A field evaluation of a new porcine circovirus type 2d and Mycoplasma hyopneumoniae bivalent vaccine in herds suffering from subclinical PCV2d infection and enzootic pneumonia.

BACKGROUND: This field efficacy study was designed to determine the efficacy of a new bivalent vaccine containing porcine circovirus type 2d (PCV2d) and Mycoplasma hyopneumoniae at three independent pig farms. METHODS: Three pig farms were selected based on their history of subclinical PCV2 infection and enzootic pneumonia. Each farm housed a total of 40, 18-day-old pigs that were randomly allocated to 1 of 2 treatment groups. Pigs were administered a 2.0 mL dose of the bivalent vaccine intramuscularly at 21 days of age in accordance with the manufacturer's recommendations, whereas unvaccinated pigs were administered a single dose of phosphate-buffered saline at the same age. RESULTS: Clinically, the average daily weight gain of vaccinated groups was significantly higher (p < 0.05) than those of unvaccinated animals during the growing (70-112 days of age), finishing (112-175 days of age) and overall (3-175 days of age) stages of production. Vaccinated animals elicited neutralizing anti-PCV2 antibodies and PCV2d-specific interferon-γ secreting cells (IFN-γ-SC), which reduced the amount of PCV2d genomic copies in blood and reduced lymphoid lesions severity when compared with unvaccinated animals. Similarly, vaccinated animals elicited M. hyopneumoniae-specific IFN-γ-SC, which reduced the amount of M. hyopneumoniae in the larynx and reduced lung lesions severity. CONCLUSIONS: The result of the field trial demonstrated that the bivalent vaccine was efficacious in the protection of swine herds suffering from subclinical PCV2d infection and enzootic pneumonia.

Discovery of the rich diversity of Mesomycoplasma hyopneumoniae through high-throughput sequencing.

Pneumonia caused by Mesomycoplasma hyopneumoniae (Mhp) is a respiratory disease with high morbidity and low mortality that typically presents in growing pigs. Although often subclinical, the disease can significantly affect the pig farming industry economically due to decreased growth rates and inefficient feed conversion. Effective control of Mhp depends on the detection of dominant strains prevalent in infected animals, which vary in virulence. However, traditional culture methods for diagnosing Mhp are laborious and slow, whereas multi-locus sequence typing, another possible method, requires identifying several genes. This study introduces a novel pair of polymerase chain reaction (PCR) primers for the rapid detection and genetic evolution analysis of Mhp strains to facilitate improved vaccine selection. The genetic evolutionary tree established using the PCR amplification fragment was highly similar to the genetic evolutionary tree established using whole-genome sequences. Analysis of 131 samples from Guangxi and Hunan slaughterhouses revealed a 30.53 % prevalence of Mhp. High-throughput sequencing has shown that Mhp has a diverse bacterial population in clinically collected samples. The prevalence of major strains may vary among regions. Additionally, the strains of Mhp vaccines sold may differ significantly from the strains prevalent on farms. In summary, this work has designed a pair of primers that will be useful for detecting the diversity of Mhp and for targeted prevention and control.

Shedding reduction and immunity modulation in piglets with an inactivated Mycoplasma hyopneumoniae vaccine encapsulated in nanostructured SBA-15 silica.

Mycoplasma (M.) hyopneumoniae is a primary etiological agent of porcine enzootic pneumonia (PEP), a disease that causes significant economic losses to pig farming worldwide. Current commercial M. hyopneumoniae vaccines induce partial protection, decline in preventing transmission of this pathogen or inducing complete immunity, evidencing the need for improving vaccines against PEP. In our study, we aimed to test the effectiveness of the SBA-15 ordered mesoporous silica nanostructured particles as an immune adjuvant of a vaccine composed of M. hyopneumoniae strain 232 proteins encapsulated in SBA-15 and administered by intramuscular route in piglets to evaluate the immune responses and immune-protection against challenge. Forty-eight 24-day-old M. hyopneumoniae-free piglets were divided into four experimental groups with different protocols, encompassing a commercial vaccine against M. hyopneumoniae, SBA-15 vaccine, SBA-15 adjuvant without antigens and a non-immunized group. All piglets were challenged with the virulent strain 232 of M. hyopneumoniae. Piglets that received the SBA-15 and commercial vaccine presented marked immune responses characterized by anti-M. hyopneumoniae IgA and IgG antibodies in serum, anti-M. hyopneumoniae IgA antibodies in nasal mucosa and showed an upregulation of IL-17 and IL-4 cytokines and downregulation of IFN-γ in lungs 35 days post-infection. Piglets immunized with SBA-15 vaccine presented a reduction of bacterial shedding compared to piglets immunized with a commercial bacterin. In addition, piglets from SBA-15 adjuvant suspension group presented increased IL-17 gene expression in the lungs without involvement of Th1 and Th2 responses after challenge. These results indicated that SBA-15 vaccine induced both humoral and cell-mediated responses in the upper respiratory tract and lungs, first site of replication and provided protection against M. hyopneumoniae infection with a homologous strain with reduction of lung lesions and bacterial shedding. Finally, these results enhance the potential use of new technologies such as nanostructured particles applied in vaccines for the pig farming industry.

Suspected aerosol transmission of swine pathogens: A field case.

A swine production system had 3 sections located a few kilometers apart. Sections A and C contained several thousand sows and nursery and finishing pigs. Section B, located between the other 2 sections, was the smallest and had 6 finishing sites and 2 sow sites. The entire system was infected with porcine reproductive and respiratory syndrome virus, Mycoplasma hyopneumoniae, and Actinobacillus pleuropneumoniae. Section B was depopulated, cleaned, disinfected, and repopulated with negative gilts. Despite extreme measures, recontamination occurred for each pathogen, with aerosol considered the most plausible contamination source.

Transmission suspectée d'agents pathogènes porcins par aérosol : un cas de terrainUn système de production porcine comportait 3 sections situées à quelques kilomètres l'une de l'autre. Les sections A et C contenaient plusieurs milliers de truies et de porcs en maternité et en finition. La section B, située entre les 2 autres sections, était la plus petite et comptait 6 sites de finition et 2 sites de truies. L'ensemble du système était infecté par le virus du syndrome reproducteur et respiratoire porcin, Mycoplasma hyopneumoniae et Actinobacillus pleuropneumoniae. La section B a été dépeuplée, nettoyée, désinfectée et repeuplée de cochettes négatives. Malgré des mesures extrêmes, une recontamination s'est produite pour chaque agent pathogène, les aérosols étant considérés comme la source de contamination la plus plausible.(Traduit par Dr Serge Messier).

[Development of an ELISA method for detection of antibodies against Mycoplasma hyopneumoniae based on Mhp336 protein].

This study aims to establish an ELISA method with high specificity for the detection of antibodies against Mycoplasma hyopneumoniae. Firstly, we constructed a recombinant strain Escherichia coli BL21(DE3)-pET-32a(+)-mhp336 to express the recombinant protein Mhp336 and used the purified Mhp336 as the coating antigen. Then, we optimized the ELISA parameters, including antigen concentration, blocking buffer, blocking time, dilution of serum, incubation time with serum, secondary antibody dilution, secondary antibody incubation time, colorimetric reaction time, and cut-off value. Afterwards, reproducibility experiments were conducted, and the cross reactivity of Mhp366 with other antisera of porcine major pathogens and the maximum dilution ratios of the sera were determined. Finally, 226 porcine serum samples were detected using the method established in this study, a commercial ELISA kit for M. hyopneumoniae antibody detection, and a convalescent serum ELISA kit for M. hyopneumoniae antibody detection. The detection results of the three methods were compared to evaluate the sensitivity and specificity of the ELISA method established in this study. For this method, the optimal antigen concentration, blocking buffer, blocking time, dilution of serum, incubation time with serum, secondary antibody dilution, secondary antibody incubation time, and colorimetric reaction time were 0.05 µg/mL, PBS containing 2.5% skim milk, 1 h, 1:500, 0.5 h, 1:10 000, 1 h, and 5 min, respectively. Validation of the ELISA method based on Mhp336 showed a cut-off value of 0.332. The coefficients of variation of both intra-batch and inter-batch kits were below 7%. The detection results of porcine serum samples indicated that the method established in this study outperformed the commercial ELISA kit and the convalescent serum ELISA kit for M. hyopneumoniae antibody detection in terms of sensitivity and specificity. We successfully established an ELISA method for detecting the antibodies against M. hyopneumoniae based on Mhp336 protein. This method demonstrated high sensitivity and specificity, serving as a tool for the prevention of mycoplasmal pneumonia of swine in pig farms.

Effect of pooled tracheal sample testing on the probability of Mycoplasma hyopneumoniae detection.

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

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.

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.

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.

[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.

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.

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.

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.

Genome-wide detection of changes in allelic frequency in Landrace pigs selected for resistance to mycoplasma pneumonia of swine.

Closed-pig line breeding could change the genetic structure at a genome-wide scale because of the selection in a pig breeding population. We investigated the changes in population structure among generations at a genome-wide scale and the selected loci across the genome by comparing the observed and expected allele frequency changes in mycoplasma pneumonia of swine (MPS)-selected pigs. Eight hundred and seventy-four Landrace pigs, selected for MPS resistance without reducing average daily gain over five generations, had 37,299 single nucleotide polymorphisms (SNPs) and were used for genomic analyses. Regarding population structure, individuals in the first generation were the most widely distributed and then converged into a specific group, as they were selected over five generations. For allele frequency changes, 96 and 14 SNPs had higher allele frequency changes than the 99.9% and 99.99% thresholds of the expected changes, respectively. These SNPs were evenly spread across the genome, and a few of these selected regions overlapped with previously detected quantitative trait loci for MPS and immune-related traits. Our results indicated that the considerable changes in allele frequency were identified in many regions across the genome by closed-pig line breeding based on estimated breeding value.

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.

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

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

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.