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Severe asthma comprises only 5% of patients with asthma, but the burden it brings to the social health system accounts for more than half of all asthmatics. Clinical evidence shows that severe asthma is often linked to the recruitment and activation of neutrophils in the airways. However, the underlying molecular and immunological mechanisms of neutrophilia in severe asthma are not clear and currently available drugs exert only limited effects on neutrophilic inflammation. Great efforts are underway to address the mystery of neutrophilic inflammation in chronic respiratory disorders. Sialic acid-binding immunoglobulin-like lectins (Siglecs) are members of the immunoglobulin gene family. Of note, Siglec-9 is uniquely expressed by human neutrophils and monocytes, as well as a minor population of natural killer cells. Engaging this structure with antibodies or glycan ligands results in programmed cell death in human neutrophils. Intriguingly, the administration of Siglec-E antibody abolished the recruitment of neutrophils in mouse models of neutrophilic pulmonary inflammation in vivo. Given that neutrophils are probably a major culprit in the generation and perpetuation of inflammation, targeting Siglec-9 could be beneficial for the treatment of severe asthma, chronic obstructive pulmonary disease, and related pulmonary disorders characteristic of neutrophilia., (© 2018 S. Karger AG, Basel.)

Lipid-associated membrane proteins (LAMPs) are important in the pathogenicity of the Mycoplasma genus of bacteria. We investigated whether Mycoplasma hyopneumoniae LAMPs have pathogenic potential by inducing apoptosis in a St. Jude porcine lung epithelial cell line (SJPL). LAMPs from a pathogenic strain of M. hyopneumoniae (strain 232) were used in the research. Our investigation made use of diamidino-phenylindole (DAPI) and acridine orange/ethidium bromide (AO/EB) staining, terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) analysis, and Annexin-V-propidium iodide staining. After LAMP treatment for 24 h, typical changes were induced, chromosomes were concentrated, apoptotic bodies were observed, the 3'-OH groups of cleaved genomes were exposed, and the percentage of apoptotic cells reached 36.5 ± 11.66%. Caspase 3 and caspase 8 were activated and cytochrome c (cyt c) was released from the mitochondria into the cytoplasm; poly ADP ribose polymerase (PARP) was digested into two fragments; p38 mitogen-activated protein kinase (MAPK) was phosphorylated; and the expression of pro-apoptosis protein Bax increased while the anti-apoptosis protein Bcl-2 decreased. LAMPs also stimulated SJPL cells to produce nitric oxide (NO) and superoxide. This study demonstrated that LAMPs from M. hyopneumoniae can induce apoptosis in SJPL cells through the activation of caspase 3, caspase 8, cyt c, Bax, and p38 MAPK, thereby contributing to our understanding of the pathogenesis of M. hyopneumoniae, which should improve the treatment of M. hyopneumoniae infections.

The aim of this study was to establish a method for sensitive and rapid diagnosis of Mycoplasma hyopneumoniae in clinical specimens. To this effect, we employed three sets of primers specifically designed for amplification of nucleic acids under isothermal conditions. After optimization of reaction conditions, M. hyopneumoniae could be successfully detected at 63°C in 45 min through use of the loop-mediated isothermal amplification (LAMP) assay. A positive reaction was identified visually as white precipitate and confirmed by gel electrophoresis. The detection limit for this assay was 10 copies/μL, as observed by electrophoretic analysis. The accuracy of the LAMP reaction was confirmed by restriction endonuclease digestion as well as by direct sequencing of the amplified product. This method can specifically detect M. hyopneumoniae; other species with high homology and other bacterial and virus strains gave negative results. To test the utility of this procedure, the LAMP assay was applied to 40 clinical samples collected from swine lung tissues experimentally challenged with M. hyopneumoniae isolates, and compared to the results from a real-time polymerase chain reaction (PCR) assay. A concordance of 100% was observed between the two assays. In conclusion, the results from our study demonstrated that the LAMP assay provided a rapid reaction and was inexpensive to perform, with no need of complex instruments or systems such as Geneamp PCR. The LAMP assay may therefore be applied in routine diagnosis in the clinical laboratory and for in-field detection of M. hyopneumoniae 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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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 x Landrace x Yorkshire (DLY) pigs, which are Western commercial pigs that have been introduced in China. A total of 209 DLY piglets and 221 JQHPL piglets from 19 Landrace x Yorkshire and 22 JQHPL M. hyopneumoniae positive gestating sows with different expected dates of confinement were selected and raised in the same M. hyopneumoniae 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.

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Aiming at the delayed sowing of winter wheat induced by the drought and water logging often occurred in Huanghuai Plains of China, six sowing dates (15 October, normal sowing; 30 October, moderate delay; 15 November, delay; 30 November, seriously delay; 15 February, early spring sowing; and 1 March, spring sowing) were designed to investigate the effects of different sowing dates on the shoot type morphology and growth characteristics of winter wheat. With the delay of sowing date, the winter wheat grew and developed faster, and the growing period of the wheat sown in early spring and spring was 115-130 days shorter than that with normal sowing. As compared with those of the wheat with normal sowing, the shoot height, spike number per unit area, and productive spikelets per unit ear of the wheat sown delayed had a decrease, leaf position and canopy moved down, and leaf area reduced. When the sowing was delayed from the date 15 October (normal sowing) to 1 March (spring sowing), the harvest index increased from 0.46 to 0.53. Delaying sowing date also resulted in the significant reduction of grain yield, with the maximum decrement as high as 43. 6%. The spring-sown winter wheat not going through vernalization could still form yield.

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.

Aims: To investigate the species-specific prevalence of vhhP2 among Vibrio harveyi isolates and the applicability of vhhP2 in the specific detection of V. harveyi from crude samples of animal and environmental origins., Methods and Results: A gene (vhhP2) encoding an outer membrane protein of unknown function was identified from a pathogenic V. harveyi isolate. vhhP2 is present in 24 V. harveyi strains isolated from different geographical locations but is absent in 24 strains representing 17 different non-V. harveyi species, including V. parahaemolyticus and V. alginolyticus. A simple polymerase chain reaction method for the identification of V. harveyi was developed based on the conserved sequence of vhhP2. This method was demonstrated to be applicable to the quick detection of V. harveyi from crude animal specimens and environmental samples. The specificity of this method was tested by applying it to the examination of two strains of V. campbellii, which is most closely related to V. harveyi. One of the V. campbellii strains was falsely identified as V. harveyi., Conclusions: vhhP2 is ubiquitously present in the V. harveyi species and is absent in most of the non-V. harveyi species; this feature enables vhhP2 to serve as a genetic marker for the rapid identification of V. harveyi. However, this method can not distinguish some V. campbellii strains from V. harveyi., Significance and Impact of the Study: the significance of our study is the identification of a novel gene of V. harveyi and the development of a simple method for the relatively accurate detection of V. harveyi from animal specimens and environmental samples.