Mycoplasma hyopneumoniae is an important respiratory pathogen that causes great economic losses to the pig industry worldwide. Although some putative virulence factors have been reported, pathogenesis remains poorly understood. Herein, we evaluated the relative abundance of proteins in virulent 168 (F107) and attenuated 168L (F380) M. hyopneumoniae strains to identify virulence-associated factors by two-dimensional electrophoresis (2-DE). Seven proteins were found to be ≥ 1.5-fold more abundant in 168, and protein–protein interaction network analysis revealed that all seven interact with putative virulence factors. Unexpectedly, six of these virulence-associated proteins are encoded by core rather than accessory genomic elements. The most differentially abundant of the seven, fructose-1,6-bisphosphate aldolase (FBA), was successfully cloned, expressed and purified. Flow cytometry demonstrated the surface localisation of FBA, recombinant FBA (rFBA) mediated adhesion to swine tracheal epithelial cells (STEC), and anti-rFBA sera decreased adherence to STEC. Surface plasmon resonance showed that rFBA bound to fibronectin with a moderately strong KD of 469 nM. The results demonstrate that core gene expression contributes to adhesion and virulence in M. hyopneumoniae, and FBA moonlights as an important adhesin, mediating binding to host cells via fibronectin.Electronic supplementary materialThe online version of this article (10.1186/s13567-018-0610-2) contains supplementary material, which is available to authorized users.
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.
ABSTRACT. 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 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.
The objective of this study was to develop a TaqMan probe-based, sensitive, specific duplex real-time PCR assay for simultaneous detection of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis. The specific primers and probes, labeled with FAM and Texas Red, respectively, were designed to amplify the p97 gene of M. hyopneumoniae and p37gene of M. hyorhinis. The duplex real-time PCR reaction mixtures were established and optimized and the sensitivity, specificity and reproducibility of the assay were assessed. The sensitivity of the duplex real-time PCR was found to be 10 copies/μL for both M. hyopneumoniae and M. hyorhinis, respectively. There was no cross reaction with other common viral and bacterial pathogens. The concentration of standard coefficient of variation of Ct values was less than 5%, indicating a good reproducibility. Clinical samples (n = 937) were tested by the duplex real-time PCR assay, including broncho-alveolar lavage fluids, nasal swabs, tissues and cell culture supernatant. Duplex real-time PCR for simultaneous detection of M. hyopneumoniae and M. hyorhinis was highly sensitive and can be utilized for diagnosing clinical samples. It is timeefficient and economic, thereby providing a new approach to control both M. hyopneumoniae and M. hyorhinis.
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