on the production of mycoplasmal protein was assessed using a 'one factor at a time' technique. Next, factors with a significant effect, including fresh yeast extract, and horse and porcine sera, were selected for further optimization using a central composite design (CCD) of RSM. The experimental results were fitted into a second order polynomial model equation. Estimated optimal condition of the factors for maximum production of mycoplasmal protein (i.e., triple-fold increase from 0.8 mg/L produced by basal mycoplasma media to 2.5 mg/L) was 10.9% fresh yeast extract, 15% horse serum, and 31.5% porcine serum (v/v). For the optimized conditions, a 2.96 mg/L experimental result was observed, similar to the estimated optimal conditions result of the CCD.

Validation of ATP luminometry for rapid and accurate titration of Mycoplasma hyopneumoniae in Friis medium and a comparison with the color changing units assay.

Limited reports are available on the growth response of Mycoplasma hyopneumoniae in Friis medium and the routinely used color changing units (CCU) assay has not yet been profoundly compared with other titration methods. Firstly, growth kinetics of 7 diverse M. hyopneumoniae isolates were followed by ATP luminometry in five Friis medium batches. Secondly, results of the CCU and ATP assays were compared hereby evaluating the methods. Growth curves of all isolates had log, stationary and senescence phases, and reached similar maximal titres when cultured in the same batch of Friis medium. Doubling times (Tds) of the isolates grown in slowly shaken cultures varied between 4.8 and 7.8 h. Maximal titres, Tds, growth phase in which the phenol red indicator turned from red to yellow due to acidification by mycoplasmal metabolism, and the length of the stationary phase varied depending on the Friis medium batch. The effect of static vs. shaking culture conditions on the Td depended on the isolate. ATP and CCU assays obtained similar growth curves, but when maximal levels were reached the CCU titre dropped earlier than the ATP titre. During log phase, CCU and ATP titres were strongly linearly linked. We developed a model enabling transformation of ATP into CCU titres or vice versa. The calculated amount of ATP per CCU (1.77 amol ATP/ml) indicated that the CCU assay likely underestimates the actual cell concentration. When titres were determined as means of 3 measurements, the ATP assay was 7 times more accurate and had 11-fold lower outliers than the CCU assay. Unlike the CCU assay, luminometry only requires one measurement to obtain sufficient accuracy. It was concluded that the ATP assay constitutes a valuable robust alternative for reproducible real-time titre assessment of freshly grown M. hyopneumoniae cultures. It is faster, more accurate and time, work and cost efficient compared to the CCU assay. The assay is preferred to better standardise and describe M. hyopneumoniae cultures used in various experiments.

Potential use of a recombinant replication-defective adenovirus vector carrying the C-terminal portion of the P97 adhesin protein as a vaccine against Mycoplasma hyopneumoniae in swine.

Mycoplasma hyopneumoniae causes severe economic losses to the swine industry worldwide and the prevention of its related disease, enzootic porcine pneumonia, remains a challenge. The P97 adhesin protein of M. hyopneumoniae should be a good candidate for the development of a subunit vaccine because antibodies produced against P97 could prevent the adhesion of the pathogen to the respiratory epithelial cells in vitro. In the present study, a P97 recombinant replication-defective adenovirus (rAdP97c) subunit vaccine efficiency was evaluated in pigs. The rAdP97c vaccine was found to induce both strong P97 specific humoral and cellular immune responses. The rAdP97c vaccinated pigs developed a lower amount of macroscopic lung lesions (18.5 + or - 9.6%) compared to the unvaccinated and challenged animals (45.8 + or - 11.5%). rAdP97c vaccine reduced significantly the severity of inflammatory response and the amount of M. hyopneumoniae in the respiratory tract. Furthermore, the average daily weight gain was slightly improved in the rAdP97c vaccinated pigs (0.672 + or - 0.068 kg/day) compared to the unvaccinated and challenged animals (0.568 + or - 0.104 kg/day). A bacterin-based commercial vaccine (Suvaxyn MH-one) was more efficient to induce a protective immune response than rAdP97c even if it did not evoke a P97 specific immune response. These results suggest that immunodominant antigens other than P97 adhesin are also important in the induction of a protective immune response and should be taken into account in the future development of M. hyopneumoniae subunit vaccines.

Long-distance airborne transport of infectious PRRSV and Mycoplasma hyopneumoniae from a swine population infected with multiple viral variants.

Airborne transport of porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae (M hyo) has been reported out to 4.7 km. This study attempted to determine whether this event could occur over longer distances and across multiple viral variants. To accomplish this goal, a mixed infection of 3 PRRSV variants (1-8-4, 1-18-2 and 1-26-2) and M hyo 232 was established in a source population of growing pigs. Over 21-day period, air samples were collected from the source population and at designated distances from the herd. Samples were tested for PRRSV RNA and M hyo DNA by PCR and if positive, further characterized. In exhaust air from the source population, PRRSV and M hyo were detected in 21 of 21 and 8 of 21 air samples, respectively. Five of 114 (4.4%) long-distance air samples were positive for PRRSV and 6 of 114 (5.2%) were positive for M hyo. The 5 PRRSV-positive samples were collected at 2.3, 4.6, 6.6 and 9.1 km from the herd. All contained infectious virus and were >99.2% homologous to PRRSV 1-8-4. No evidence of PRRSV 1-18-2 or 1-26-2 was detected in long-distance samples. All 6 M hyo-positive samples were 99.9% homologous to M hyo 232 and 3 samples (collected at 3.5, 6.8 and 9.2km from the herd) were infectious. These results indicate that airborne transport of PRRSV 1-8-4 and M hyo 232 occurs over longer distances than previously reported and that both pathogens remained infectious.

Optimization of culture media of pathogenic Mycoplasma hyopneumoniae by a response surface methodology

Composition of culture medium for mass production of Mycoplasma hyopneumoniae was optimized using a response surface methodology (RSM). Initially, the influence of glucose, thallium acetate, fresh yeast extract, horse serum, and porcine serum on the production of mycoplasmal protein was assessed using a 'one factor at a time' technique. Next, factors with a significant effect, including fresh yeast extract, and horse and porcine sera, were selected for further optimization using a central composite design (CCD) of RSM. The experimental results were fitted into a second order polynomial model equation. Estimated optimal condition of the factors for maximum production of mycoplasmal protein (i.e., triple-fold increase from 0.8 mg/L produced by basal mycoplasma media to 2.5 mg/L) was 10.9% fresh yeast extract, 15% horse serum, and 31.5% porcine serum (v/v). For the optimized conditions, a 2.96 mg/L experimental result was observed, similar to the estimated optimal conditions result of the CCD.

Global transcriptional analysis of Mycoplasma hyopneumoniae following exposure to norepinephrine.

Mycoplasma hyopneumoniae, a component of the porcine respiratory disease complex, colonizes the respiratory tract of swine by binding to the cilia of the bronchial epithelial cells. Mechanisms of pathogenesis are poorly understood for M. hyopneumoniae, but previous work has indicated that it responds to the environmental stressors heat shock, iron deprivation and oxidative compounds. For successful infection, M. hyopneumoniae must effectively resist host responses to the colonization of the respiratory tract. Among these are changes in hormonal levels in the mucosal secretions. Recent work in the stress responses of other bacteria has included the response to the catecholamine norepinephrine. The idea that M. hyopneumoniae can respond to a host hormone, however, is novel and has not previously been demonstrated. To test this, organisms in the early exponential phase of growth were exposed to 100 muM norepinephrine for 4 h, and RNA samples from these cultures were collected and compared to RNA samples from control cultures using two-colour PCR-based M. hyopneumoniae microarrays. The M. hyopneumoniae response included slowed growth and changes in mRNA transcript levels of 84 genes, 53 of which were upregulated in response to norepinephrine. A larger proportion of the genes upregulated than those downregulated were involved with transcription and translation. The downregulated genes were mostly involved with metabolism, which correlated with the reduction in growth of the mycoplasma. Approximately 51 % of the genes were hypothetical with no known function. Thus, in response to norepinephrine, M. hyopneumoniae appears to upregulate protein expression while downregulating general metabolism.

Effect of sow vaccination against Mycoplasma hyopneumoniae on sow and piglet colonization and seroconversion, and pig lung lesions at slaughter.

The objectives of the present study were to compare Mycoplasma hyopneumoniae (Mh) colonization and serologic status on Mh vaccinated and non-vaccinated sows and to assess the effect of sow vaccination on colonization and serologic status of their piglets at weaning as well as presence of enzootic pneumonia (EP) lung lesions at slaughter. Fifty sows (25 vaccinated and 25 unvaccinated) as well as five of their piglets were included in the study. Blood samples and nasal swabs from sows at 7 weeks pre-farrowing and 1 week post-farrowing and from piglets at 3-4 weeks of age were taken. Nasal swabs and sera were tested by a nested polymerase chain reaction (nPCR) to detect Mh DNA and by an enzyme-linked immunosorbent assay (ELISA) test to detect antibodies to the pathogen, respectively. Finally, at 23 weeks of age, pigs were sent to the slaughter where the extension of EP-compatible gross lesions was assessed. Vaccination with two doses of Mh vaccine resulted in a significantly higher (p<0.05) percentage of seropositive sows than in the non-vaccinated group at 1 week post-farrowing. On the contrary, no statistical significant differences were found in the number of nasal nPCR positive sows among different treatments (p>0.05). At 3-4 weeks of age, a significantly higher percentage (p<0.001) of seropositive piglets came from vaccinated than from non-vaccinated sows. Although the number of Mh infected piglets coming from non-vaccinated sows was higher than the one from vaccinated sows, the difference was not statistically significant (p>0.05). Overall, piglets from vaccinated sows had a significant lower (p<0.05) mean of EP-compatible lung lesions (1.83+/-2.8) than piglets from non-vaccinated sows (3.02+/-3.6). Under the conditions described in this study, sow vaccination did not affect sow or piglet colonization but increased the percentage of seropositive sows and piglets at weaning and reduced significantly the mean EP-compatible lung lesion scoring at slaughter.

Application of flow cytometry for the determination of minimal inhibitory concentration of several antibacterial agents on Mycoplasma hyopneumoniae.

AIM: In this study, flow cytometry was evaluated for the determination of the minimal inhibitory concentrations (MICs) of nine antibacterial agents (enrofloxacin, ciprofloxacin, oxytetracycline, chloramphenicol, tylosin, lincomycin, gentamycin, spectinomycin and streptomycin) against M. hyopneumoniae. METHODS AND RESULTS: Flow cytometry was able to detect Mycoplasma hyopneumoniae inhibition at 12 h postincubation, whereas the results obtained by the traditional method were only obtained at 48 h, when a visible change in the medium had occurred. At 48 h, both methods gave the same result for eight antibacterial agents, whereas flow cytometry gave slightly higher MIC values for one antibacterial agent (tylosin). This was attributed to the fact that the M. hyopneumoniae growth that had occurred in those tubes was not enough to visibly change the colour of the medium. A good relationship was found between the flow cytometry and the traditional method. CONCLUSION: Flow cytometry was found to be a good method for the determination of antimicrobial MICs in M. hyopneumoniae. SIGNIFICANCE AND IMPACT OF THE STUDY: The flow cytometric method allows the determination of the response of M. hyopneumoniae to each of the antibacterial agents in near real time, and has potential for the identification and study of resistant subpopulations.

Interactions of highly and low virulent Mycoplasma hyopneumoniae isolates with the respiratory tract of pigs.

Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia, a chronic nonfatal disease affecting pigs of all ages. To obtain better insight in the mechanisms responsible for differences in virulence between highly and low virulent M. hyopneumoniae isolates, 23 caesarean-derived, colostrum-deprived piglets were randomly assigned to three groups. Groups 1 and 2 consisted of nine animals each, which were intratracheally inoculated at 1 week of age with a highly or a low virulent isolate of M. hyopneumoniae, respectively. The remaining five animals were inoculated with sterile culture medium. Animals were euthanized at 5, 10, 15 and 28 days post-inoculation (DPI). Animals inoculated with the highly virulent isolate had more neutrophils in BAL fluid at 10, 15 and 28DPI compared to the other groups. At 10 and 15DPI, animals in the highly virulent group had significantly higher concentrations of TNF-alpha in BAL fluid. IL-1beta concentration in this group was higher at 5 and 28DPI compared to the other groups. From 10DPI onwards, significantly higher titres of M. hyopneumoniae were detected in the BAL fluid of animals inoculated with the highly virulent isolate compared to animals inoculated with the low virulent isolate. Additionally, the in vitro generation time of the highly virulent M. hyopneumoniae isolate was significantly shorter than that of the low virulent isolate. The present study indicates that the difference in pathogenicity between the highly and low virulent isolates is associated with a faster in vitro growth, a higher capacity to multiply in the lungs and the induction of a more severe inflammation process by the highly virulent isolate.

Isolation of Mycoplasma hyopneumoniae from different sampling sites in experimentally infected and contact SPF piglets.

The purpose of this study was to determine the optimal route of infection and the optimal sampling sites for the recovery of M. hyopneumoniae, the etiological agent of enzootic porcine pneumonia. Virulence of two strains, BQ 14 and 116, isolated in France in 1975 and 2003, respectively, was also compared. Groups of specific pathogen free piglets were experimentally infected by the intratracheal or intranasal route. One non-inoculated pig was placed in each group of infected pigs to study direct transmission. Two groups were kept uninfected. Coughing was recorded daily. Blood samples, nasal, tonsillar and tracheal swabs and tracheobronchiolar washings were collected weekly. Pigs were killed 27-37 days post-infection. Lung lesions were scored and swabs were collected from nasal cavities, tonsils, trachea, lung, liver and spleen. All the samples, collected from live and dead pigs, were cultured for M. hyopneumoniae recovery. Results showed that both experimentally infected pigs and contact pigs developed enzootic pneumonia, whatever the route of infection and the strain tested. Direct contact transmission occurred quickly. No difference between the two routes of infection or between the two strains tested was evidenced, but high individual variations were observed between pigs. Tracheal swabs and tracheobronchiolar washings were the most effective samples to detect M. hyopneumoniae compared to nasal or tonsillar swabs. Our results also suggested that tracheobronchiolar washings could have an influence on the lesion extent observed at necropsy. M. hyopneumoniae could be re-isolated from liver and spleen of experimentally infected pigs and contact pigs.

Transcriptional profiling of Mycoplasma hyopneumoniae during heat shock using microarrays.

Bacterial pathogens undergo stress during host colonization and disease processes. These stresses result in changes in gene expression to compensate for potentially lethal environments developed in the host during disease. Mycoplasma hyopneumoniae colonizes the swine epithelium and causes a pneumonia that predisposes the host to enhanced disease from other pathogens. How M. hyopneumoniae responds to changing environments in the respiratory tract during disease progression is not known. In fact, little is known concerning the capabilities of mycoplasmas to respond to changing growth environments. With limited genes, mycoplasmas are thought to possess only a few mechanisms for gene regulation. A microarray consisting of 632 of the 698 open reading frames of M. hyopneumoniae was constructed and used to study gene expression differences during a temperature shift from 37 degrees C to 42 degrees C, a temperature swing that might be encountered during disease. To enhance sensitivity, a unique hexamer primer set was employed for generating cDNA from only mRNA species. Our analysis identified 91 genes that had significant transcriptional differences in response to heat shock conditions (P < 0.01) with an estimated false-discovery rate of 4 percent. Thirty-three genes had a change threshold of 1.5-fold or greater. Many of the heat shock proteins previously characterized in other bacteria were identified as significant in this study as well. A proportion of the identified genes (54 of 91) currently have no assigned function.

Dynamics and persistence of Mycoplasma hyopneumoniae infection in pigs.

The purpose of this study was to describe the dynamics (shedding and transmission) of Mycoplasma hyopneumoniae infection within a population of swine and to determine the duration of the infection (persistence) through the identification of the agent in bronchial samples. Sixty-three 2-month-old pigs were used in this study. The pigs (n = 28) were experimentally infected by the intratracheal route with M. hyopneumoniae and considered as seeder pigs. The remaining pigs (n = 32) were not inoculated and randomly allocated to 2 different groups: direct contact exposure pigs (n = 12) and indirect contact exposure pigs (n = 20). Blood samples and nasal swabs were collected throughout the study on days 0, 28, 35, 42, 49, 63, 91, and 119 postinfection. To assess the duration of M. hyopneumoniae infection, 9 seeder and 6 contact exposure pigs were slaughtered at days 155 (group 1), 170 (group 2), and 185 (group 3) postinfection. Direct contact pigs showed evidence of infection on day 28 by polymerase chain reaction (PCR) and on day 35 by serology. The indirect contact exposure pigs presented a very delayed and slow seroconversion pattern; they did not present evidence of transmission until 42 d after the infection of seeder pigs. Identification of M. hyopneumoniae in bronchial swabs was confirmed by nested-PCR from days 155 to 185 postinfection. At the last slaughter date, 77.7% and 100% of the seeders and contact exposure pigs, respectively, tested positive. The results of this study reconfirmed direct infection of M. hyopneumoniae and suggest that indirect transmission can occur in a population. Finally, duration of the infection in this study was longer than previously described.