Divergent clinical outcomes depending on the sequential infection order of porcine circovirus type 2 and Mycoplasma hyopneumoniae.

This study compared the different sequential order of infection of porcine circovirus type 2d (PCV2d) and Mycoplasma hyopneumoniae. Thirty-six pigs were allocated randomly across six different groups. Pigs underwent various inoculation sequences: M. hyopneumoniae administered 14 days before PCV2d, simultaneous PCV2d-M. hyopneumoniae, PCV2d given 14 days before M. hyopneumoniae, PCV2d only, M. hyopneumoniae only, or a mock inoculum. Overall, the pigs inoculated with M. hyopneumoniae 14 days prior to PCV2d (Mhyo-PCV2 group) and those inoculated simultaneously with PCV2d and M. hyopneumoniae (PCV2+Mhyo group) displayed notably higher clinical disease severity and experienced a significant decrease of their average daily weight gain than pigs inoculated with PCV2d 14 days prior to M. hyopneumoniae (PCV2-Mhyo group). M. hyopneumoniae infection potentiated PCV2 blood and lymph node viral loads, as well as PCV2-associated lesions, while the infection of PCV2d did not impact the intensity of M. hyopneumoniae infection. Tumor necrosis factor-α (TNF-α) sera levels were significantly increased in the Mhyo-PCV2 and PCV2+Mhyo groups as compared to the PCV2-Mhyo, PCV2, and Mhyo groups. The most important information was that the potentiation effect of M. hyopneumoniae on PCV2d was found only in pigs inoculated with either M. hyopneumoniae followed by PCV2d (Mhyo-PCV2 group) or a simultaneous inoculation of PCV2d and M. hyopneumoniae (PCV2+Mhyo group). The sequential infection order of PCV2d and M. hyopneumoniae resulted in divergent clinical outcomes.

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

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

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

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

Chimeric proteins of Mycoplasma hyopneumoniae as vaccine and preclinical model for immunological evaluation.

Mycoplasma hyopneumoniae (M. hyopneumoniae) is a primary agent of porcine enzootic pneumonia, a disease that causes significant economic losses to pig farming worldwide. Commercial vaccines induce partial protection, evidencing the need for a new vaccine against M. hyopneumoniae. In our work, three chimeric proteins were constructed, composed of potentially immunogenic domains from M. hyopneumoniae proteins. We designed three chimeric proteins (Q1, Q2, and Q3) based on bioinformatics analysis that identified five potential proteins with immunogenic potential (MHP418, MHP372, MHP199, P97, and MHP0461). The chimeric proteins were inoculated in the murine model to evaluate the immune response. The mice vaccinated with the chimeras presented IgG and IgG1 against proteins of M. hyopneumoniae. There was induction of IgG in mice immunized with Q3 starting from 30 days post-vaccination, and groups Q1 and Q2 showed induction at 45 days. Mice of the group immunized with Q3 showed the production of IgA. In addition, the mice inoculated with chimeric proteins showed a proinflammatory cytokine response; Q1 demonstrated higher levels of TNF, IL-6, IL2, and IL-17. In contrast, animals immunized with Q2 showed an increase in the concentrations of TNF, IL-6, and IL-4, whereas those immunized with Q3 exhibited an increase in the concentrations of TNF, IL-6, IL-10, and IL-4. The results of the present study indicate that these three chimeric proteins can be used in future vaccine trials with swine because of the promising antigenicity.

[Advances in innate immune responses induced by Mycoplasma hyopneumoniae infection].

Mycoplasma hyopneumoniae is the pathogen causing swine mycoplasmal pneumonia. The lack of well-established animal models of M. hyopneumoniae infection has delayed the progress of M. hyopneumoniae-related anti-infection immunity studies. This paper reviews the inflammatory response, the recognition of M. hyopneumoniae by the innate immune system, and the role of innate immune cells, complement system, antimicrobial peptides, autophagy, and apoptosis in M. hyopneumoniae infection. The aim was to elucidate the important roles played by the components of the innate immune system in the control of M. hyopneumoniae infection, and prospect key research directions of innate immune response of M. hyopneumoniae infection in the future.

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

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

Mycoplasma hyopneumoniae infection dynamics in naïve replacement gilts introduced to positive farms.

This study was designed to characterize the dynamics of infection of Mycoplasma hyopneumoniae in naïve replacement gilts after introduction to positive systems. Ninety-eight naïve gilts were monitored in three positive commercial farms (A, B, and C). The näive gilts were housed for 21 days in pens adjacently located to older gilt cohorts (named seeders), which have been naturally exposed to the positive farms. The infection dynamics was evaluated by PCR and ELISA, from laryngeal swabs and serum samples, respectively. Samples were collected at 150 (arrival), 165, 180, 210, 240, 270, 300 days of age (doa), and pre-farrowing. Infection occurred rapidly on farms A and B, taking 25.2 and 23.9 days for 95% of gilts to be PCR positive, respectively. There was no influence on the number of seeders at the time of exposure, but their absence (farm C) could explain the extended period it took for gilts to get infected (69.4 days). On average, it took 162.2 days after the first PCR detection for 85% of gilts to stop shedding the bacterium. The serology results were consistent with the herd infection curve. At pre-farrowing, 100% of gilts seroconverted and 36.7% remained PCR positive. A total of 1.33% of piglets were positive at weaning. Fifteen variants were detected among the three farms by MLVA. The acclimation protocol was efficient and easy to perform, and the presence of seeders was likely critical for early acclimation for M. hyopneumoniae.

Cross-protection of a porcine circovirus types 2a/b (PCV-2a/b) and Mycoplasma hyopneumoniae trivalent vaccine against a dual PCV-2e and Mycoplasma hyopneumoniae challenge.

The purpose of this experimental study was to determine the cross-protection of a new trivalent vaccine containing porcine circovirus types 2a/b (PCV-2a/b) and Mycoplasma hyopneumoniae. Pigs were vaccinated intramuscularly at 21 days of age, then challenged at 42 days of age with a dual PCV-2e and M. hyopneumoniae challenge. Growth performance was significantly improved during the experimental period (21 to 63 days of age) in vaccinated-challenged pigs compared to unvaccinated-challenged pigs. Pigs that were vaccinated and challenged elicited a significant amount of PCV-2e- and M. hyopneumoniae-specific interferon-γ secreting cells (IFN-γ-SC) and reduced the levels of PCV-2e viremia and laryngeal shedding. The results of the present study demonstrated that a trivalent vaccine provided cross-protection against a dual PCV-2e and M. hyopneumoniae challenge.

Le but de cette étude expérimentale était de déterminer la protection croisée d'un nouveau vaccin trivalent contenant le circovirus porcin de types 2a/b (PCV-2a/b) et Mycoplasma hyopneumoniae. Les porcs ont été vaccinés par voie intramusculaire à l'âge de 21 jours, puis provoqués à l'âge de 42 jours avec double provocation par PCV-2e et M. hyopneumoniae. Les performances de croissance ont été significativement améliorées au cours de la période expérimentale (21 à 63 jours) chez les porcs vaccinés-provoqués par rapport aux porcs non-vaccinés-provoqués. Les porcs qui ont été vaccinés et provoqués ont produit une quantité importante de cellules sécrétant de l'interféron-γ spécifiques au PCV-2e et à M. hyopneumoniae (IFN-γ-SC) et ont réduit les niveaux de virémie du PCV-2e et d'excrétion laryngée. Les résultats de la présente étude ont démontré qu'un vaccin trivalent offrait une protection croisée contre une double provocation par le PCV-2e et M. hyopneumoniae.(Traduit par Docteur Serge Messier).

A new strategy: high level expression and immunogenicity analysis of triplicate repeated multigenes in Mycoplasma hyopneumoniae.

Highly immunogenic nucleotide fragments from 3 genes of Mycoplasma hyopneumoniae strain 232 were selected using information software technology. After repeating each fragment three times, a total of 9 nucleotide fragments were joined together to form a new nucleotide sequence called Mhp2321092bp. Mhp2321092bp was directly synthesized and cloned into a pET100 vector and expressed in Escherichia coli. After purification, the proteins were successfully validated by SDS-PAGE and Western blotting using mouse His-tag antibody and pig anti-Mhp serum. BALB/c mice were intraperitoneally injected with purified proteins in the high-dose (100 µg), medium-dose group (50 µg) and low-dose (10 µg) groups. Mice in each group were injected on day 1, day 8 and day 15 of feeding, respectively. Serum samples were collected from all mice on the day before immunization and on day 22 after immunization. The antibody level in the mouse serum was detected using western blotting using purified expressed proteins as antigens. IL-2, TNF-α and IFN-γ were simultaneously detected in the mouse serum by ELISA. The results showed that the 60 kDa protein was successfully expressed and reacted specifically with the specific serum Mhp His-Tag mouse monoclonal antibody and pig anti-Mhp serum. From day 0 to day 22 of immunization, IFN-γ increased from 269.52 to 467.74 pg/mL, IL-2 increased from 14.03 to 145.16 pg/mL, and TNF-α increased from 6.86 to 12.37 pg/mL. The IgG antibody in mice increased significantly from 0 day to day 22 after immunization. This study suggests that the expressed recombinant protein may serve as one of the novel vaccine candidates for Mhp.

Regulatory functional role of NLRP3 inflammasome during Mycoplasma hyopneumoniae infection in swine.

Mycoplasma hyopneumoniae causes enzootic pneumonia, a highly contagious respiratory disease in swine that causes significant economic losses worldwide. It is unknown whether the nucleotide oligomerization domain-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome regulates the immune response in swine during M. hyopneumoniae infection. The current study utilized an in vivo swine model of M. hyopneumoniae infection to investigate the regulatory functional role of the NLRP3 inflammasome during M. hyopneumoniae infection. Notable histopathological alterations were observed in M. hyopneumoniae-infected swine tissues, which were associated with an inflammatory response and disease progression. Swine M. hyopneumoniae infection was associated with an increase in the expression of the NLRP3 inflammasome, which stimulated pro-inflammatory cytokines such as tumor necrosis factor-alpha, interleukin 18, and interleukin 1 beta (IL-1ß). The impact of the NLRP3 inhibitor, MCC950 on NLRP3 and pro-inflammatory cytokines in M. hyopneumoniae-infected swine was examined to investigate the relationship between the NLRP3 inflammasome and M. hyopneumoniae infection. Taken together, our findings provide strong evidence that the NLRP3 inflammasome plays a critical regulatory functional role in M. hyopneumoniae infection in swine.

Our study highlights the importance of controlling the innate immune defense against respiratory mycoplasma invasion to suppress mycoplasma growth and minimize lung tissue damage. Using an in vivo swine model, we investigated the regulatory functional role of the NLR family pyrin domain containing 3 (NLRP3) inflammasome during acute Mycoplasma hyopneumoniae infection. Furthermore, we also found that NLRP3 expression levels have the potential to serve as a novel diagnostic marker for detecting M. hyopneumoniae infection in the respiratory tract of pigs. The NLRP3 inhibitor, MCC950, was used to investigate how NLRP3 inhibition affects the expression of inflammatory cytokines, and it was found that the NLRP3 inhibitor significantly reduced the mRNA and protein expression of NLRP3, indicating its specific targeting of the NLRP3 inflammasome during M. hyopneumoniae infection in swine. The findings suggest that MCC950 is a promising therapeutic option for treating NLRP3-related disorders, including porcine enzootic pneumonia.

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

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

Hijacking of Host Plasminogen by Mesomycoplasma (Mycoplasma) hyopneumoniae via GAPDH: an Important Virulence Mechanism To Promote Adhesion and Extracellular Matrix Degradation.

Mesomycoplasma hyopneumoniae is the etiological agent of mycoplasmal pneumonia of swine (MPS), which causes substantial economic losses to the world's swine industry. Moonlighting proteins are increasingly being shown to play a role in the pathogenic process of M. hyopneumoniae. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in glycolysis, displayed a higher abundance in a highly virulent strain of M. hyopneumoniae than in an attenuated strain, suggesting that it may have a role in virulence. The mechanism by which GAPDH exerts its function was explored. Flow cytometry and colony blot analysis showed that GAPDH was partly displayed on the surface of M. hyopneumoniae. Recombinant GAPDH (rGAPDH) was able to bind PK15 cells, while the adherence of a mycoplasma strain to PK15 was significantly blocked by anti-rGAPDH antibody pretreatment. In addition, rGAPDH could interact with plasminogen. The rGAPDH-bound plasminogen was demonstrated to be activated to plasmin, as proven by using a chromogenic substrate, and to further degrade the extracellular matrix (ECM). The critical site for GAPDH binding to plasminogen was K336, as demonstrated by amino acid mutation. The affinity of plasminogen for the rGAPDH C-terminal mutant (K336A) was significantly decreased according to surface plasmon resonance analysis. Collectively, our data suggested that GAPDH might be an important virulence factor that facilitates the dissemination of M. hyopneumoniae by hijacking host plasminogen to degrade the tissue ECM barrier. IMPORTANCE Mesomycoplasma hyopneumoniae is a specific pathogen of pigs that is the etiological agent of mycoplasmal pneumonia of swine (MPS), which is responsible for substantial economic losses to the swine industry worldwide. The pathogenicity mechanism and possible particular virulence determinants of M. hyopneumoniae are not yet completely elucidated. Our data suggest that GAPDH might be an important virulence factor in M. hyopneumoniae that facilitates the dissemination of M. hyopneumoniae by hijacking host plasminogen to degrade the extracellular matrix (ECM) barrier. These findings will provide theoretical support and new ideas for the research and development of live-attenuated or subunit vaccines against M. hyopneumoniae.

Ureaplasma diversum-Ureaplasma sp. alone or concomitantly with Mycoplasma hyopneumoniae in pig lungs with and without pneumonia: A descriptive exploratory study in abattoirs.

Occurrence of Ureaplasma diversum (U. diversum) has been associated with reproductive failures in cattle and detected in pigs with and without pneumonia. However, its role in the porcine respiratory disease complex (PRDC) is unclear. A cross-sectional study was conducted in abattoirs, inspecting 280 pig lungs from eight herds. All the lungs were inspected, processed and classified according to the histopathological analysis. Moreover, bronchoalveolar lavage (BAL) specimens were collected and processed by PCR for detection of U. diversum and Mycoplasma hyopneumoniae (M. hyopneumoniae). Ureaplasma sp.-U. diversum and M. hyopneumoniae were detected in 17.1% and 29.3% of the analyzed BAL specimens, respectively. The concomitant presence of both microorganisms was detected in 12.5% of the inspected lungs. Both agents were found in lungs with and without pneumonia. M. hyopneumoniae was detected in 31.8% of pig lungs with enzootic pneumonia-like lesions, while Ureaplasma sp.-U. diversum was detected in 27.5% of lungs with these lesions. This descriptive exploratory study provides information for future experimental and field-based studies to better define the pathogenic role of this organism within the PRDC.

Efficacy evaluation of a novel oral silica-based vaccine in inducing mucosal immunity against Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae, the main etiological agent of Porcine Enzootic Pneumonia, is widely spread in swine production worldwide. Its prevention is of great interest for the productive system, since its colonization in the lung tissue leads to intense production losses. This study aimed to compare the M. hyopneumoniae shedding and acute-phase response in 30 pigs submitted to different vaccination protocols: an experimental oral vaccine using a nanostructured mesoporous silica (SBA-15) as adjuvant (n = 10); an intramuscular commercially available vaccine at 24 days of age (n = 10); and a control group (n = 10) following experimental challenge with M. hyopneumoniae. Laryngeal and nasal swabs were collected weekly and oral fluids were collected at 7, 10, 14, 17, 23, 28, 35, 42, and 49 days post-infection to monitor pathogen excretion by qPCR. Nasal swabs were also used to detect anti-M. hyopneumoniae IgA by ELISA. Blood samples were collected for monitoring acute phase proteins. The antibody response was observed in both immunized groups seven days after vaccination, while the control group became positive for this immunoglobulin at 4 weeks after challenge. Lung lesion score was similar in the immunized groups, and lower than that observed in the control. SBA-15-adjuvanted oral vaccine provided immunological response, decreased shedding of M. hyopneumoniae and led to mucosal protection confirmed by the reduced pulmonary lesions. This study provides useful data for future development of vaccines against M. hyopneumoniae.

Use of Nanostructured Silica SBA-15 as an Oral Vaccine Adjuvant to Control Mycoplasma hyopneumoniae in Swine Production.

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.

Long-term follow-up of Mycoplasma hyopneumoniae-specific immunity in vaccinated pigs.

Mycoplasma hyopneumoniae is the primary agent of enzootic pneumonia in pigs. To minimize the economic losses caused by this disease, M. hyopneumoniae vaccination is commonly practiced. However, the persistence of M. hyopneumoniae vaccine-induced immunity, especially the cell-mediated immunity, till the moment of slaughter has not been investigated yet. Therefore, on two commercial farms, 25 pigs (n = 50) received a commercial bacterin intramuscularly at 16 days of age. Each month, the presence of M. hyopneumoniae-specific serum antibodies was analyzed and the proliferation of and TNF-α, IFN-γ and IL-17A production by different T cell subsets in blood was assessed using recall assays. Natural infection with M. hyopneumoniae was assumed in both farms. However, the studied pigs remained M. hyopneumoniae negative for almost the entire trial. Seroconversion was not observed after vaccination and all pigs became seronegative at two months of age. The kinetics of the T cell subset frequencies was similar on both farms. Mycoplasma hyopneumoniae-specific cytokine-producing CD4+CD8+ T cells were found in blood of pigs from both farms at one month of age but decreased significantly with increasing age. On the other hand, T cell proliferation after in vitro M. hyopneumoniae stimulation was observed until the end of the fattening period. Furthermore, differences in humoral and cell-mediated immune responses after M. hyopneumoniae vaccination were not seen between pigs with and without maternally derived antibodies. This study documents the long-term M. hyopneumoniae vaccine-induced immune responses in fattening pigs under field conditions. Further research is warranted to investigate the influence of a natural infection on these responses.

Effects of a water-soluble formulation of tylvalosin on disease caused by porcine reproductive and respiratory syndrome virus alone in sows or in combination with Mycoplasma hyopneumoniae in piglets.

BACKGROUND: The effect of a water-soluble formulation of tylvalosin (Aivlosin® 625 mg/g granules) on disease caused by porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae (Mhyop) was investigated in two animal studies. In a PRRSV challenge model in pregnant sows (n = 18), six sows received water medicated at target dose of 5 mg tylvalosin/kg body weight/day from 3 days prior to challenge until the end of gestation. Six sows were left untreated, with a third group remaining untreated and unchallenged. Sows were challenged with PRRSV-2 at approximately 85 days of gestation. Cytokines, viremia, viral shedding, sow reproductive parameters and piglet performance to weaning were evaluated. In a dual infection study (n = 16), piglets were challenged with Mhyop on days 0, 1 and 2, and with PRRSV-1 on day 14 and euthanized on day 24. From day 10 to 20, eight piglets received water medicated at target dose of 20 mg tylvalosin/kg body weight/day and eight piglets were left untreated. Cytokines, viremia, bacteriology and lung lesions were evaluated. RESULTS: In the PRRSV challenge study in pregnant sows, tylvalosin significantly reduced the levels of serum IL-8 (P < 0.001), IL-12 (P = 0.032), TNFα (P < 0.001) and GM-CSF (P = 0.001). IL-8 (P = 0.100) tended to be lower in uterus of tylvalosin sows. All piglets from tylvalosin sows surviving to weaning were PRRSV negative in faecal swabs at weaning compared to 33.3% PRRSV positive piglets from untreated sows (P = 0.08). In the dual challenge study in piglet, tylvalosin reduced serum IL1ß, IL-4, IL-6, IL-8, IL-10, IL-12, IL-1α, IL-13, IL-17A, IL-18, GM-CSF, TGFß1, TNFα, CCL3L1, MIG, PEPCAM-1 (P < 0.001) and increased serum IFNα, IL-1ra and MIP-1b (P < 0.001). In the lungs, tylvalosin reduced IL-8, IL-10 and IL-12 compared to untreated pigs (P < 0.001) and tended to reduce TNFα (P = 0.082). Lung lavage samples from all tylvalosin treated piglets were negative for Mhyop (0 cfu/mL) compared to the untreated piglets which had mean Mhyop counts of 2.68 × 104 cfu/mL (P = 0.023). CONCLUSION: Overall, tylvalosin reduced both local and systemic proinflammatory cytokines after challenge with respiratory pathogens in sows and in piglets. Tylvalosin was effective in reducing Mhyop recovery from the lungs and may reduce virus shedding in piglets following transplacental PRRSV infection in sows.

Condenações de carcaça suínas devido a doenças respiratórias revisão bibliográfica / Pork carcass condemnation due to respiratory diseases bibliographic review / Condena de canales de cerdo por enfermedades respiratorias revisión bibliográfica

As doenças respiratórias são um problema significativo na produção suína e podem levar à condenação de carcaças no abate. Entre os agentes causadores dessas doenças destacam-se o Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae e a Pasteurella multocida. O Actinobacillus pleuropneumoniae é um patógeno altamente contagioso, que ocasiona hemorragia, pleuropneumonia purulenta e fibrosa. A Pleuropneumonia é amplamente distribuída e gera graves prejuízos para a suinocultura. O Mycoplasma hyopneumoniae ocasionador da pneumonia por micoplasma, doença respiratória crônica. As infecções originadas podem regular negativamente o sistema imunológico do hospedeiro e aumentar a infecção e assim a replicação de outros patógenos. A Pasteurella multocida é o agente causador de uma ampla gama de infecções levando a alto impacto econômico. Patógeno comensal e oportunista da boca, nasofaringe e trato respiratório superior. A identificação precoce e o manejo adequado desses agentes causadores de doenças respiratórias são fundamentais para minimizar a incidência de carcaças suínas. A adoção de medidas preventivas, como a vacinação e práticas de manejo adequadas, pode ajudar a prevenir a propagação dessas doenças e garantir a produção de carne suína segura e de alta qualidade para o consumo humano.(AU)

Respiratory diseases are a significant problem in pork production and can lead to condemnation of carcasses at slaughter. Among the causative agents of these diseases are Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae and Pasteurella multocida. Actinobacillus pleuropneumoniae is a highly contagious pathogen that causes hemorrhage, purulent and fibrous pleuropneumonia. Pleuropneumonia is widely distributed and causes serious damage to pig farming. Mycoplasma hyopneumoniae causes mycoplasma pneumonia, a chronic respiratory disease. Originating infections can down-regulate the host's immune system and increase infection and thus replication of other pathogens. Pasteurella multocida is the causative agent of a wide range of infections leading to high economic impact. Commensal and opportunistic pathogen of the mouth, nasopharynx and upper respiratory tract. Early identification and proper management of these agents that cause respiratory diseases are essential to minimize the incidence of swine carcasses. Adopting preventive measures, such as vaccination and proper management practices, can help prevent the spread of these diseases and ensure the production of safe, high-quality pork for human consumption.(AU)

Las enfermedades respiratorias son un problema importante en la producción porcina y pueden provocar el decomiso de las canales en el matadero. Entre los agentes causantes de estas enfermedades se encuentran Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae y Pasteurella multocida. Actinobacillus pleuropneumoniae es un patógeno altamente contagioso que causa hemorragia, pleuroneumonía purulenta y fibrosa. La pleuroneumonía está ampliamente distribuida y causa graves daños a la cría de cerdos. Mycoplasma hyopneumoniae causa neumonía por micoplasma, una enfermedad respiratoria crónica. Las infecciones que se originan pueden regular a la baja el sistema inmunitario del huésped y aumentar la infección y, por lo tanto, la replicación de otros patógenos. Pasteurella multocida es el agente causal de una amplia gama de infecciones que tienen un alto impacto económico. Patógeno comensal y oportunista de la boca, nasofaringe y tracto respiratorio superior. La identificación temprana y el manejo adecuado de estos agentes causantes de enfermedades respiratorias son fundamentales para minimizar la incidencia de las canales porcinas. La adopción de medidas preventivas, como la vacunación y prácticas de manejo adecuadas, puede ayudar a prevenir la propagación de estas enfermedades y garantizar la producción de carne de cerdo segura y de alta calidad para el consumo humano.(AU)

Identification of the multiple roles of enolase as an plasminogen receptor and adhesin in Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is the etiological agent underlying porcine enzootic pneumonia, a chronic respiratory disease worldwide. The recruitment of plasminogen to the surface and subsequently promotion of plasmin conversion by the surface-located receptor, have been reported to assist the adhesion and invasion of Mycoplasmas. The surface localization and plasminogen-binding ability of M. hyopneumoniae enolase were previously confirmed; however, the biological functions were not be determined, especially the role as a plasminogen receptor. Here, using ELISA and SPR analyses, we confirmed the stable binding of M. hyopneumoniae enolase to plasminogen in a dose-dependent manner. The facilitation of the activation of plasminogen in the presence of tPA and direct activation of plasminogen at low efficiency without tPA addition by M. hyopneumoniae enolase were also determined using a plasmin-specific chromogenic substrate. Notably, the C-terminal and N-terminal regions located in M. hyopneumoniae enolase play an important role in plasminogen binding and activation. Additionally, we demonstrate that M. hyopneumoniae enolase can competitively inhibit the adherence of M. hyopneumoniae to PK15 cells. These results provide insight into the role of enolase in M. hyopneumoniae infection, a mechanism that manipulates the proteolytic system of the host.

Porcine antibody profiles of 33 Mycoplasma hyopneumoniae fusion proteins from M. hyopneumoniae natural infection but not vaccination.

BACKGROUND: Mycoplasma hyopneumoniae, the primary pathogen responsible for porcine enzootic pneumonia, reduces average daily weight gain and causes substantial economic losses to the pig industry worldwide. Vaccination is the most common strategy to control this disease but offers partial protection. Therefore, developing next-generation vaccines by screening protective antigens is crucial. OBJECTIVES: The aim of this study was to evaluate the antibody response to 33 recombinant proteins in pigs naturally infected with M. hyopneumoniae. METHODS: The genes encoding 33 (hypothetical) membrane proteins or secretory proteins were ligated into pGEX-6P-1, pGEX-6P-2, pGEX-5X-3 or pGEX-4T-3 vectors and transformed into Escherichia coli BL21(DE3) or E. coli XL-1 Blue to construct recombinant bacteria and to express the recombinant proteins. The recombinant bacteria expressing the target proteins reacted with porcine convalescent sera and negative sera to screen immunodominant proteins by ELISA. Then, recombinant bacteria expressing immunodominant proteins were used to identify the discriminating immunodominant proteins that were recognised by convalescent sera nut not hyperimmune sera. RESULTS: All recombinant bacteria could express the target recombinant proteins in soluble form. Twenty-one proteins were shown to present immunodominant antigens, and four proteins were not recognised by convalescent sera. Moreover, six proteins were considered discriminating and reacted with convalescent sera but not with hyperimmune sera. CONCLUSIONS: The identified immunodominant proteins were antigenic and expressed during bacterial infection, suggesting that these proteins, especially those capable of discriminating between sera, can be used to identify protective antigens with the view to develop more effective vaccines against M. hyopneumoniae infection.