Intradermal vaccination with Porcilis® Begonia can clinically protect against fatal PRV challenge with the highly virulent ZJ01 field strain.

Since pseudorabies (PR) re-emerged and rapidly spread in China at the end of 2011, researchers have focused on effective vaccine strategies to prevent and control pseudorabies virus (PRV) infection in pig herds. Due to the extensive application of an attenuated vaccine based on the Bartha-K61 strain isolated in Hungary in 1961 and the variation of the PRV strain, it has been suggested that traditional vaccines based on the Bartha-K61 strain offer only partial protection against variant strains. It was therefore evaluated whether the Porcilis® Begonia vaccine, which is based on the NIA-3 strain with deletions in the gE and TK genes, is efficacious against experimental infection with the virulent, contemporary Chinese PRV strain ZJ01. In this study, piglets were vaccinated with Porcilis® Begonia through either the intradermal (ID) route or the intramuscular (IM) route and subsequently challenged intranasally with strain ZJ01 at 4 weeks post-vaccination. An unvaccinated challenge group and an unvaccinated/nonchallenged group were also included in the study. All animals were monitored for 14 days after challenge. Vaccinated and negative control pigs stayed healthy during the study, while the unvaccinated control animals developed lesions associated with PRV ZJ01 challenge, and 44% of these pigs died before the end of the experiment. This study demonstrated that ID or IM vaccination of pigs with a vaccine based on the NIA-3 strain Porcilis® Begonia clinically protects against fatal PRV challenge with the ZJ01 strain.

Preparation of the porcine secretory component and a monoclonal antibody against this protein.

Secretory component (SC) is a component of secretory IgA that is designated sIgA to distinguish it from IgA. The monoclonal antibody (MAb) against SC has been shown to be an excellent tool for the detection of the level of sIgA and for the evaluation of the efficacy of mucosal immunity. To prepare a monoclonal antibody against porcine SC, a recombinant porcine SC was expressed and purified. To develop this recombinant SC, the gene encoding the porcine SC was ligated into the pCold I vector. The recombinant vector was then transformed into Escherichia coli BL 21 (DE3), and gene expression was successfully induced by isopropyl-ß-D-thiogalactoside (IPTG). After affinity purification with Ni-NTA resin and gel recovery, the recombinant SC protein was used to immunize BALB/c mice. Finally, three hybridoma cell lines showing specific recognitions of both recombinant SC and native SC were used as stable secretors of MAbs against porcine SC and were confirmed to have no reaction to porcine IgA or IgG. The successful preparations of recombinant SC protein and MAbs provide valuable materials that can be used in the mucosal infection diagnosis for porcine disease and mucosal immune evaluation for porcine vaccine, respectively.

Development of a loop-mediated isothermal amplification assay for rapid detection of Mycoplasma hyorhinis.

BACKGROUND: To establish a method for sensitive and rapid diagnosis of Mycoplasma hyorhinis in clinical specimens, a simple, sensitive loop-mediated isothermal amplification (LAMP) assay was designed and evaluated. METHODS: Three sets of four special primers, recognizing distinct sequences of the target, were designed for sensitive, specific amplification of nucleic acid under isothermal conditions. The LAMP assay was carried out using 35 clinical specimens of bronchoalveolar lavage fluid (BALF) from pigs. For comparison, these specimens were also tested using conventional PCR, real-time PCR, and nested PCR assays. RESULTS: After optimization of the reaction condition and reaction system, the LAMP reaction successfully detected Mycoplasma hyorhinis within 40 minutes at 61 degrees C. The LAMP assay achieved a sensitivity of 10(1) copies per microL at 61 degrees C in 40 minutes, compared to real-time PCR and nested PCR, and was over 10(3) times more sensitive than conventional PCR. In the test for the specificity of the LAMP assay, only Mycoplasma hyorhinis genomic DNA was positive and no other microorganisms were positive with the primers, indicating that the LAMP assay is specific to Mycoplasma hyorhinis. Mycoplasma hyorhinis was detected in 32 samples using the LAMP and real-time PCR assays and in 27 and 11 samples using the nested PCR assay and conventional PCR assay, respectively. All the positive samples detected by real-time PCR, nested PCR and conventional PCR assays were positive in the LAMP assay. CONCLUSIONS: The LAMP assay is inexpensive, easy to perform, shows a rapid reaction and does not require complex instruments like PCR. Therefore, LAMP is a simple, accurate, fast, and economical assay suitable as an alternative in veterinary practices.

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.

In vitro protective efficacy of Lithium chloride against Mycoplasma hyopneumoniae infection.

Mycoplasma hyopneumoniae (M. hyopneumoniae) infection affects the swine industry. Lithium chloride (LiCl), is a drug used to treat bipolar disorder and has also shown activity against bacterial and viral infections. Herein, we evaluated the antibacterial activity of LiCl on PK-15 cells infected with M. hyopneumoniae. Incubation of LiCl (40mM) with cells for 24h, did not significantly affect the cell viability. The qRT-PCR showed ~80% reduction in M. hyopneumoniae genome when LiCl added post-infection. A direct effect of LiCl on bacteria was also observed. However, treatment of cells with LiCl prior infection, does not protect against the infection. Anti-bacterial activity of LiCl was further confirmed by IFA, which demonstrated a reduction in the bacterial protein. With 40mM LiCI, the apoptotic cell death, production of nitric oxide and superoxide anion induced by M. hyopneumoniae, were prevented by ~80%, 60% and 58% respectively. Moreover, caspase-3 activity was also reduced (82%) in cells treated with 40mM LiCl. LiCl showed activity against various strains of M. hyopneumoniae examined in our study. Collectively, our data showed that LiCl inhibited the infection of M. hyopneumoniae through anti-apoptotic mechanism.

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.

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.

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.