Yuan Gan

Development and validation of a SIgA-ELISA for the detection of Mycoplasma hyopneumoniae infection

An alternative indirect enzyme-linked immunosorbent assay (ELISA) for the detection of Mycoplasma hyopneumoniae secretory IgA (SIgA) antibody (SIgA-ELISA) was developed using an adhesin (P97R1) of M. hyopneumoniae produced in Escherichia coli. The SIgA-ELISA assay was validated by the comparison with a nested-PCR assay and a commercial M. hyopneumoniae antibody detection kit (IgG-ELISA). Two hundred and sixty nasal swab samples, bronchoalveolar lavage fluids or serum samples were prepared for SIgA-ELISA validation from a M. hyopneumoniae-free farm, a M. hyopneumoniae vaccinated farm and two M. hyopneumoniae contaminated farms. The results showed that the SIgA-ELISA assay could distinguish the M. hyopneumoniae infection from M. hyopneumoniae vaccinated pigs, which was impossible for the current commercial M. hyopneumoniae antibody detection kits. The diagnostic sensitivity (DSN), specificity (DSP) and accuracy of the SIgA-ELISA were 97.0%, 94.4% and 95.8%, respectively and were compared with nested-PCR on 260 field nasal swab samples. The results of repeatability tests revealed that the coefficients of variation of swab samples within and between runs were less than 10%. This SIgA-ELISA is a needle-free detection methodology for large-scale surveys of M. hyopneumoniae infection.

Effect of different adjuvant formulations on the immunogenicity and protective effect of a live Mycoplasma hyopneumoniae vaccine after intramuscular inoculation

Mycoplasma hyopneumoniae (M. hyopneumoniae) vaccine strain 168 is an intrapulmonically injected attenuated live vaccine that is available in the Chinese market. The aim of this study was to develop suitable adjuvants for this live vaccine to provide effective protection after intramuscular inoculation. Several adjuvant components were screened to assess their toxicity for the live vaccine, and various adjuvant formulations were then designed and prepared. Vaccines supplemented with these adjuvants were used to immunize mice intramuscularly to assess the capacity of the adjuvants to induce a specific immune response. The screened formulations were then evaluated in pigs. Seven of the eight adjuvant components did not affect the viability of the live vaccine, and seven different adjuvant formulations were then designed. In mice, the ISCOM-matrix adjuvant and the levamisole-chitosan mixture adjuvant significantly enhanced serum IgG responses against M. hyopneumoniae, while lymphocyte proliferation was enhanced by the ISCOM-matrix adjuvant, the carbomer-astragalus polysaccharide mixture adjuvant and an oil-in-water emulsion adjuvant. These four adjuvants were evaluated in pigs. Enhancement of specific lymphocyte proliferation responses was observed in the groups vaccinated with the ISCOM-matrix adjuvant and the carbomer-astragalus polysaccharide mixture adjuvant. Significant enhancement of serum IgG antibody production was observed before challenge in pigs vaccinated with the carbomer-astragalus polysaccharide mixture adjuvant and the levamisole-chitosan mixture adjuvant, while after challenge, all of the animals that received vaccines containing adjuvants had higher antibody concentrations against M. hyopneumoniae than unvaccinated animals. Animals inoculated with a vaccine containing the ISCOM-matrix adjuvant (median score 3.57) or the carbomer-astragalus polysaccharide mixture adjuvant (median score 5.28) had reduced lesion scores compared to unvaccinated animals (median score 14.81). These studies will help in the development of appropriate adjuvants for intramuscular administration of this live M. hyopneumoniae vaccine.

Development and validation of an attenuated Mycoplasma hyopneumoniae aerosol vaccine

Mycoplasma hyopneumoniae (M. hyopneumoniae) causes a chronic respiratory disease with high morbidity and low mortality in swine, and has been presented as a major cause of growth retardation in the swine industry. Aerosol vaccination presents a needle free, high throughput, and efficient platform for vaccine delivery, and has been widely applied in poultry vaccination. However, aerosol vaccines have rarely been used in swine vaccination primarily because the long and curving respiratory track of swine presents a barrier for vaccine particle delivery. To develop an effective M. hyopneumoniae aerosol vaccine, three major barriers need to be overcome: to optimize particle size for aerosol delivery, to maintain the viability of mycoplasma cells in the vaccine, and to optimize the environmental conditions for vaccine delivery. In this study, an aerosol mycoplasma vaccine was successfully developed based on a conventional live attenuated M. hyopneumoniae vaccine. Specifically, the Pari LCD nebulizer was used to produce an aerosol vaccine particle size less than 5 μm; and a buffer with 5% glycerol was developed and optimized to prevent inactivation of M. hyopneumoniae caused by aerosolization and evaporation. Before nebulization, the room temperature and relative humidity were control to 20-25 °C and 70-75%, respectively, which helped maintain the viability of aerosol vaccine. Animal experiments demonstrated that this newly developed aerosol vaccine was effectively delivered to swine low respiratory track, being confirmed by nested-PCR, in situ hybridization and scanning electron microscope. Moreover, M. hyopneumoniae specific sIgA secretion was detected in the nasal swab samples at 14 days post-immunization. To our knowledge, this is the first report on a live M. hyopneumoniae aerosol vaccine.

Immune Responses to the Attenuated Mycoplasma hyopneumoniae 168 Strain Vaccine by Intrapulmonic Immunization in Piglets

To investigate the immune responses to the attenuated Mycoplasma hyopneumoniae 168 strain vaccine, 8-15 d old piglets were immunized with M. hyopneumoniae 168 strain vaccine by intrapulmonic route. And the specific IgG antibody in serum, lymphoproliferation, IFNγ, and specific secretory IgA (SIgA) antibody in bronchoalveolar lavage fluid were detected on 30 and 60 d post-immunization (DPI), respectively. On 60 DPI, all the pigs except for those in health control group were challenged with a field M. hyopneumoniae strain JS. Necropsy was performed on 30 d post-challenge (DPC). The results showed that IFNγ and specific SIgA were stimulated on surface of respiratory tract after immunization. And peripheral blood mononuclear cells could also be proliferated about 1.81 and 2.12 fold on 30 and 60 DPI when stimulated by M. hyopneumoniae protein in vitro. However, no serum IgG antibody against M. hyopneumoniae was detected during the whole immune phage. After challenge, vaccinated pigs were observed with only very slight histological lesion in individual lobes. None of vaccinated pigs showed any clinical signs. While the unvaccinated pigs from challenge control group showed varying degrees of clinical sign and severe macroscopical lesion of mycoplasmal pneumonia of swine (MPS). The result suggested that the attenuated M. hyopneumoniae 168 strain vaccine inoculated by intrapulmonic route could activate the systemic cellular immunity, the local mucosal immunity and IFNγ secretion in respiratory tract to against M. hyopneumoniae infection in piglets.

Protective efficacy of a live attenuated Mycoplasma hyopneumoniae vaccine with an ISCOM-matrix adjuvant in pigs

An attenuated Mycoplasma hyopneumoniae vaccine that requires intrathoracic administration is commercially available for use against mycoplasmal pneumonia in China. Given the limitations of such a route of administration, this study was undertaken to assess the capacity of an ISCOM-matrix adjuvant to enhance immunogenicity following intramuscular use. Immune responses in pigs following vaccination and subsequent intra-tracheal bacterial inoculation were examined using lymphocyte proliferation, serology and mucosal IgA in both nasal and saliva swabs. Vaccination induced clear lymphocyte proliferation, but only slight serum antibody responses although these were significantly increased following experimental infection. Mucosal IgA was not detected in either nasal or salivary secretions. Following bacterial challenge, animals vaccinated with the adjuvant-containing live vaccine exhibited less severe pulmonary lesions (median score 3.67) than unvaccinated pigs (median score 13.58). The degree of ciliary loss on the respiratory tract surface was reduced in vaccinated pigs compared with experimentally infected controls. The findings indicated that the adjuvant vaccine administered IM provided protection against experimentally induced mycoplasmal pneumonia and could have commercial potential.

The effects of Mycoplasma hyopneumoniae on porcine circovirus type 2 replication in vitro PK-15 cells.

Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS). Mycoplasma hyopneumoniae (Mhp) is a very well-known co-factor that potentially enhances PCV2 replication and thus the development of PMWS. However, co-infection with Mhp and PCV2 in vivo under different conditions can produce divergent clinical signs and lesions. In this study, PCV2 replication could be enhanced by subsequent co-inoculation with Mhp (PCV2+Mhp) in a time and dose dependent method, but not by prior (Mhp+PCV2) or simultaneous (Mhp/PCV2) co-inoculation. Furthermore, different magnitudes of PCV2-infected cells, varying from 150% ± 14% to 351% ± 28%, were detected when co-infected with different Mhp strains. The relative percentage of PCV2-infected cells greatly decreased from 351% ± 28 to 141% ± 18 when the Mhp strain was treated with UV light for 12 h. These results offer the evidences to better understand the complex clinical syndromes in Mhp/PCV2 co-infection cases, and the occurrence of PMWS.

Use of serological and mucosal immune responses to Mycoplasma hyopneumoniae antigens P97R1, P46 and P36 in the diagnosis of infection.

Currently available ELISAs used to diagnose Mycoplasma hyopneumoniae infection in pigs have high specificity but low sensitivity. To develop more sensitive assays, the kinetics of specific serum IgG and respiratory mucosal sIgA responses against three M. hyopneumoniae antigens, namely, P97R1 (an adhesin protein), P46 (a membrane protein), and P36 (a cytosolic protein), were characterised over 133 days following experimental infection. Immunoglobulin G against the three proteins remained at high concentrations from 28 to 133 days post-infection (dpi), although IgG against P97R1 was detected earlier and was more reactive than the other two antigens under assessment. Mucosal sIgA appeared earlier than serum IgG but did not persist as long; sIgA concentrations against P97R1 were the highest. Seroconversion was detected 2 weeks earlier with the P97R1-based ELISA than with a commercially available ELISA. On analysis of serum samples from five pig farms that did not use a M. hyopneumoniae vaccine, the P97R1-based IgG ELISA demonstrated a 73.6% coincidence rate with the commercial kit. Moreover, this more specific P97R1-based ELISA detected more positive samples than the commercial kit (52.8% vs. 39.2%). It was concluded that the systemic immune response to M. hyopneumoniae infection in pigs was delayed in onset but persistent whereas the mucosal response developed more rapidly but was less sustained. The P97R1 antigen was identified as a suitable serological marker for diagnosing M. hyopneumoniae infection in pigs, particularly early stage infection.

Establishment and comparison of air-liquid interface culture systems for primary and immortalized swine tracheal epithelial cells

BackgroundAir-liquid interface (Ali) systems allow the establishment of a culture environment more representative of that in vivo than other culture systems. They are useful for performing mechanistic studies of respiratory epithelial cells as drug permeation barriers and can be used to study the interactions between hosts and respiratory pathogens. However, there have been few studies concerning Ali cultures of primary swine tracheal epithelial cells (STECs) and an immortalized STEC line, and the differences between these two systems remain poorly defined.ResultsIn this study, we established Ali culture systems for primary STECs and for immortalized STEC line, and we systematically compared the differentiation capacities and immunological functions of these systems for the first time. Under Ali culture conditions, immortalized STEC line and primary STECs could survive for at least forty days, formed tight junctions and differentiated into stratified cells. They both possessed complete abilities to produce mucin and inflammatory cytokines and develop cilia. However, in contrast to primary STECs, which had a heterogeneous morphology, Ali-cultured immortalized STEC line appeared to be a homogenous population. The formation of tight junctions in Ali-cultured primary STECs was superior to that in immortalized STEC line. In addition, cilia in Ali-cultured immortalized STEC line were more pronounced, but their duration of expression was shorter than in primary STECs.ConclusionsAli-cultured primary STECs and immortalized STEC line systems possessing complete abilities to undergo ciliary differentiation and inflammatory cytokine production were established for the first time in this study, and several differences in morphology and the formation of tight junctions and cilia were observed between these two systems. These two systems will be important tools for drug screening studies, as well as for detailed analyses of the interactions between hosts and respiratory pathogens.

Application of a sIgA-ELISA method for differentiation of Mycoplasma hyopneumoniae infected from vaccinated pigs

In order to evaluate the sIgA-ELISA method reported previously for differentiating Mycoplasma hyopneumoniae (M. hyopneumoniae) infected from vaccinated pigs, dynamics of anti-M. hyopneumoniae secretory IgA (sIgA) antibody secretion in nasal mucus and IgG antibodies in serum from 10 pigs experimentally infected with M. hyopneumoniae or vaccinated with an inactivated vaccine were examined using sIgA-ELISA and a commercial M. hyopneumoniae antibody detection kit (IgG-ELISA), respectively. In addition, nasal swabs and serum samples from 2368 pigs of different ages originating from 10 pig farms with different M. hyopneumoniae infection and vaccination status were examined using the two ELISA. In the experimental model, anti-M. hyopneumoniae IgG antibodies were detected in both, the challenge group and the vaccine group. Anti-M. hyopneumoniae sIgA antibodies were detected in the challenge group from 7 days post challenge onwards, but not in the vaccine group. According to the data obtained from pig farms maintaining administration of inactivated vaccine, the prevalence of anti-M. hyopneumoniae sIgA antibody positive pigs was significantly lower than that of IgG antibody positive pigs. In non-vaccinating herds, the prevalence of sIgA antibodies was correlated with the severity of clinical symptoms typical for porcine enzootic pneumonia. In all suckling pigs, no matter vaccinated or not, the prevalence of anti-M. hyopneumoniae sIgA antibody positives was significantly lower than that of IgG antibody positives. These results prove that the sIgA-ELISA is a valuable method enabling the surveillance of M. hyopneumoniae infections in pig herds without interference due to maternally derived antibodies or antibodies induced by administration of inactivated vaccines.

Establishment and characterization of a telomerase-immortalized porcine bronchial epithelial cell line: XIE et al.

Primary porcine bronchial epithelial cells (PBECs) are an ideal model to study the molecular and pathogenic mechanisms of various porcine respiratory pathogens. However, the short lifespan of primary PBECs greatly limit their application. Here, we isolated and cultured primary PBECs and established immortalized PBECs by transfecting primary PBECs with the pEGFP‐hTERT recombinant plasmid containing human telomerase reverse transcriptase (hTERT). Immortalized PBECs (hTERT‐PBECs) retained the morphological and functional features of primary PBECs as indicated by cytokeratin 18 expression, telomerase activity assay, proliferation assays, karyotype analysis, and quantitative reverse‐transcriptase polymerase chain reaction. Compared to primary PBECs, hTERT‐PBECs had higher telomerase activity, extended replicative lifespan, and displayed enhanced proliferative activity. Moreover, this cell line is not transformed in vitro and does not exhibit a malignant phenotype in vivo, suggesting that it can be safely used in further studies. Besides, hTERT‐PBECs were susceptible to swine influenza virus of H3N2 subtype and porcine circovirus type 2. In conclusion, the immortalized hTERT‐PBECs represent a valuable in vitro model, which can be widely used in the study of porcine respiratory pathogenic infections.