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The present study aimed to investigate the optimal inactivants and inactivation conditions for preparing inactivated vaccines of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis. Mycoplasma inactivation was performed using formaldehyde, thimerosal, β-propiolactone (BPL), and binary ethylenimine (BEI) and compared. The results showed that M. hyopneumoniae was completely inactivated when incubated with 0.01 % formaldehyde for 24 h or 0.02 % formaldehyde for 12 h at 37 °C, with 0.0008 % thimerosal for 12 h at 37 °C, with 0.02 % BPL for 24 h or 0.1 % BPL for 12 h at 4 °C, or with 0.004 % BEI for 24 h or 0.5 % BEI for 12 h at 37 °C. M. hyorhinis was completely inactivated when incubated with 0.01 % formaldehyde for 24 h or 0.02 % formaldehyde for 12 h at 37 °C, with 0.004 % thimerosal for 24 h or 0.02 % thimerosal for 12 h at 37 °C, with 0.1 % BPL for 12 h at 4 °C, or with 0.004 % BEI for 24 h or 0.5 % BEI for 12 h at 37 °C. Next, the immunogenicity of the mycoplasmas after inactivation was evaluated by immunizing BALB/c mice. Immunization of mice with a high dose (106 color-changing units [CCU] per dose) of M. hyopneumoniae and M. hyorhinis vaccines inactivated with all inactivants led to high levels of serum IgG antibodies. M. hyopneumoniae vaccines inactivated with formaldehyde induced significantly higher titers of antibodies than vaccines inactivated with other inactivants, whereas M. hyorhinis vaccines inactivated with BEI induced significantly higher titers of antibodies than vaccines inactivated with thimerosal. However, in mice immunized with a low dose of mycoplasmas (104 CCU per dose), only M. hyopneumoniae vaccines inactivated with formaldehyde and BEI and M. hyorhinis vaccines inactivated with formaldehyde, BPL, and BEI led to significant levels of serum IgG antibodies. Among these groups, the antibody levels in the formaldehyde-inactivated vaccine group were higher than those in the other inactivant groups. This study provides a reliable basis for inactivation during large-scale production of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis inactivated vaccines.

Vaccination against Mycoplasma hyopneumoniae is still carried out worldwide, but unfortunately current commercial vaccines only provide partial protection. Therefore, two M. hyopneumoniae strains were genetically modified by transposon-mediated gene disruption of mmsA and mnuA , encoding methylmalonate semialdehyde dehydrogenase and membrane nuclease A, respectively. We investigated how immune responses elicited by these genetically modified M. hyopneumoniae strains protected pigs against challenge infection. An endotracheal single dose vaccination with genetically modified M. hyopneumoniae strain 1 (Δ mmsA ) or 2 (Δ mnuA ), or physiological saline solution (Control) was followed by challenge infection. Piglets from Δ mnuA had a higher respiratory disease score post-vaccination, but this group coughed significantly less after challenge. Significantly fewer DNA copies of the challenge strains were observed in broncho-alveolar lavage fluid (BAL) from Δ mnuA after challenge. Two weeks post-challenge, significantly more BAL IgG and BAL IgA was observed in Δ mnuA , but at euthanasia significantly more IgA and less pro-inflammatory cytokines were detected in BAL from both vaccinated groups. Furthermore, a significantly lower percentage of IFN-γ + and TNF-α + IFN-γ + CD8 + T cells was observed after administration of Δ mnuA. The percentage of IFN-γ + CD8 + T cells was significantly lower in Δ mmsA at euthanasia. To conclude, the results of this exploratory study show that a single endotracheal administration of Δ mnuA resulted in coughing post-vaccination, but reduced clinical signs post-challenge and challenge strain DNA load in BAL. Therefore, a strain mutated in the mnuA gene might be an interesting mutant strain that could be promising as a potential live vaccine candidate strain as it can reduce M. hyopneumoniae infection burden under field conditions.

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.

Mycoplasma ( M .) hyopneumoniae is the main pathogen of porcine enzootic pneumonia (PEP). Its controlling is challenging, and requires alternative strategies. This study aimed to develop an oral vaccine against M. hyopneumoniae using a nanostructured mesoporous silica (SBA-15) as an adjuvant, and compare its effect with an intramuscular (IM) commercial vaccine (CV). Fifty 24 day-old M . hyopneumoniae -free piglets composed five equal groups for different immunization protocols, consisting of a CV and/or oral immunization (OI). Control piglets did not receive any form of immunization. All piglets were challenged with M. hyopneumoniae strain 232 on D49 by tracheal route. IgA antibody response in the respiratory tract, bacterial shedding and serum IgG were evaluated. The piglets were euthanized on 28 (D77) and 56 (D105) days post-infection. Lung lesions were macroscopically evaluated; lung fragments and bronchoalveolar fluid (BALF) were collected for estimation of bacterial loads by qPCR and/or histopathology examination. All immunization protocols induced reduction on Mycoplasma -like macroscopic lung lesions. IgA Ab responses anti- M. hyopneumoniae , the expression of IL-4 cytokine and a lower expression of IL-8 were induced by CV and OI vaccines, while IgG was induced only by CV. Oral immunization using silica as a carrier-adjuvant can be viable in controlling M. hyopneumoniae infection.

is the etiological agent of mycoplasmal pneumonia of swine (MPS). Commercial vaccines provide partial protection and do not prevent the colonization and transmission of . The bottleneck in the development of more effective vaccines for MPS is the stimulation of effective immune responses in the host. The purpose of the present study was to evaluate the immune responses of immunodominant proteins Mhp170, Mhp274 and Mhp336 in BALB/c mice. The recombinant Mhp170 (rMhp170), Mhp274 (rMhp274), and Mhp336 (rMhp336) proteins were purified from recombinant bacteria. Fifty-two six-week-old SPF female BALB/c mice were divided into five groups: a commercial inactivated vaccine-immunized group, three recombinant protein-inoculated groups, and a PBS-treated group. The physical parameters and body weights of the mice were observed during the experiment. The lung/body coefficient and macroscopic and microscopic lung lesions were evaluated. IgG and its isotypes IgG1 and IgG2a in serum and BALF and sIgA in BALF were assessed. The levels of IFN-γ, IL-4, and IL-17, in the supernatants of splenocytes and in serum were measured, and the mRNA levels of three cytokines in splenocytes were analyzed. Finally, lymphocyte proliferation after stimulation with corresponding proteins or crude extract of J strain was assessed. We successfully constructed recombinant bacteria expressing rMhp170, rMhp274, and rMhp336. None of the mice from all groups presented adverse reactions and macroscopic and microscopic lung lesions. rMhp170 and rMhp274 were capable of inducing the production of IgG, IgG1 and IgG2 in serum and BALF, the secretion of IFN-γ, IL-4 and IL-17 in serum, the expression of IFN-γ, IL-4 and IL-17 mRNAs in splenocytes, and high levels of lymphocyte proliferation. Moreover, rMhp274 significantly increased sIgA in BALF. Nevertheless, rMhp336 induced only IgG, IgG1 and IgG2 production in sera; the secretion of IFN-γ and IL-4 in sera and BALF; the expression of IFN-γ and IL-4 mRNAs in the splenocyte population; and lymphocyte proliferation. Mhp170 and Mhp274 induced Th1/Th2/Th17 immune responses, and Mhp336 stimulated mixed Th1/Th2-type immune responses, in mice. Our data suggest that Mhp274 is a potential viable candidate for the development of a subunit vaccine for MPS.

Mycoplasma hyopneumoniae (Mhyo) and porcine circovirus type 2 (PCV2) are critical pathogens in the swine industry, both contributing significantly to the porcine respiratory disease complex (PRDC). Given their impact, it is logical to control these pathogens simultaneously. Consequently, combined vaccinations against Mhyo and PCV2 are gaining popularity in swine health management. We present the efficacy of the first commercial combined vaccine prepared of a genotype PCV2d strain and Mhyo and tested against experimental challenge infections with target pathogens in comparative trials with other commercial products. In these studies, three-week-old piglets were vaccinated according to the manufacturers’ instructions. Five weeks later, they were challenged with two Mhyo strains over three consecutive days or with a PCV2d strain once. Positive controls included challenged pigs without prior vaccination, while non-vaccinated/non-challenged pigs served as negative controls. The key parameters measured were lung lesion scores and seroconversion for Mhyo, and viraemia, rectal shedding, lymph node and lung viral content, and seroconversion for PCV2. Findings and conclusion: The results showed no compromising effects between the vaccine components and highlighted significant differences in efficacy among the various products tested. Additionally, oral fluid sampling demonstrated a strong correlation with the viraemia and fecal shedding of PCV2, underscoring the diagnostic and animal welfare benefits of this sampling method.

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.

Background A field efficacy trial was conducted on three farms to evaluate a novel combined vaccine containing porcine circovirus types 2a/d (PCV2a/d), Mycoplasma hyopneumoniae, and M. hyorhinis. Methods Three farms with a history of subclinical PCV2 infection, enzootic pneumonia, and polyserositis were enrolled. Each farm included 40 piglets (18 days old), which were randomly allocated to vaccinated or unvaccinated groups. Pigs in the vaccinated group received a single 2‐mL intramuscular dose of the combined vaccine at 21 days of age, while unvaccinated pigs received phosphate‐buffered saline. Results Vaccination significantly improved (p < 0.05) body weight and average daily weight gain on all three farms compared with unvaccinated pigs. Vaccinated pigs mounted protective humoral and cellular immune responses against PCV2, M. hyopneumoniae, and M. hyorhinis. Furthermore, vaccination reduced viral and mycoplasmal loads in serum, laryngeal, and nasal samples and decreased the severity of associated lesions. Conclusions The combined vaccine demonstrated strong efficacy under field conditions, providing protection against subclinical PCV2 infection, enzootic pneumonia caused by M. hyopneumoniae, and polyserositis caused by M. hyorhinis. These findings support its potential as an effective intervention for improving both health and productivity in swine herds. A field trial on three commercial farms evaluated a combined vaccine targeting PCV2a/d, Mycoplasma hyopneumoniae, and Mycoplasma hyorhinis. Vaccinated pigs showed improved average daily weight gain, stronger immune responses against these pathogens, and significantly reduced viral and mycoplasmal loads and lesion severity, supporting its efficacy under field infection conditions.

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.

•The new bivalent vaccine reduced the levels of PCV2 viremia and mycoplasma nasal shedding.•The new bivalent vaccine reduced lung and lymphoid lesions.•The new bivalent vaccine protected pigs against either PCV2 or M. hyopneumoniae challenge or both. The objective of this study was to evaluate the efficacy of a new bivalent vaccine (Fostera™ PCV MH, Zoetis) of porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae in growing pigs under experimental conditions. A total of 80 pigs were randomly divided into 8 groups (10 pigs per group). The pigs were administered the bivalent vaccine intramuscularly as a 2.0mL dose at 21 days of age based on the manufacturer's instructions. Three weeks after vaccination, the pigs were inoculated with either PCV2 (intranasal route) or M. hyopneumoniae (intratracheal route) or both. Regardless of the type of inoculation, vaccinated pigs after challenge exhibited effective reduction of clinical signs, PCV2 viremia levels and mycoplasma nasal shedding, and lung and lymphoid lesion when compared to unvaccinated challenged pigs. Vaccinated challenged pigs had significantly higher (P<0.05) levels of PCV2-specific neutralizing antibodies, and numbers of PCV2-and M. hyopneumoniae-specific interferon-γ secreting cells compared to unvaccinated challenged pigs. This study demonstrates that the bivalent vaccine is able to protect pigs against either PCV2 or M. hyopneumoniae infection or both based on clinical, microbiological, immunological, and pathological evaluation.