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

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

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

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

Influence of genetics and the pre-vaccination blood transcriptome on the variability of antibody levels after vaccination against Mycoplasma hyopneumoniae in pigs.

BACKGROUND: The impact of individual genetic and genomic variations on immune responses is an emerging lever investigated in vaccination strategies. In our study, we used genetic and pre-vaccination blood transcriptomic data to study vaccine effectiveness in pigs. RESULTS: A cohort of 182 Large White pigs was vaccinated against Mycoplasma hyopneumoniae (M. hyo) at weaning (28 days of age), with a booster 21 days later. Vaccine response was assessed by measuring seric M. hyo antibodies (Ab) at 0 (vaccination day), 21 (booster day), 28, 35, and 118 days post-vaccination (dpv). Inter-individual variability of M. hyo Ab levels was observed at all time points and the corresponding heritabilities ranged from 0.46 to 0.57. Ab persistence was higher in females than in males. Genome-wide association studies with a 658 K SNP panel revealed two genomic regions associated with variations of M. hyo Ab levels at 21 dpv at positions where immunity-related genes have been mapped, DAB2IP on chromosome 1, and ASAP1, CYRIB and GSDMC on chromosome 4. We studied covariations of Ab responses with the pre-vaccination blood transcriptome obtained by RNA-Seq for a subset of 82 pigs. Weighted gene correlation network and differential expression analyses between pigs that differed in Ab responses highlighted biological functions that were enriched in heme biosynthesis and platelet activation for low response at 21 dpv, innate antiviral immunity and dendritic cells for high response at 28 and 35 dpv, and cell adhesion and extracellular matrix for high response at 118 dpv. Sparse partial least squares discriminant analysis identified 101 genes that efficiently predicted divergent responders at all time points. We found weak negative correlations of M. hyo Ab levels with body weight traits, which revealed a trade-off that needs to be further explored. CONCLUSIONS: We confirmed the influence of the host genetics on vaccine effectiveness to M. hyo and provided evidence that the pre-vaccination blood transcriptome co-varies with the Ab response. Our results highlight that both genetic markers and blood biomarkers could be used as potential predictors of vaccine response levels and more studies are required to assess whether they can be exploited in breeding programs.

Development of an indirect ELISA for detection of anti-Mycoplasma hyopneumoniae IgG in naturally infected pathogen-induced convalescent sera.

BACKGROUND: Immunization of pigs with an inactivated Mycoplasma hyopneumoniae vaccine (bacterin) generates hyperimmune serum that contains high concentrations of anti-M. hyopneumoniae IgG. Commercially available IgG-ELISA kits cannot distinguish between anti-M. hyopneumoniae IgG in inactivated bacterin-induced hyperimmune sera and convalescent sera resulting from natural M. hyopneumoniae infection. Establishment of an ELISA to detect anti-M. hyopneumoniae IgG in convalescent sera will facilitate the evaluation of the M. hyopneumoniae status of pig farms. RESULTS: In this study, we expressed and purified recombinant Mhp366-N protein, which contains an epitope recognized by M. hyopneumoniae convalescent sera but not hyperimmune sera, for use as a coating antigen. For the M. hyopneumoniae convalescent serum IgG-ELISA, 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 0.25 µg/mL, 2.5 % skim milk, 1 h, 1:500, 0.5 h, 1:10,000, 1 h and 15 min, respectively. Validation of the M. hyopneumoniae convalescent serum IgG-ELISA showed a cut-off value of 0.323, the intra-assay CV ranged from 3.27 to 7.26 %, the inter-assay CV ranged from 3.46 to 5.93 %, and the assay was able to differentiate convalescent sera from antibodies to 7 other porcine respiratory pathogens. The convalescent serum IgG-ELISA detected no anti-M. hyopneumoniae IgG in hyperimmune serum samples while a commercial IgG-ELISA identified 95/145 of these sera as positive. The accuracy of the M. hyopneumoniae convalescent serum IgG-ELISA was comparable to the sIgA-ELISA but better than the commercial IgG-ELISA. CONCLUSIONS: The convalescent serum IgG-ELISA is a reproducible, sensitive, and specific indirect ELISA to detect anti-M. hyopneumoniae IgG in naturally infected pathogen-induced convalescent sera. This ELISA could be used to carry out large scale surveillance of M. hyopneumoniae infection in pig farms regardless of vaccination status.

Antibody responses to porcine reproductive and respiratory syndrome virus, influenza A virus, and Mycoplasma hyopneumoniae from weaning to the end of the finisher stage in fourteen groups of pigs in Ontario, Canada.

BACKGROUND: Respiratory diseases are among the most important factors affecting swine farm productivity in Canada. The objectives of this study were to investigate antibody responses to porcine reproductive and respiratory syndrome virus (PRRSV), influenza A virus (IAV), and Mycoplasma hyopneumoniae (M. hyopneumoniae) from weaning to the end of the finisher stage on a subset of commercial swine farms in Ontario, Canada, and to examine the association between nursery diet and antibody responses. RESULTS: Overall, older pigs were more likely to test seropositive for PRRSV and less likely to test seropositive for M. hyopneumoniae (p <  0.001). Pigs were more likely to test seropositive for IAV at weaning and the end of the grower and finisher stages compared to the end of nursery (p <  0.001). Pigs that were seropositive for IAV were more likely to test seropositive for both PRRSV and M. hyopneumoniae (p <  0.001). Two, 9, and 4 groups that had more than 20% of pigs seropositive to PRRSV, IAV, and M. hyopneumoniae, respectively, from the end of nursery to the end of finisher were classified as seropositive. Pigs fed a plant-based (low complexity) diet during nursery were more likely to be seropositive for PRRSV (p <  0.001) but there were no significant differences in seropositivity to IAV or M. hyopneumoniae due to nursery diet complexity. CONCLUSIONS: This study provides information regarding changes in serum antibody in pigs across different stages of production and highlights periods of vulnerability. Additionally, these findings may encourage further research into the effects of nursery diet complexity on disease susceptibility and immune response.

Genomic Variability and Post-translational Protein Processing Enhance the Immune Evasion of Mycoplasma hyopneumoniae and Its Interaction With the Porcine Immune System.

Mycoplasma hyopneumoniae (M. hyopneumoniae, Mhp) is a geographically widespread and economically devastating pathogen that colonizes ciliated epithelium; the infection of Mhp can damnify the mucociliary functions as well as leading to Mycoplasma pneumonia of swine (MPS). MPS is a chronic respiratory infectious disease with high infectivity, and the mortality can be increased by secondary infections as the host immunity gets down-regulated during Mhp infection. The host immune responses are regarded as the main driving force for the disease development, while MPS is prone to attack repeatedly in farms even with vaccination or other treatments. As one of the smallest microorganisms with limited genome scale and metabolic pathways, Mhp can use several mechanisms to achieve immune evasion effect and derive enough nutrients from its host, indicating that there is a strong interaction between Mhp and porcine organism. In this review, we summarized the immune evasion mechanisms from genomic variability and post-translational protein processing. Besides, Mhp can induce the immune cells apoptosis by reactive oxygen species production, excessive nitric oxide (NO) release and caspase activation, and stimulate the release of cytokines to regulate inflammation. This article seeks to provide some new points to reveal the complicated interaction between the pathogen and host immune system with Mhp as a typical example, further providing some new strategies for the vaccine development against Mhp infection.

IL-12p40 gene expression in lung and hilar lymph nodes of MPS-resistant pigs.

Mycoplasma pneumonia of swine (MPS) is caused by Mycoplasma hyopneumoniae (M.hp) and is a common chronic respiratory disease of pigs. Recently, a genetically selected variant of the Landrace pig (Miyagino L2) has a lower incidence of pulmonary MPS lesions. We investigated the pathological and immunological characteristics of MPS resistance in these pigs (n = 24) by comparing with the normal landrace pig (control: n = 24). The pathological MPS lung lesion score in MPS-selected landrace pigs was significantly lower than in the control. The gene expression of interleukin (IL)-12p40, which acts as a chemoattractant and a component of the bioactive cytokines IL-12 and IL-23, was significantly higher at the hilar lymph nodes, lung, and spleen in MPS-selected landrace pigs than in control landrace pigs, and these were negatively correlated with the macroscopic MPS lung lesion score. In summary, we demonstrate that resistance against MPS in Miyagino L2 pigs is associated with IL-12p40 up-regulation, in comparison with normal landrace pigs without the MPS vaccine. In addition, a comparative study of macroscopic MPS lung lesions and IL-12p40 gene expression in lung and hilar lymph nodes may lead to beneficial selection traits for the genetic selection for MPS resistance in pigs.

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

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

Kinome profiling of peripheral blood mononuclear cells collected prior to vaccination reveals biomarkers and potential mechanisms of vaccine unresponsiveness in pigs.

Inter-individual variance in host immune responses following vaccination can result in failure to develop protective immunity leaving individuals at risk for infection in addition to compromising herd immunity. While developing more efficacious vaccines is one strategy to mitigate this problem, predicting vaccine responsiveness prior to vaccination could inform which individuals require adjunct disease management strategies. To identify biomarkers of vaccine responsiveness, a cohort of pigs (n = 120) were vaccinated and pigs representing the high (n = 6; 90th percentile) and low (n = 6; 10th percentile) responders based on vaccine-specific antibody responses following vaccination were further analyzed. Kinase-mediated phosphorylation events within peripheral blood mononuclear cells collected prior to vaccination identified 53 differentially phosphorylated peptides when comparing low responders with high responders. Functional enrichment analysis revealed pro-inflammatory cytokine signaling pathways as dysregulated, and this was further substantiated by detection of higher (p < 0.01) concentrations of interferon-gamma in plasma of low responders compared to high responders prior to vaccination. In addition, low responder pigs with high plasma interferon-gamma showed lower (p < 0.01) birth weights than high responder pigs. These associations between vaccine responsiveness, cytokine signaling within peripheral immune cells, and body weight in pigs provide both evidence and insight into potential biomarkers for identifying low responders to vaccination.

Cytokine expression and Mycoplasma hyopneumoniae burden in the development of lung lesions in experimentally inoculated pigs.

This study aimed to assess immunopathological factors and M. hyopneumoniae (M. hyo) load in macroscopic lesion formation at four timepoints after experimental infection of swine. To do this, 24 M. hyo-free pigs were divided into two groups: non-inoculated control (n = 8) and inoculated (n = 16). At day 0 post-infection (dpi), animals of infected group were intratracheally inoculated with 5 mL of lung inoculum containing 107 CCU (Color Changing Units) ∕mL of M. hyo strain 232, while control group was mock infected with 5 mL of sterilized Friis medium. At 14, 28, 42 and 56 dpi, four animals from the infected group and two from the control group were euthanized and necropsied. The extent of macroscopic lung lobe lesions was visually assessed, scored and lesion samples (qPCR, histopathology and gene expression) were collected. The macroscopic lesion score and estimated M. hyo load (in copies/µL) at the different timepoints were: 14 dpi: 18.5 %-1.55 × 103 copies∕µL; 28dpi: 15.8 %-8.4 × 103 copies∕µL; 42 dpi: 7.0 %-3.2 × 104 copies∕µL and 56 dpi: 6.3 %-1.11 × 105 copies∕µL; Significant and positive correlations between macroscopic lung lesion and the pathogen load were found (coefficient range: 0.77-0.99). The cytokine's IL-6 (0.73) and INF-γ (-0.69) gene expression were significantly (p < 0.05) correlated to macroscopic lung lesion score while IL-8, TNF- α, IL-1α and IL-1ß were associated to other pathological effects such as losses in average daily weight gain and microscopic lesion score. The results provide a better understanding about the pathogenicity of M. hyo strain 232 and the host-pathogen interactions, which may be helpful for the development of new treatments or control measures.

Seroprevalences of multi-pathogen and description of farm movement in pigs in two provinces in Vietnam.

BACKGROUND: In Vietnam, lack of animal health information is considered a major challenge for pig production. The main objective of this study was to assess the seroprevalences of five pathogens [porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), mycoplasma hyopneumoniae (M. hyo), Japanese encephalitis virus (JEV) and leptospirosis] and to better characterize the farm movements through a survey. RESULTS: A total of 600 samples were collected from 120 farms from Bac Giang and Nghe An. Among unvaccinated herds, the highest seroprevalence was found for JE with 73.81% (95% CI: 68.39-78.74) in Bac Giang and 53.51% (95% CI 47.68-59.27) in Nghe An. Seroprevalences for PCV2 and M.hyo were 49.43% (95% CI: 45.06-53.80) and 46.06% (95% CI: 41.48-50.69) among unvaccinated animals. Accumulative co-infections for JE (86.25%) showed the highest level followed by M. hyo (66.25%) and PCV2 (62.50%). Three co-infections with JE had the highest positive rate (28.75%) followed by four co-infections (25.0%). Medium farms had relatively higher herd prevalences for all pathogens, except from leptospirosis. Overall, farmers exported/imported their pigs at the most 1-2 times every 6 months. Some respondents (5% for exportation and 20% for importation) had moved pigs more than 6 times over the last 6 months. CONCLUSIONS: Our study provided another pool of evidence that showed that PCV2, PRRS and H. hyo are endemic in pigs in Vietnam. Given the economic impacts of these pathogens elsewhere, the findings confirm the need for studies to evaluate the association between antibody response and clinical relevance as well as to assess the economic impact of co-infections at farm level. We also found that high seroprevalences of JE and leptospirosis were detected in pigs. From a pubic health point of view, it is crucial to raise public awareness especially for high risk occupations (mainly pig farm workers).

Evaluation of the efficacy of a trivalent vaccine mixture against a triple challenge with Mycoplasma hyopneumoniae, PCV2, and PRRSV and the efficacy comparison of the respective monovalent vaccines against a single challenge.

BACKGROUND: The objective of this study was to assess the efficacy of a trivalent vaccine mixture and compare it to the respective monovalent vaccines against Mycoplasma hyopneumoniae, porcine circovirus type 2 (PCV2), and porcine reproductive and respiratory syndrome virus (PRRSV). RESULTS: Pigs that were triple challenged with M. hyopneumoniae, PCV2, and PRRSV following vaccination with the trivalent vaccine mixture exhibited a significantly better growth performance when compared to unvaccinated and challenged pigs. A statistical difference was not found when comparing pig populations which were vaccinated with the trivalent vaccine followed by a triple challenge and pigs vaccinated with monovalent M hyopneumoniae vaccine followed by mycoplasmal single challenge in the following areas: M. hyopneumoniae nasal shedding, the number of M. hyopneumoniae-specific interferon-γ secreting cells (IFN-γ-SC), and mycoplasmal lung lesion scores. Pigs vaccinated with the trivalent vaccine mixture followed by a triple challenge resulted in a similar reduction of PCV2 viremia, an increase in the number of PCV2-specific IFN-γ-SC and reduction in interstitial lung lesion scores when compared to pigs vaccinated with a PCV-2 vaccine and challenged with PCV2 only. Lastly, there was a significant difference in the reduction of PRRSV viremia, an increase in PRRSV-specific IFN-γ-SC and a reduction of interstitial lung lesion scores between pigs vaccinated with the trivalent vaccine mixture followed by a triple challenge and pigs vaccinated with a monovalent PRRSV vaccine followed by PRRSV challenge only. CONCLUSION: The trivalent vaccine mixture was efficacious against a triple challenge of M. hyopneumoniae, PCV2, and PRRSV. The trivalent vaccine mixture, however, did not result in equal protection when compared against each respective monovalent vaccine, with the largest vaccine occurring within PRRSV.

Toll-Like Receptor 2 (TLR2) and TLR4 Mediate the IgA Immune Response Induced by Mycoplasma hyopneumoniae.

IgA plays an important role in mucosal immunity against infectious pathogens; however, the molecular mechanism of IgA secretion in response to infection remains largely unknown, particularly in Mycoplasma spp. In this study, we found that the levels of IgA in the peripheral blood serum, bronchoalveolar lavage fluid, nasal mucosa, trachea, hilar lymph nodes, and lung tissues of pigs increased significantly after infection with Mycoplasma hyopneumoniae Furthermore, IgA and CD11c were detected in the lungs and hilar lymph nodes by immunohistochemical analysis, and colocalization of these two markers indicates that CD11c+ cells play an important role in IgA mucosal immunity induced by M. hyopneumoniae To investigate the regulatory mechanism of IgA, we separated mouse dendritic cells (DCs) from different tissues and mouse macrophages from the lungs and then cultured mouse B cells together with either DCs or macrophages in vitro In the mouse lung-DC/B (LDC/B) cell coculture, IgA secretion was increased significantly after the addition of whole-cell lysates of M. hyopneumoniae The expression of both Toll-like receptor 2 (TLR2) and TLR4 was also upregulated, as determined by mRNA and protein expression analyses, whereas no obvious change in the expression of TLR3 and TLR7 was detected. Moreover, the IgA level decreased to the same as the control group when TLR2 or TLR4 was inhibited instead of TLR8 or TLR7/9. In conclusion, M. hyopneumoniae can stimulate the response of IgA through TLR2 and TLR4 in a mouse LDC/B cell coculture model, and the coculture model is an ideal tool for studying the IgA response mechanism, particularly that with Mycoplasma spp.

A Porcine circovirus type 2b (PCV2b)-based experimental vaccine is effective in the PCV2b-Mycoplasma hyopneumoniae coinfection pig model.

Porcine circovirus type 2 (PCV2) is one of the major swine pathogens causing high economic losses due to PCV2-associated disease (PCVAD). PCV2 infection is not only immunosuppressive by damaging lymphoid tissues but is also exacerbated by co-infections with other pathogens including Mycoplasma hyopneumoniae. While PCV2 can be divided into several genotypes, currently only PCV2a, PCV2b and PCV2d are globally prevalent and considered of major importance. Most commercial PCV2 vaccines are based on PCV2a isolates; however, the high prevalence of PCV2b and PCV2d in the global pig population is raising concerns among pig veterinarians. The objective of this study was to evaluate the efficacy of an experimental PCV2b-based subunit vaccine in a combined PCV2b and M. hyopneumoniae coinfection model. Briefly, a total of 49 PCV2- and M. hyopneumoniae-free 3-week-old pigs were randomly divided into four groups: A non-vaccinated, non-infected NEG-CONTROL group, a non-vaccinated, PCV2b-infected, POS-CONTROL group, and two vaccinated and PCV2b-infected groups (SINGLE-VAC, DUAL-VAC). SINGLE-VAC and DUAL-VAC pigs were vaccinated at 3 weeks of age and DUAL-VAC pigs received a booster dose at 5 weeks of age. All pigs, except NEG-CONTROLs, were experimentally infected with M. hyopneumoniae 28 days after initial vaccination and challenged with PCV2b one week later. The pigs were necropsied 21 days after PCV2b challenge. Prior to PCV2b challenge, both vaccinated groups had detectable humoral and cell-medicated immune responses to PCV2. Vaccination significantly reduced PCV2b viremia and also reduced or eliminated PCV2-associated lymphoid lesions compared to the POS-CONTROL pigs. Under the study conditions, an experimental PCV2b vaccine protected conventional growing pigs against PCV2b viremia and associated lesions in a coinfection model with some advantages of the two-dose regimen versus the one dose regimen. Both protocols induced neutralizing antibodies against PCV2a and PCV2d prior to challenge.

Development of an indirect ELISA for detecting humoral immunodominant proteins of Mycoplasma hyopneumoniae which can discriminate between inactivated bacterin-induced hyperimmune sera and convalescent sera.

BACKGROUND: Mycoplasma hyopneumoniae (M. hyopneumoniae) is the primary pathogen of porcine enzootic pneumonia, which has been associated with economic losses due to reduced daily weight gain and feed efficiency. Although it has a small genome and no more than 1000 genes, M. hyopneumoniae can be cultured in cell free media. However, some proteins were not expressed or were only expressed in negligible amounts under culture conditions. Nevertheless, some of these proteins can be expressed at a high level and induce a strong and rapid immune response after M. hyopneumoniae infection. The unexpressed or less expressed proteins may play critical roles in pathogenesis and/or immune response. In order to find the differentially expressed proteins of M. hyopneumoniae between culture condition and infected animals, we established an indirect ELISA for the detection of humoral immunodominant proteins which can discriminate between inactivated bacterin-induced hyperimmune sera and convalescent sera by using Mhp366 protein which did not react with sera from bacterin-immunized pigs, but revealed a strong immunoreaction with porcine convalescent sera. RESULTS: The checkerboard titration method was done by using porcine convalescent sera as positive sera and inactivated bacterin-induced hyperimmune sera as negative sera. The bacterial lysates of fusion proteins and free GST protein without dilution were the optimal coating antigens. The optimal blocking buffer was PBS with 10% FBS and 2.5% skimmed milk. In the checkboard ELISAs, when the sera were diluted at 1:500 and the HRP-labeled rabbit anti-pig IgG were diluted at 1:20000, most positive result was obtained for the assay. CONCLUSIONS: This established indirect ELISA can be used as a tool for the detection of humoral immunodominant proteins of M. hyopneumoniae which can discriminate between inactivated bacterin-induced hyperimmune sera and convalescent sera.

Mucosal and systemic immune responses induced by intranasal immunization of recombinant Bacillus subtilis expressing the P97R1, P46 antigens of Mycoplasma hyopneumoniae.

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

Systems Immunology Characterization of Novel Vaccine Formulations for Mycoplasma hyopneumoniae Bacterins.

We characterized five different vaccine candidates and a commercial vaccine in terms of safety, immunogenicity and using a systems vaccinology approach, with the aim to select novel vaccine candidates against Mycoplasma hyopneumoniae. Seven groups of six M. hyopneumoniae-free piglets were primo- and booster vaccinated with the different experimental bacterin formulations, the commercial vaccine Hyogen® as a positive control or PBS as a negative control. The experimental bacterin was formulated with cationic liposomes + c-di-AMP (Lipo_AMP), cationic liposomes + Toll-like receptor (TLR) 2/1, TLR7, and TLR9 ligands (TLR ligands; Lipo_TLR), micro-particles + TLR ligands (PLGA_TLR), squalene-in-water emulsion + TLR ligands (SWE_TLR), or DDA:TDB liposomes (Lipo_DDA:TDB). Lipo_DDA:TDB and Lipo_AMP were the most potent in terms of serum antibody induction, and Lipo_DDA:TDB, Lipo_AMP, and SWE_TLR significantly induced Th1 cytokine-secreting T-cells. Only PLGA_TLR appeared to induce Th17 cells, but was unable to induce serum antibodies. The transcriptomic analyses demonstrated that the induction of inflammatory and myeloid cell blood transcriptional modules (BTM) in the first 24 h after vaccination correlated well with serum antibodies, while negative correlations with the same modules were found 7 days post-vaccination. Furthermore, many cell cycle and T-cell BTM upregulated at day seven correlated positively with adaptive immune responses. When comparing the delivery of the identical TLR ligands with the three formulations, we found SWE_TLR to be more potent in the induction of an early innate immune response, while the liposomal formulation more strongly promoted late cell cycle and T-cell BTM. For the PLGA formulation we found signs of a delayed and weak perturbation of these BTM. Lipo_AMP was found to be the most potent vaccine at inducing a BTM profile similar to that correlating with adaptive immune response in this and other studies. Taken together, we identified four promising vaccine candidates able to induce M. hyopneumoniae-specific antibody and T-cell responses. In addition, we have adapted a systems vaccinology approach developed for human to pigs and demonstrated its capacity in identifying early immune signatures in the blood relating to adaptive immune responses. This approach represents an important step in a more rational design of efficacious vaccines for pigs.

Cathepsin L promotes secretory IgA response by participating in antigen presentation pathways during Mycoplasma Hyopneumoniae infection.

Cathepsin L (CTSL) has been proved to help contain leishmaniasis and mycoplasma infection in mice by supporting cellular immune responses, but the regulatory functions of CTSL on mucosal immune responses haven't been tested and remain undefined. Here, we investigated the effects of CTSL on SIgA responses and invariant chain (Ii) degradations in the co-cultured swine dendritic cells (DCs) and B cells system in vitro. When the cells system were transfected with vector CTSL-GFP or incubated with recombinant CTSL (rCTSL) before they were infected with Mycoplasma hyopneumoniae (M.hp), SIgA significantly increased and Ii chain was degraded into smaller intermediates, while SIgA decreased when CTSL was knockdown or inhibited with E64. To confirm the SIgA responses promoted by CTSL contribute to the resistance to mycoplasma pneumonia, pigs injected with rCTSL before they were challenged with M.hp, showed milder clinical symptoms and histopathological damage of lungs, less mycoplasma burden together with higher secretion of SIgA, percentages of CD4+ T cells and level of MHC II molecules comparing with the group without rCTSL. Collectively, these results suggested that rCTSL could provide effective protection for piglets against mycoplasma pneumonia by enhancing M.hp-specific mucosal immune responses through its role in antigen presentation by processing the invariant chain.

Pathogenicity study of Mycoplasma hyorhinis and M. flocculare in specific-pathogen-free pigs pre-infected with M. hyopneumoniae.

Mycoplasma (M.) hyopneumoniae is the initiator agent of the porcine respiratory disease complex (PRDC) and the etiological agent of enzootic pneumonia. M. hyorhinis and M. flocculare are also found in extensive gross pneumonia-like lesions, but their role is not known. We investigated the pathogenicity of M. hyorhinis and M. flocculare in specific-pathogen-free pigs pre-infected or not with M. hyopneumoniae. Mono-inoculated pigs with M. flocculare showed no clinical signs, hematological changes or macroscopic lesions upon necropsy. Mono-inoculated pigs with M. hyorhinis showed, overall seven days after inoculation, an increase in mean temperature with increases in white blood cell (monocyte) counts and in concentrations of pig major acute phase protein, whereas the average daily weight gain (ADWG) decreased compared with non-infected animals. M. hyorhinis was detected in serous membranes (polyserositis) but not in bronchi. Co-infected pigs with M. hyopneumoniae and M. hyorhinis or M. flocculare showed lower ADWG during the third week of the experiment and higher haptoglobin concentrations in contrast to pigs only mono-infected with M. hyopneumoniae. In pigs co-infected with M. hyopneumoniae and M. hyorhinis, it was interesting to observe that (i) M. hyorhinis was detected in bronchi of six pigs, (ii) M. hyopneumoniae was detected in polyserositis and (iii) there was a slight delay in the production of anti-M. hyopneumoniae IgG. The extent of pneumonia was not statistically different between groups. These results suggest that mycoplasmal associations appear to induce an additive effect and increase the inflammatory status in pigs, probably involving in the impairment of the immune system.

Analysis of swine antigen-specific antibody responses to Mycoplasma hyopneumoniae infection determined by protein microarray.

Pigs harbor several different species of mycoplasmas, of which Mycoplasma hyopneumoniae presents the most significant economic impact on the swine industry. While ELISAs are the predominant diagnostic assay to measure antibody responses during infection with M. hyopneumoniae, the assay itself is only a rough estimate of the total antibody response. It lends little information on pathogen-wide antigen-specific responses. In addition, antibody responses to M. hyopneumoniae as measured by ELISA are slow to develop in infected swine. Our goal was to determine if a protein microarray could be more sensitive and informative of the serological responses of pigs to M. hyopneumoniae infection. The gene sequences of approximately 50 M. hyopneumoniae surface proteins or protein fragments were cloned, mutated to remove UGA codons, expressed in Escherichia coli and purified. The arrays were used to interrogate pig sera from various sources. Sera from naturally-infected swine gave some variability in antigen-specific responses, but, unexpectedly, the responses against the C-terminal portion of the major adhesin P97 was weak in all animals, including those that were experimentally infected. In two of four 118-day experimentally-infected caesarian-derived colostrum-deprived pigs, the strongest antibody responses occurred on days 30 and 54 against members of the P97/P102 paralog families. Our Day 0 results in the other two animals indicate that although thought to be mycoplasma free by all known criteria (serology and PCR), they may have harbored an inapparent Mycoplasma infection. In summary, the protein microarray has the potential to identify new targets for assay development to enhance sensitivity of antibody-based assays.