Mycoplasma hyopneumoniae inhibits the unfolded protein response to prevent host macrophage apoptosis and M2 polarization.

Enzootic pneumonia caused by Mycoplasma hyopneumoniae (M. hyopneumoniae) has inflicted substantial economic losses on the global pig industry. The progression of M. hyopneumoniae induced-pneumonia is associated with lung immune cell infiltration and extensive proinflammatory cytokine secretion. Our previous study established that M. hyopneumoniae disrupts the host unfolded protein response (UPR), a process vital for the survival and immune function of macrophages. In this study, we demonstrated that M. hyopneumoniae targets the UPR- and caspase-12-mediated endoplasmic reticulum (ER)-associated classical intrinsic apoptotic pathway to interfere with host cell apoptosis signaling, thereby preserving the survival of host tracheal epithelial cells (PTECs) and alveolar macrophages (PAMs) during the early stages of infection. Even in the presence of apoptosis inducers, host cells infected with M. hyopneumoniae exhibited an anti-apoptotic potential. Further analyses revealed that M. hyopneumoniae suppresses the three UPR branches and their induced apoptosis. Interestingly, while UPR activation typically drives host macrophages toward an M2 polarization phenotype, M. hyopneumoniae specifically obstructs this process to maintain a proinflammatory phenotype in the host macrophages. Overall, our findings propose that M. hyopneumoniae inhibits the host UPR to sustain macrophage survival and a proinflammatory phenotype, which may be implicated in its pathogenesis in inducing host pneumonia.

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.

Immune responses induced by a combined vaccination with a recombinant chimera of Mycoplasma hyopneumoniae antigens and capsid virus-like particles of porcine circovirus type 2.

BACKGROUND: Mycoplasma hyopneumoniae (Mhp) and porcine circovirus type 2 (PCV2) are two important pathogens causing Mycoplasma pneumonia of swine (MPS) and porcine circovirus diseases and porcine circovirus-associated diseases (PCVDs/PCVADs), respectively, and resulted in considerable economic loss to the swine industry worldwide. Currently, vaccination is one of the main measures to control these two diseases; however, there are few combination vaccines that can prevent these two diseases. To determine the effect of combination immunization, we developed capsid-derived (Cap) virus-like particles (VLPs) of PCV2 and a new recombinant chimera composed of the P97R1, P46, and P42 antigens of Mhp. Then we investigated the immune responses induced by the immunization with this combination vaccine in mice and piglets. RESULTS: The high level antibodies against three protein antigens (P97R1, P46, and P42 of Mhp) were produced after immunization, up to or higher than 1:400,000; the antibody levels in Pro group continuously increased throughout the 42 days for all the antigens tested. The lymphocyte proliferative response in PCV2 group was stronger than that in PBS, VP, Mhp CV in mice. The antibody levels for Cap remained stable and reached the peak at 35 DAI. The IFN-γ and IL-4 in sera were significantly enhanced in the Pro group than that in the negative control-VP group on Day 14 and 28 post-the first immunization in piglets. CONCLUSIONS: Above all, the combination immunization could induce humoral and cellular immune responses against all four antigens in mice and piglets. Therefore, our approach is a simple and effective vaccination strategy to protect pigs against MPS and PCVD/PCVAD.

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.

Genetic variation of Mycoplasma hyopneumoniae from Brazilian field samples.

BACKGROUND: Porcine enzootic pneumonia is a worldwide problem in swine production. The infected host demonstrates a respiratory disease whose etiologic agent is Mycoplasma hyopneumoniae (Mhp). A total of 266 lung samples with Mycoplasma-like lesions were collected from two slaughterhouses. We analyzed the genetic profile of Mhp field samples using 16 genes that encode proteins involved in the mechanisms of bacterial pathogenesis and/or the immune responses of the host. Bioinformatic analyses were performed to classify the Mhp field samples based on their similarity according to the presence of the studied genes. RESULTS: Our results showed variations in the frequency of the 16 studied genes among different Mhp field samples. It was also noted that samples from the same farm were genetically different from each other and samples from different regions could be genetically similar, which is evidence of the presence of different genetic profiles among the Mhp field strains that circulate in Brazilian swine herds. CONCLUSION: This work demonstrated the genetic diversity of several Mhp field strains based on 16 selected genes related to virulence and/or immune response in Brazil. Our findings demonstrate the difference between Mhp field strains could influence the virulence, and we hypothesize that the most frequent genes in Mhp field strains could possibly be used as vaccine candidates. Based on our results, we suspect that Mhp genetic variability may be associated with the frequency of genes among the field strains and we have demonstrated that some Mhp field samples could not have many important genes described in the literature.

Surface proteins mhp390 (P68) contributes to cilium adherence and mediates inflammation and apoptosis in Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia (EP) and responsible for major economic losses in global swine industry. After colonization of the respiratory epithelium, M. hyopneumoniae elicits a general mucociliary clearance loss, prolonged inflammatory response, host immunosuppression and secondary infections. Until now, the pathogenesis of M. hyopneumoniae is not completely elucidated. This present study explores the pathogenicity of mhp390 (P68, a membrane-associated lipoprotein) by elucidating its multiple functions. Microtitrer plate adherence assay demonstrated that mhp390 is a new cilia adhesin that plays an important role in binding to swine tracheal cilia. Notably, mhp390 could induce significant apoptosis of lymphocytes and monocytes from peripheral blood mononuclear cells (PBMCs), as well as primary alveolar macrophages (PAMs), which might weaken the host immune response. In addition, mhp390 contributes to the production of proinflammatory cytokines, at least partially, via the release of IL-1ß and TNF-α. To the best of our knowledge, this is the first report of the multiple functions of M. hyopneumoniae mhp390, which may supplement known virulence genes and further develop our understanding of the pathogenicity of M. hyopneumoniae.

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.

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.

Development of a Combined Genetic Engineering Vaccine for Porcine Circovirus Type 2 and Mycoplasma Hyopneumoniae by a Baculovirus Expression System.

Mycoplasma hyopneumoniae (Mhp) and porcine circovirus type 2 (PCV2) are the main pathogens for mycoplasmal pneumonia of swine (MPS) and post-weaning multisystemic wasting syndrome (PMWS), respectively. Infection by these pathogens often happens together and causes great economic losses. In this study, a kind of recombinant baculovirus that can display P97R1P46P42 chimeric protein of Mhp and the capsid (Cap) protein of PCV2 was developed, and the protein location was identified. Another recombinant baculovirus was constructed without tag proteins (EGFP, mCherry) and was used to evaluate the immune effect in experiments with BALB/c mice and domestic piglets. Antigen proteins P97R1P46P42 and Cap were expressed successfully; both were anchored on the plasma membrane of cells and the viral envelope. It should be emphasized that in piglet immunization, the recombinant baculovirus vaccine achieved similar immunological effects as the mixed commercial vaccine. Both the piglet and mouse experiments showed that the recombinant baculovirus was able to induce humoral and cellular responses effectively. The results of this study indicate that this recombinant baculovirus is a potential candidate for the further development of more effective combined genetic engineering vaccines against MPS and PMWS. This experiment also provides ideas for vaccine development for other concomitant diseases using the baculovirus expression system.

Determination of Mycoplasma hyopneumoniae-Specific IgG, IgG1, and IgG2a Titers in BALB/c Mice Induced by Mineral Oil-Based Oil-in-Water Emulsion Adjuvants Prepared Using a Self-Emulsifying Drug Delivery System.

We prepared mineral oil-based emulsion adjuvants by employing simple self-emulsifying drug delivery system (SEDDS). Mineral oil emulsions (3%, 5%, and 7%) were prepared using deionized water and C-971P NF and C-940 grade carbomer solutions with concentrations 0.01% (w/v) and 0.02% (w/v). In total, 15 emulsions were prepared and mixed with a solution containing inactivated Mycoplasma hyopneumoniae (J101 strain) antigen and porcine circovirus type 2 antigen to prepare vaccines. Droplet sizes in the submicron range and zeta potential values between - 40 and 0 mV were maintained by most emulsion adjuvants for a period of 6 months. Emulsion adjuvants were regarded safe, and their M. hyopneumoniae-specific IgG, IgG1, and IgG2a titers were either better or comparable to those of aluminum gel.

The immune mechanism of Mycoplasma hyopneumoniae 168 vaccine strain through dendritic cells.

BACKGROUND: Mycoplasma hyopneumoniae (Mhp) causes porcine enzootic pneumonia, a disease that cause major economic losses in the pig industry. Dendritic cells (DCs), the most effective antigen-presenting cells, are widely distributed beneath respiratory epithelium, DCs uptake and present antigens to T cells, to initiate protective immune responses in different infections. In this study, we investigated the role of porcine DCs in vaccine Mhp-168 exposure. RESULTS: The antigen presenting ability of DCs were improved by vaccine Mhp-168 exposure. DCs could activate T-cell proliferation by up-regulating the antigen presenting molecule MHCII expression and co-stimulatory molecule CD80/86. However, the up-regulation of IL-10 and accompany with down-regulation of IFN-γ gene level may account for the limitation of attenuated Mhp-168 strain use as vaccine alone. CONCLUSION: These findings are benefit for exploring the protection mechanisms and the possible limitations of this attenuated Mhp-168 vaccine.

Efficacy of one dose vaccination against experimental infection with two Mycoplasma hyopneumoniae strains.

BACKGROUND: Mycoplasma hyopneumoniae (M. hyopneumoniae) is the primary agent of enzootic pneumonia in pigs. Pigs are often infected with different M. hyopneumoniae strains. This study assessed the efficacy of vaccination against experimental infection with two genetically different M. hyopneumoniae strains in weaned piglets. At 33 days of age (D0), 45 M. hyopneumoniae-free piglets were randomly assigned to three different groups: 1) negative control group (NCG; n = 5): not vaccinated, not infected, 2) positive control group (PCG; n = 20): not vaccinated, infected, and 3) vaccination group (VG; n = 20): single vaccination with an inactivated whole-cell M. hyopneumoniae vaccine (Hyogen®, Ceva) (D1), infected. The PCG and VG were endotracheally inoculated with 7 × 107 CCU in 7 ml of the highly virulent M. hyopneumoniae strain F7.2C (D24) and 7 × 107 CCU in 7 ml low virulent strain F1.12A (D25). A respiratory disease score (RDS) was assessed from D24 until D53. At D53 (euthanasia), macroscopic lung lesions (MLL) were scored, log copies of M. hyopneumoniae DNA (qPCR) and IL-1 and IL-6-concentrations (ELISA) on bronchoalveolar lavage fluid were determined. RESULTS: The RDS and MLL at euthanasia were respectively 0, 1.20 and 0.55 (P < 0.001) and 0, 7.56 and 0.68 (P < 0.001) for NCG, PCG and VG, respectively. The qPCR results for PCG and VG were 3.99 and 1.78 log copies (P < 0.001), respectively, with a significant difference between PCG and VG. The IL-1 and IL-6 results at euthanasia for NCG, PCG and VG were 17.61, 1283.39 and 53.04 pg/ml (P < 0.001) and 148.10, 493.35 and 259.80 pg/ml (P = 0.004), respectively with a significant difference between PCG and VG. CONCLUSIONS: Vaccination with Hyogen® in pigs was efficacious against an experimental challenge with both a low and highly virulent M. hyopneumoniae strain as the vaccinated pigs coughed significantly less, and showed significantly less lung lesions compared to the non-vaccinated challenged pigs: the vaccinated animals showed a 52.9% lower RDS and 91.0% lower MLL compared to the PCG. In the bronchoalveolar lavage fluid collected at the necropsy of the vaccinated pigs, a significantly lower amount of M. hyopneumoniae-DNA and a significantly lower IL-1 and IL-6 concentration was found compared to the pigs of the PCG.

A randomized controlled study on the efficacy of a novel combination vaccine against enzootic pneumonia (Mycoplasma hyopneumoniae) and porcine Circovirus type 2 (PCV2) in the presence of strong maternally derived PCV2 immunity in pigs.

BACKGROUND: Mycoplasma hyopneumoniae (M. hyo) and Porcine Circovirus Type 2 (PCV2) are major pathogens that cause significant health problems in swine worldwide. Maternal derived immunity (MDI) has been suggested as a significant immediate defence factor for newborn piglets and may interfere with piglet's vaccination-induced immunity. The study aimed to assess the efficacy of a novel combination vaccine (consisting of PCV2 subunits and inactivated M. hyo strain J), against PCV2 and M. hyo natural infection [Porcilis® PCV M Hyo (MSD Animal Health, Boxmeer, the Netherlands)], in the presence of strong maternally derived PCV2 immunity (antibody titre averaged 11.08 log2), under field conditions. The study was performed according to a controlled, randomized and blinded design in a Greek swine unit with Enzootic Pneumonia (EP) and subclinical PCV2 infection. In total, 600 healthy three-week-old suckling piglets were allocated randomly, either to treatment (vaccinated with the test product) or control group (injected with sterile buffered saline). RESULTS: Vaccination significantly reduced the severity of lung lesions at slaughter (lesions of cranio-ventral pulmonary consolidation) (P < 0.001). The overall mean lung lesion score (LLS) was 9.6 in the vaccinated group and 12.2 in controls. The level of PCV2 viraemia was significantly reduced in vaccinated pigs. Furthermore, 25 g higher average daily weight gain (ADWG) was observed during the finishing phase (P < 0.001) and 18 g greater ADWG overall (P < 0.001). CONCLUSIONS: Results of LLS, PCV2 viremia and ADWG support the test product's efficacy in the face of strong maternally derived PCV2 immunity.

Parenteral adjuvant potential of recombinant B subunit of Escherichia coli heat-labile enterotoxin.

BACKGROUND: The B subunit of Escherichia coli heat-labile enterotoxin (LTB) is a potent mucosal immune adjuvant. However, there is little information about LTB's potential as a parenteral adjuvant. OBJECTIVES: We aimed at evaluating and better understanding rLTB's potential as a parenteral adjuvant using the fused R1 repeat of Mycoplasma hyopneumoniae P97 adhesin as an antigen to characterise the humoral immune response induced by this construct and comparing it to that generated when aluminium hydroxide is used as adjuvant instead. METHODS: BALB/c mice were immunised intraperitoneally with either rLTBR1 or recombinant R1 adsorbed onto aluminium hydroxide. The levels of systemic anti-rR1 antibodies (total Ig, IgG1, IgG2a, and IgA) were assessed by enzyme-linked immunosorbent assay (ELISA). The ratio of IgG1 and IgG2a was used to characterise a Th1, Th2, or mixed Th1/Th2 immune response. FINDINGS: Western blot confirmed rR1, either alone or fused to LTB, remained antigenic; anti-cholera toxin ELISA confirmed that LTB retained its activity when expressed in a heterologous system. Mice immunised with the rLTBR1 fusion protein produced approximately twice as much anti-rR1 immunoglobulins as mice vaccinated with rR1 adsorbed onto aluminium hydroxide. Animals vaccinated with either rLTBR1 or rR1 adsorbed onto aluminium hydroxide presented a mixed Th1/Th2 immune response. We speculate this might be a result of rR1 immune modulation rather than adjuvant modulation. Mice immunised with rLTBR1 produced approximately 1.5-fold more serum IgA than animals immunised with rR1 and aluminium hydroxide. MAIN CONCLUSIONS: The results suggest that rLTB is a more powerful parenteral adjuvant than aluminium hydroxide when administered intraperitoneally as it induced higher antibody titres. Therefore, we recommend that rLTB be considered an alternative adjuvant, even if different administration routes are employed.

Maternal immunity enhances Mycoplasma hyopneumoniae vaccination induced cell-mediated immune responses in piglets.

BACKGROUND: Passively acquired maternal derived immunity (MDI) is a double-edged sword. Maternal derived antibody-mediated immunity (AMI) and cell-mediated immunity (CMI) are critical immediate defenses for the neonate; however, MDI may interfere with the induction of active immunity in the neonate, i.e. passive interference. The effect of antigen-specific MDI on vaccine-induced AMI and CMI responses to Mycoplasma hyopneumoniae (M. hyopneumoniae) was assessed in neonatal piglets. To determine whether CMI and AMI responses could be induced in piglets with MDI, piglets with high and low levels of maternal M. hyopneumoniae-specific immunity were vaccinated against M. hyopneumoniae at 7 d of age. Piglet M. hyopneumoniae-specific antibody, lymphoproliferation, and delayed type hypersensitivity (DTH) responses were measured 7 d and 14 d post vaccination. RESULTS: Piglets with M. hyopneumoniae-specific MDI failed to show vaccine-induced AMI responses; there was no rise in M. hyopneumoniae antibody levels following vaccination of piglets in the presence of M. hyopneumoniae-specific MDI. However, piglets with M. hyopneumoniae-specific MDI had primary (antigen-specific lymphoproliferation) and secondary (DTH) M. hyopneumoniae-specific CMI responses following vaccination. CONCLUSIONS: In this study neonatal M. hyopneumoniae-specific CMI was not subject to passive interference by MDI. Further, it appears that both maternal derived and endogenous CMI contribute to M. hyopneumoniae-specific CMI responses in piglets vaccinated in the face of MDI.

Innate and adaptive immune responses of snatch-farrowed porcine-colostrum-deprived pigs to Mycoplasma hyopneumoniae vaccination.

BACKGROUND: The snatch-farrowed porcine-colostrum-deprived (SF-pCD) pig model, in which neonates are raised on commercially available bovine colostrum, is an alternative model for porcine infectious disease research. It is not known if SF-pCD pigs possess growth performance and immunity comparable to conventional, farm-raised pigs. The current experiment compared growth performance and immune responses of SF-pCD pigs to their farm-raised siblings following Mycoplasma hyopneumoniae (Mhyo) vaccination. Twelve SF-pCD and 13 farm-raised siblings were vaccinated on day 7 (D7) and D26 of age. Body weights were measured once or twice weekly and average daily gain (ADG) was calculated. Peripheral blood mononuclear cells (PBMC) were isolated on D40. Cytokine secretion from PBMC stimulated with Mhyo antigen or phorbol myristate acetate plus ionomycin (PMA/Iono) was assessed using a multiplexed fluorescent microsphere immunoassay (FMIA). Additionally, interferon gamma (IFNγ) secretion from stimulated PBMC was assessed using ELISPOT. Mhyo IgG titers were measured by an ELISA in D40 sera. RESULTS: Growth performance did not differ between groups before weaning, but SF-pCD pigs had higher ADG after weaning. In response to Mhyo stimulation, numbers of IFNγ secreting PBMC and levels of interleukin 8 (IL8) and IL10 in PBMC supernatants were significantly higher in SF-pCD pigs, as were Mhyo antibody levels in sera, and levels of IL1ß, IL8 and IL12 in supernatants of PMA/Iono stimulated PBMC. CONCLUSIONS: Under the conditions of this experiment, SF-pCD pigs demonstrated superior growth performance and enhanced humoral and cell-mediated immunity following vaccination. Whether or not this reflects greater resistance or tolerance to infection is unknown but the ability to react positively to the vaccination provides evidence that SF-pCD pigs are a suitable alternative model for swine disease research.

Comparative analysis of mucosal immunity to Mycoplasma hyopneumoniae in Jiangquhai porcine lean strain and DLY piglets.

The Jiangquhai porcine lean strain (JQHPL) is a new pork meat-type strain that has been developed in recent years from the parent lines Duroc, Fengjing, and Jiangquhai pigs (DurocxFengjing pigxJiangquhai pig). Enzootic pneumonia (EP) in pigs induced by Mycoplasma hyopneumoniae (M. hyopneumoniae) is a chronic respiratory disease of pigs, generating high economic losses in the swine industry. Here, we investigated the degree of resistance to M. hyopneumoniae for the Jiangquhai porcine lean strain and the Duroc x Landrace x Yorkshire (DLY) pigs, which are Western commercial pigs that have been introduced in China. A total of 209 DLY piglets and 221 JQHPL piglets from 19 Landrace x Yorkshire and 22 JQHPL M. hyopneumoniae positive gestating sows with different expected dates of confinement were selected and raised in the same M. hyopneumoniae positive farrowing barn. When the oldest suckling piglets were 37 days old, nasal swabs were collected from all the piglets (ranging from 4 to 37 days old) to detect the M. hyopneumoniae pathogen using n-PCR and M. hyopneumoniae specific SIgA using ELISA. Positive M. hyopneumoniae infection rates in both the strains increased with age; however, positive rates for JQHPL were lower compared to DLY at 14 to 35 days old. The level of the specific SIgA rose rapidly in JQHPL respiratory tracts, particularly in piglets 21 to 35 days in age compared to DLY piglets of the same age; however, the level of the specific SIgA in DLY also marginally increased. In conclusion, JQHPL pigs exhibits higher resistance to M. hyopneumoniae compared to DLY. It is possible that this characteristic is caused by the faster and stronger mucosal immunity phenotype of the JQHPL strain.

Detection of anti-Mycoplasma hyopneumoniae antibodies in backyard pigs in the state of Paraná, Brazil.

Mycoplasma hyopneumoniae is the causative bacterium of porcine enzootic pneumonia and one of the primary etiologic agents of the porcine respiratory disease complex. Most Brazilian commercial pig farms are positive for this pathogen. However, the prevalence of the pathogen in backyard pig farms has not been described, to our knowledge. Therefore, we aimed to determine the prevalence of M. hyopneumoniae in backyard pig farms in the state of Paraná, Brazil. In January-March 2020, we collected 585 serum samples from pigs in 187 non-vaccinated herds. We tested the sera with an indirect ELISA for anti-M. hyopneumoniae antibodies and found that 182 of 585 (31.1%) samples were positive, and were found in 109 of 187 (58.3%) herds assessed.

Comparative assessment of the performance of a commercial fluorescent microsphere immunoassay and three commercial ELISAs for Mycoplasma hyopneumoniae serum antibody detection.

Mycoplasma hyopneumoniae (M. hyopneumoniae) is a significant porcine respiratory disease complex pathogen, prompting many swine farms and production systems to pursue M. hyopneumoniae elimination strategies. Antibody testing is cost-effective in demonstrating sustained freedom from M. hyopneumoniae, often replacing PCR testing on deep tracheal swabs. The process typically involves testing a subpopulation of the herd using an M. hyopneumoniae screening antibody ELISA, with non-negative results further assessed through confirmatory testing, such as PCR. Recently, a commercial (Biovet) fluorescent microsphere immunoassay (FMIA) for detecting M. hyopneumoniae antibodies has been introduced as an alternative to ELISA. Its performance was compared to three commercial ELISAs (Idexx, Hipra, and Biochek) using experimental serum samples from pigs inoculated with M. hyopneumoniae, M. hyorhinis, M. hyosynoviae, M. flocculare, or mock-inoculated with Friis medium. FMIA consistently detected M. hyopneumoniae at earlier time points than the ELISAs, although two false-positive results were encountered using the manufacturer's recommended cutoff. ROC analysis allowed for the evaluation of various cutoffs depending on testing objectives. Poisson regression of misclassification error counts detected no difference in the Biovet FMIA and Hipra ELISA but significantly fewer misclassification errors than Idexx and Biocheck ELISAs. This study showed FMIA as a suitable alternative to traditional ELISAs for screening purposes due to its superior antibody detection rate at early stages. Alternatively, adopting a more stringent cutoff to improve diagnostic specificity could position the FMIA as a viable confirmatory test option. Overall, FMIA is an optimal choice for M. hyopneumoniae antibody surveillance testing, offering versatility in testing strategies (e.g., triplex FMIA M. hyopneumoniae/PRRSV types 1 and 2) and contributing to improved diagnostic capabilities in porcine health management.