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Vaccination with inactivated bacterin is the most popular and practical measure to control enzootic pneumonia. After immunisation with inactivated bacterin, Mycoplasma hyopneumoniae colonised on the respiratory tract and lung stimulates the humoural immune responses and produces IgG and IgA antibodies. ELISA is a widely used serological method to detect M. hyopneumoniae antibodies. However, commercial IgG‐ELISA kit cannot distinguish between inactivated bacterin‐induced hyperimmune sera and convalescent sera stimulated by natural infection. SIgA‐ELISA method needs to collect nasal swabs, but collecting nasal swabs is not easy to operate. Establishment of a discriminative ELISA detecting humoural IgG from convalescent sera but not hyperimmune sera facilitates to evaluate the natural infection of M. hyopneumoniae after inactivated bacterin vaccination. We expressed and purified a recombinant protein named Mhp366‐N which contains an epitope recognised by the convalescent sera but not hyperimmune sera. The developed discriminative IgG‐ELISA could discriminate between inactivated bacterin‐induced hyperimmune sera and convalescent sera and was reproducible, sensitive and specific to M. hyopneumoniae antibody produced by natural infection. Compared to SIgA‐ELISA method, discriminative IgG‐ELISA was more convenient to detect IgG antibody from sera than IgA from nasal swabs, although it has limited sensitivity in the early stages of infection. Additionally, to some extent, it has a potential to avoid the interference of maternally derived IgG antibodies. The established discriminative IgG‐ELISA was efficient to judge the serological IgG antibodies induced from natural infection or inactivated vaccine stimulation and provided a useful method to investigate and evaluate the live organism infection after the application of inactivated bacterin., An indirect IgG‐ELISA was developed to evaluate M. hyopneumoniae infection but not bacterin vaccination. The method was reproducible, sensitivity and specificity. This method was comparable to SIgA‐ELISA but more convenient.

Autophagy is an important cellular homeostatic mechanism for recycling of degradative proteins and damaged organelles. Autophagy has been shown to play an important role in cellular responses to bacteria and bacterial replication. However, the role of autophagy in Mycoplasma hyopneumoniae infection and the pathogenic mechanism is not well characterized. In this study, we showed that M. hyopneumoniae infection significantly increases the number of autophagic vacuoles in host cells. Further, we found significantly enhanced expressions of autophagy marker proteins (LC3-II, ATG5, and Beclin 1) in M. hyopneumoniae-infected cells. Moreover, immunofluorescence analysis showed colocalization of P97 protein with LC3 during M. hyopneumoniae infection. Interestingly, autophagic flux marker, p62, accumulated with the induction of infection. Conversely, the levels of p62 and LC3-II were decreased after treatment with 3-MA, inhibiting the formation of autophagosomes, during infection. In addition, accumulation of autophagosomes promoted the expression of P97 protein and the survival of M. hyopneumoniae in PK-15 cells, as the replication of M. hyopneumoniae was down-regulated by adding 3-MA. Collectively, these findings provide strong evidence that M. hyopneumoniae induces incomplete autophagy, which in turn enhances its reproduction in host cells. These findings provide novel insights into the interaction of M. hyopneumoniae and host.

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.

Background: Mycoplasma hyopneumoniae is the primary pathogen of mycoplasmal pneumonia of swine (MPS). Vaccination with inactivated bacterin is the most popular and practical measure to control MPS. However, these commercial vaccines have a limited effect on the transmission of microorganism and cannot prevent colonization. Therefore, after immunization with inactivated bacterin, M. hyopneumoniae colonized on the respiratory tract and lungs stimulates the humoral immune responses and produce IgG and IgA antibodies. ELISA is a widely used serological method to detect M. hyopneumoniae antibodies. However, commercial IgG ELISA kit cannot distinguish between inactivated bacterin-induced hyperimmune sera and convalescent sera stimulated by natural infection. SIgA ELISA method is laborious for nasal swab collection, and the amount of each swab sample obtained from the nasal cavity is less compared to the serum sample. Establishment of a discriminative ELISA detecting humoral IgG from convalescent sera but not hyperimmune sera is facilitating to evaluate the natural infection of Mycoplasma hyopneumoniae after inactivated bacterins vaccination. Result: We expressed and purified a recombinant protein named Mhp366-N which contains an epitope recognized by the convalescent sera but not hyperimmune sera. The developed discriminative IgG-ELISA could discriminate between inactivated bacterin-induced hyperimmune sera and convalescent sera, and was reproducible, sensitive, and specific to M. hyopneumoniae antibody produced by natural infection. Compared to SIgA-ELISA method, discriminative IgG-ELISA was more convenient to detect IgG antibody from sera than IgA from nasal swabs, although it is limited sensitivity in the early stages of infection. Additionally, to some extent, it has a potential to avoid the interference of maternally derived IgG antibodies. Conclusions: The established discriminative IgG-ELISA was efficient to judge the serological IgG antibodies induced from natural infection or inactivated vaccine stimulation and provided a useful method to investigate and evaluate the live organism infection after the application of inactivated bacterin.

Mycoplasma hyopneumoniae is the respiratory pathogen of porcine enzootic pneumonia, a chronic respiratory infectious disease that causes substantial pecuniary losses to pig husbandry worldwide. Commercial bacterins only provide incomplete protection and do not prevent the colonization and transmission of M. hyopneumoniae. Identification of new protective antigens is a key imperative for the development of more effective novel vaccine. The objective of this study was to evaluate antibody responses of 27 recombinant proteins in convalescent sera obtained from pigs that were naturally infected with M. hyopneumoniae. Fifteen proteins were identified as serological immunodominant antigens, while 3 proteins were not recognized by any convalescent serum. Moreover, Mhp462, a leucine aminopeptidase, was found to be a discriminative serological immunodominant antigen which reacted with convalescent sera but not with hyperimmune sera. The serological immunodominant proteins were antigenic and were expressed during infection; this suggests that these proteins (especially the discriminative one) are potential candidate antigens for the development of next generation vaccines against M. hyopneumoniae., Biotechnology; Microbiology; Infectious disease; Vaccination; Bacteria; Microbial biotechnology; Veterinary medicine; Mycoplasma hyopneumoniae, Serological immunodominant protein, Convalescent sera, Hyperimmune sera, ELISA

In order to evaluate the prevalence of the Mycoplasma mycoides cluster in goats in Chongqing, China, an epidemiological survey in this area was carried out. A total of 68 samples were subjected to bacteria isolation on Hartley’s medium. Four isolates (three from lung tissue and one from nasal discharges) were recovered from the samples and identified as the Mycoplasma species by their morphological and biochemical characteristics. They were further confirmed by PCR using 16S rRNA specific primer pairs and by restriction enzyme analysis. In vitro antimicrobial susceptibility of the isolates indicated that some strains had developed resistance to the antibiotics tested. This is the first report on the isolation, identification, and molecular characterisation of Mycoplasma species isolated from goats in Chongqing. This study also revealed a prevalence of Mycoplasma species infection in goats in this area.

In this study, we investigated 72 Escherichia coli O157:H7 strains from humans and animals to determine stx2 and its variants by polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism. Most isolates were found to carrystx2 or its variants and the stx2c was the dominant subtype; for the prevalence ofstx2c in stx2-positive isolates was 89.6% (42/47). All stx2 and stx2c harboring isolates obtained from humans had caused diarrhea or hemolytic-uremic syndrome. Three strains isolated from a piglet and two cattle carried the stx2c gene with an IS1203v inserted sequence. Sequencing of this region revealed that, this 1.3 kb insertion was very similar to a previously identified IS 1203v sequence and the insertion interrupted the carboxyl end of the B subunit coding region of stx2c gene. The corresponding positions in the stx2c gene sequence in which the IS 1203v sequence was inserted was varied. The isolates possessing IS1203v were inactive in the Vero cell toxicity assay. Key words: Shiga toxin-producing Escherichia coli; stx2; PCR-RFLP; insertion sequence; cytotoxicity.

Helicobacter pylori (H. pylori) is a major human pathogenic bacterium in gastric mucosa. However, the regulatory mechanism of H. pylori-induced immune response is not clear. MicroRNAs (miRNAs) have recently emerged as key post-transcriptional regulators of gene expression, and their role in H. pylori infection is just beginning to be explored. Here, we first reported that H. pylori infection up-regulated the expression of miR-146a in gastric epithelial cells as well as in gastric mucosal tissues in NF-κB-dependent manner. In turn, miR-146a may downregulate the expression of target genes, interleukin-1 receptor-associated kinase 1 (IRAK1) and TNF receptor-associated factor 6 (TRAF6). Furthermore, miR-146a negatively regulated H. pylori-triggered interleukin (IL)-8, growth-related oncogene (GRO)-α, and macrophage inflammatory protein (MIP) -3α through diminishing NF-κB activity. In conclusion, H. pylori-induced miR-146a plays a potential role in a negative feedback loop to modulate the inflammation by targeting IRAK1 and TRAF6.

Prevalence, presence of virulence and adherence associated genes, genetic diversity, biochemical characteristics, and antibiotic susceptibility were determined for Escherichia coli O157 isolated over 4 months in Chongqing city and Three-Gorge Reservoir Areas. 11 isolates of E. coli O157 were isolated from 1504 samples and 7 of them are O157:H7 and 4 are O157:H? All O157:H7 isolates had eaeA, ehxA, EspA and Tccp genes, but did not have stx1 and stx2. All O157:H? isolates did not have stx1, stx2, eaeA, ehxA, EspA and Tccp genes except for the isolate obtained from Yunyang county which had stx1. When eaeA and ehxA presented in isolates were digested by restriction enzymes, the numbers and the sizes of the segments were the same as the control E. coli O157 strains. This suggests that eaeA and ehxA exhibit poor polymorphism. Most E. coli O157 isolates showed identical biochemical activities to the standard strains for sorbitol and rhamnose, and all E. coli O157:H7 obtained from feces at the same dairy cattle farm had similar biochemical characteristics. Antibiotic susceptibility demonstrated resistance of the isolates to penicillin, ampicillin, bacitracin, cefuroxime, erythromycin, gentamycin and tetracycline, indicating the isolates obtained in this study had a multi-drug resistance.

The haem oxygenase ChuZ from Campylobacter jejuni, a major enteric pathogen in humans, is part of the iron-acquisition mechanism that is involved in bacterial survival and persistence in hosts. The ChuZ-haemin complex has been purified and crystallized and diffraction data have been collected to 2.4 Å resolution. The ChuZ-haemin complex crystals belonged to space group C222(1), with unit-cell parameters a = 106.474, b = 106.698, c = 52.464 Å, α = β = γ = 90°. The asymmetric unit contained one ChuZ monomer, with a Matthews coefficient of 2.58 Å(3) Da(-1).

Glycogen has been localized both inside and outside Chlamydia trachomatis organisms. We now report that C. trachomatis glycogen synthase (GlgA) was detected in both chlamydial organism-associated and -free forms. The organism-free GlgA molecules were localized both in the lumen of chlamydial inclusions and in the cytosol of host cells. The cytosolic GlgA displayed a distribution pattern similar to that of a known C. trachomatis-secreted protease, CPAF. The detection of GlgA was specific since the anti-GlgA antibody labeling was only removed by preabsorption with GlgA but not CPAF fusion proteins. GlgA was detectable at 12h and its localization into host cell cytosol only became apparent at 24h after infection. The cytosolic localization of GlgA was conserved among all C. trachomatis serovars. However, the significance of the GlgA secretion into host cell cytoplasm remains unclear since, while expression of chlamydial GlgA in HeLa cells increased glycogen stores, it did not affect a subsequent infection with C. trachomatis. Similar to several other C. trachomatis-secreted proteins, GlgA is immunogenic in women urogenitally infected with C. trachomatis, suggesting that GlgA is expressed and may be secreted into host cell cytosol during C. trachomatis infection in humans. These findings have provided important information for further understanding C. trachomatis pathogenic mechanisms.

Staphylococcus aureus is an opportunistic bacterial pathogen responsible for a diverse spectrum of human diseases and a leading cause of nosocomial and community-acquired infections. Development of a vaccine against this pathogen is an important goal. The fibronectin binding protein A (FnBPA) of S. aureus is one of multifunctional 'microbial surface components recognizing adhesive matrix molecules' (MSCRAMMs). It is one of the most important adhesin molecules involved in the initial adhesion steps of S. aureus infection. It has been studied as potential vaccine candidates. However, FnBPA is a high-molecular-weight protein of 106 kDa and difficulties in achieving its high-level expression in vitro limit its vaccine application in S. aureus infection diseases control. Therefore, mapping the immunodominant regions of FnBPA is important for developing polyvalent subunit fusion vaccines against S. aureus infections. In the present study, we cloned and expressed the N-terminal and C-terminal of FnBPA. We evaluated the immunogenicity of the two sections of FnBPA and the protective efficacy of the two truncated fragments vaccines in a murine model of systemic S. aureus infection. The results showed recombinant truncated fragment F130-500 had a strong immunogenicity property and survival rates significantly increased in the group of mice immunized with F130-500 than the control group. We futher identified the immunodominant regions of FnBPA. The mouse antisera reactions suggest that the region covering residues 110 to 263 (F1B110-263) is highly immunogenic and is the immunodominant regions of FnBPA. Moreover, vaccination with F1B110-263 can generate partial protection against lethal challenge with two different S. aureus strains and reduced bacterial burdens against non-lethal challenge as well as that immunization with F130-500. This information will be important for further developing anti- S. aureus polyvalent subunit fusion vaccines.