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Abstract Agaric is a dual‐purpose fungus for medicine and food, which is considered a healthcare product. However, in cultivation and production, agaric is contaminated by a variety of molds, resulting in a decline in quality and yield. Fusarium, as one of the poisonous filamentous fungi, seriously threatens the healthy development of the agaric industry and even affects human health. Therefore, it is necessary to establish timely and accurate identification and rapid detection of pathogens and crops. In this study, the visible near infrared hyperspectral imaging was used to obtain the image and spectral information, and the best classification model was selected and established to quickly distinguish Fusarium oxysporum from Fusarium verticillioides, which was also successfully applied for the preliminary identification of agaric infection. This approach could provide a nondestructive manner for rapid identification and detection of pathogenic fungi contamination in agaric.

African swine fever (ASF) is an infectious disease caused by African swine fever virus (ASFV) that is highly contagious and has an extremely high mortality rate (infected by virulent strains) among domestic and wild pigs, causing huge economic losses to the pig industry globally. In this study, SDS−PAGE gel bands hybridized with ASFV whole virus protein combined with ASFV-convalescent and ASFV-positive pig serum were identified by mass spectrometry. Six antigens were detected by positive serum reaction bands, and eight antigens were detected in ASFV-convalescent serum. In combination with previous literature reports and proteins corresponding to MHC-II presenting peptides screened from ASFV-positive pig urine conducted in our lab, seven candidate antigens, including KP177R (p22), K78R (p10), CP204L (p30), E183L (p54), B602L (B602L), EP402R-N (CD2V-N) and F317L (F317L), were selected. Subunit-Group 1 was prepared by mixing above-mentioned seven ASFV recombinant proteins with MONTANIDETM1313 VG N mucosal adjuvant and immunizing pigs intranasally and intramuscularly. Subunit-Group 2 was prepared by mixing four ASFV recombinant proteins (p22, p54, CD2V-N1, B602L) with Montanide ISA 51 VG adjuvant and immunizing pigs by intramuscular injection. Anticoagulated whole blood, serum, and oral fluid were collected during immunization for flow cytometry, serum IgG as well as secretory sIgA antibody secretion, and cytokine expression testing to conduct a comprehensive immunogenicity assessment. Both immunogen groups can effectively stimulate the host to produce ideal humoral, mucosal, and cellular immune responses, providing a theoretical basis for subsequent functional studies, such as immunogens challenge protection and elucidation of the pathogenic mechanism of ASFV.

Abstract Mycoplasma hyopneumoniae, the causative agent of swine respiratory disease, demonstrates differences in virulence. However, factors associated with this variation remain unknown. We herein evaluated the association between differences in virulence and genotypes as well as phenotype (i.e., biofilm formation ability). Strains 168 L, RM48, XLW-2, and J show low virulence and strains 232, 7448, 7422, 168, NJ, and LH show high virulence, as determined through animal challenge experiments, complemented with in vitro tracheal mucosa infection tests. These 10 strains with known virulence were then subjected to classification via multilocus sequence typing (MLST) with three housekeeping genes, P146-based genotyping, and multilocus variable-number tandem-repeat analysis (MLVA) of 13 loci. MLST and P146-based genotyping identified 168, 168 L, NJ, and RM48 as the same type and clustered them in a single branch. MLVA assigned a different sequence type to each strain. Simpson’s index of diversity indicates a higher discriminatory ability for MLVA. However, no statistically significant correlation was found between genotypes and virulence. Furthermore, we investigated the correlation between virulence and biofilm formation ability. The strains showing high virulence demonstrate strong biofilm formation ability, while attenuated strains show low biofilm formation ability. Pearson correlation analysis revealed a significant positive correlation between biofilm formation ability and virulence. To conclude, there was no association between virulence and our genotyping data, but virulence was found to be significantly associated with the biofilm formation ability of M. hyopneumoniae.

IntroductionAflatoxins (AFT) identified as a Group 1 human carcinogen naturally contaminate various types of food and could increase the risk of hepatocellular carcinoma (HCC) through dietary intake. Chongqing municipality is located in Southwest China with subtropical monsoon climate which is conducive to AFT contamination in crops. However, the burden of HCC caused by the dietary exposure of the population in Chongqing to AFT has not been quantified.MethodsThe burden of HCC was estimated in terms of Disability Adjusted Life Year (DALY) using FDA-iRISK software. Dietary exposure to AFT in three food categories including grain and its products, nuts and seeds, and spices was assessed.ResultsThe lifetime average daily dose (LADD) of AFT exposure for the population ranged from 2.40 to 8.25 ng/kg bw/day and 9.51 to 15.10 ng/kg bw/day at the mean and heavy (P95) AFT contamination levels, respectively. Among the three food categories, grain and its products contributed most to AFT exposure of the population. The estimated DALYs related to HCC induced by AFT were 162,000–556,000 and 641,000-1,020,000; the DALY rates were 6.47–22.20 and 25.59–40.72 per 100,000 persons per year; and the population attribution fractions (PAF) were 1.68–5.78% and 6.66–10.60%.DiscussionAlthough the burden of HCC caused by dietary AFT was estimated to be relatively low among the population, the overall health burden might be underestimated owing to the uncertainties of this dataset. Thus, the overall health burden associated with AFT intake should still be of concern in further studies.

Abstract Urbanization has become a prominent anthropogenic activity globally, resulting in the substantial modification of temperature and rainfall in and around urban areas. Studies also indicate that rainfall exhibits an asymmetrical shift from light to extreme rainfall, causing both floods and droughts over different parts of the globe. However, to what extent urbanization influences the asymmetrical changes in rainfall and urban drought remains understudied. Accordingly, we provided an investigation of the urbanization effects on both rainfall and drought events from both statistical and model‐based perspectives. Results showed that urbanization generally increased heavy rainfall and decreased light rainfall in the rainy season over five urban agglomerations (i.e., Yangtze River Delta [YRD], Middle Region of Yangtze River [MRYR], Chengdu‐Chongqing, Guizhou, and Yunnan) in the Yangtze River Basin during 1981–2020. Specifically, urbanization contributed 42.7% and 30.8% of the increases of heavy rainfall in MRYR and YRD while 30.6% contribution to the decrease of light rainfall. Interestingly, results suggested that the urban regions were more prone to drought due to urbanization. Nevertheless, we also found that the urbanization effects showed some variabilities across different areas due to the interactions with complex terrains and climate patterns. Further model‐based simulations using the Weather Research and Forecasting model confirmed our findings and helped understand the potential underlying mechanism. The current research is expected to provide scientific knowledge for better urban planning and preparedness for urbanization‐induced hazards.

Primary porcine tracheal epithelial cells (PTECs) are an appropriate model for studying the molecular mechanism of various porcine respiratory diseases, including swine-origin mycoplasmas, which are isolated from respiratory tract of pigs and mainly found on the mucosal surface surrounding swine trachea. However, the short proliferation ability of primary PTECs greatly limits their lifespan. In this study, primary PTECs were carefully isolated and cultured, and immortal PTECs were constructed by transfecting primary PTECs with the recombinant constructed plasmid pEGFP-hTERT containing human telomerase reverse transcriptase (hTERT). Immortal PTECs (hTERT-PTECs) maintained both the morphological and functional characteristics of primary PTECs, as indicated by the expression of cytokeratin 18, cell-cycle analysis, proliferation assay, Western blotting, telomerase activity assay, karyotype analysis and quantitative RT-PCR. Compared to primary PTECs, hTERT-PTECs had an extended replicative lifespan, higher telomerase activity, and enhanced proliferative activity. In addition, this cell line resulted in a lack of transformed and grown tumors in nude mice, suggesting that it could be safely applied in further studies. Moreover, hTERT-PTECs were vulnerable to all swine-origin mycoplasmas through quantitative analysis as indicated by 50% color changing unit (CCU50) calculation, and no significant differences of adhesion ability between primary and immortal PTECs were observed. For the representative swine mycoplasma Mycoplasma hyopneumoniae (Mhp), except for DNA copies quantitative real-time PCR assay, indirect immunofluorescence assay and Western blotting analysis also depicted that hTERT-PTECs was able to adhere to different Mhp strains of different virulence. In summary, like primary PTECs, hTERT-PTECs could be widely used as an adhesion cell model for swine-origin mycoplasmas and in infection studies of various porcine respiratory pathogens.

ABSTRACT Lung inflammation induced by Mycoplasma hyorhinis infection accounts for significant economic losses in the swine industry. Increasing evidence suggests that there is cross talk between the lungs and the gut, but little is known about the effect of the lung inflammation caused by M. hyorhinis infection on gut microbiota and intestinal barrier function. Here, we investigated changes in the fecal microbiotas of pigs with M. hyorhinis infection and the microbial regulatory role of such infection in intestinal barrier function. We infected pigs with M. hyorhinis and performed 16S rRNA gene sequencing analyses of fecal samples, data-independent acquisition (DIA) quantitative proteomic analyses of intestinal mucosa, and analyses of barrier dysfunction indicators in serum. We found that pigs with M. hyorhinis infection exhibit lung and systemic inflammation, as reflected by the histopathological changes and activation of the TLR4/MyD88/NF-κB p65 signaling pathway in lung tissue, as well as the increased concentrations of serum inflammatory cytokines. Gut microbiotas tended to become disturbed, as evidenced by the enrichment of opportunistic pathogens. The increased diamine oxidase activities and d-lactate concentrations in serum and the decreased relative mRNA expression of Occludin, ZO-1, and Mucin2 indicated the impairment of intestinal barrier function. Quantitative proteomic analyses showed a variety of altered proteins involved in immunomodulatory and inflammatory functions. There was a positive correlation between the abundance of opportunistic pathogens and inflammatory-cytokine concentrations, as well as intestinal immunomodulatory proteins. Our results suggest that lung inflammation induced by M. hyorhinis infection can contribute to the dysbiosis of gut microbiota and intestinal barrier dysfunction, and dysbiosis of gut microbiota was associated with systemic inflammation and intestinal immune status. IMPORTANCE Cumulative evidence suggests that bacterial pneumonia may contribute to the dysbiosis of the gut microbiota and other gastrointestinal symptoms. Our experiment has demonstrated that lung inflammation induced by M. hyorhinis infection was associated with gut microbiota dysbiosis and intestinal barrier dysfunction, which may provide a theoretical basis for exploring the gut-lung axis based on M. hyorhinis infection.

ABSTRACT Mycoplasma hyorhinis is a widespread pathogen in pig farms worldwide. Although the majority of M. hyorhinis-colonized pigs have no apparent clinical disease, the pathogen can induce diseases such as polyserositis, arthritis, and eustachitis in some cases. To explore the mechanisms for the occurrence of these diseases, we challenged 4 groups of Bama miniature pigs with M. hyorhinis isolated from pigs without clinical symptoms (non-clinical origin [NCO] strain) or with typical clinical symptoms (clinical origin [CO] strain) and investigated the impacts of different strains and inoculation routes (intranasal [IN], intravenous [IV] + intraperitoneal [IP], and IV+IP+IN) on disease induction. Another group of pigs was set as a negative control. Pigs inoculated with the CO strain through a combined intravenous and intraperitoneal (IV+IP) route showed a significant decrease in average daily weight gain (ADWG), serious joint swelling, and lameness compared with the pigs in the negative-control group. Furthermore, this group developed moderate-to-severe pericarditis, pleuritis, peritonitis, and arthritis, as well as high levels of IgG and IgM antibodies. Pigs inoculated IV+IP with the NCO strain developed less marked clinical, pathological changes and a weaker specific antibody response compared with the pigs inoculated with the CO strain. The challenging results of the NCO strain via different routes (IV+IP, IV+IP+IN, and IN) indicated that the combined route (IV+IP) induced the most serious disease compared to the other inoculation routes. Intranasal inoculation induced a smaller decrease in ADWG without obvious polyserositis or arthritis. These data suggest that differences in both strain virulence and inoculation route affect the consequences of M. hyorhinis infection. IMPORTANCE Mycoplasma hyorhinis is a widespread pathogen in pig farms worldwide. The mechanisms or conditions that lead to the occurrence of disease in M. hyorhinis-infected pigs are still unknown. The objective of this study was to evaluate the impact of differences in the virulence of strain and the inoculation route on the consequences of M. hyorhinis infection.

Mycoplasma hyorhinis is a common pathogen of swine and is also associated with various human tumors. It causes systemic inflammation, typically polyserositis and polyarthritis, in some infected pigs. However, the pathogenic mechanism of M. hyorhinis remains unclear. DnaK is a highly conserved protein belonging to the heat-shock protein 70 family of molecular chaperones, which plays important roles as a moonlighting protein in various bacteria. In the present study, we identified the surface exposure of M. hyorhinis DnaK. Two virulent strains expressed more DnaK on their surface than the avirulent strain. Thereafter, the potential moonlighting functions of DnaK were investigated. Recombinant M. hyorhinis DnaK (rMhr-DnaK) was found to be able to adhere to swine PK-15 cells and human NCI-H292 cells. It also bound to four extracellular matrix components—fibronectin, laminin, type IV collagen, and vitronectin—in a dose-dependent manner. ELISA demonstrated an interaction between rMhr-DnaK and plasminogen, which was significantly inhibited by a lysine analog, ε-aminocaproic acid. rMhr-DnaK-bound plasminogen was activated by tissue-type plasminogen activator (tPA), and the addition of rMhr-DnaK significantly enhanced the activation. Finally, a DnaK-specific antibody was detected in the serum of pigs immunized with inactivated vaccines, which indicated good immunogenicity of it. In summary, our findings imply that DnaK is an important multifunctional moonlighting protein in M. hyorhinis and likely participates extensively in the infection and pathogenesis processes of M. hyorhinis.

Abstract Mycoplasma hyopneumoniae (M. hyopneumoniae), is the primary aetiological agent of enzootic pneumonia leading to chronic respiratory disease prevalent worldwide. Conventional pigs are the only animals used for pathogenicity studies and vaccine evaluations of M. hyopneumoniae. Considering that the challenge animals have better genetic stability and a smaller body size to operate with, an alternative experimental animal model of M. hyopneumoniae infection with Bama miniature pigs was established. Nine seven-week-old snatch-farrowed, porcine colostrum-deprived (SF-pCD) Bama miniature pigs and nine conventional pigs were randomly divided into two infected groups (Bama miniature-infected (BI) and conventional-infected groups (CI), BI and CI, n = 6) and two control groups (Bama miniature control (BC) and conventional control (CC) groups, BC and CC, n = 3). Every piglet was tracheally inoculated with 5 × 108 CCU/mL containing 10% suspension of a stock of frozen lung homogenate from SF-pCD pigs infected with virulent strain JS or sterilized KM2 medium. Typical lung lesions appeared in all infected pigs after necropsy, and the mean gross lung lesions was 17.3 and 13.7 in groups of BI and CI. Serum IgG and nasal sIgA antibody titres were increased significantly. Cilia shedding and mucus staining increased greatly in JS-infected bronchi. Obvious reddish gross lesions and M. hyopneumoniae antigen were detected, especially apparently observed in group of BI. Moreover, DNA copies of M. hyopneumoniae from bronchoalveolar lavage fluid (BALF) of each JS-infected piglet reached more than 108, and M. hyopneumoniae could be re-isolated from each infected BALF. These results indicate that Bama miniature pigs could be used as an alternative and more maneuverable experimental infection model for M. hyopneumoniae and display typical clinical and pathological features consistent with those in conventional pigs.

Abstract Background Rhomboid domain containing 1 (RHBDD1) plays a crucial role in tumorigenesis. Silibinin, which is a natural extract from milk thistle, has shown anti-tumor effects against various tumors. Here, we investigate whether silibinin affects the function of RHBDD1 in non-small cell lung cancer (NSCLC) cell proliferation, migration and invasion. Methods The Oncomine database and an immunohistochemistry (IHC) assay were used to determine the RHBDD1 expression levels in lung cancer tissues. The associations between RHBDD1 and overall survival rate or clinicopathological parameters were respectively assessed using the Kaplan-Meier overall survival analysis or Chi-squared test. CCK-8 and Transwell assays were applied to analyze cell proliferation, migration and invasion. A549 cells were incubated with increasing concentrations of silibinin. RHBDD1 knockdown and overexpression were achieved via transfection with si-RHBDD1 or RHBDD1 overexpression plasmid, respectively. Western blotting was performed to measure the expressions of epithelial–mesenchymal transition (EMT) markers. Results We found that overexpression of RHBDD1 in lung cancer tissues correlates with a poor prognosis of survival. Clinical specimen analysis showed that upregulation of RHBDD1 correlates remarkably well with TNM stage and lymph node metastasis. Silibinin suppresses A549 cell proliferation, migration, invasion and EMT in a dose-dependent manner. Importantly, RHBDD1 was downregulated in silibinin-treated A549 cells. RHBDD1 overexpression reversed the suppressive effects of silibinin on A549 cell proliferation, migration, invasion and EMT expression, while its knockdown enhanced them. Conclusions These findings shown an anti-tumor impact of silibinin on NSCLC cells via repression of RHBDD1.

Objective The high resolution mass spectra database of common alkaloids were established and applied to the automatic screening of poisoning samples. Methods The accurate molecular mass and appropriate collision energy of each alkaloid was obtained by injecting alkaloid standard solutions into the Q exactive mass spectrometry directly or separated by high performance liquid chromatography and mass spectrometry analysis. The chromatographic analysis was performed on a HSS T3 column (2.1 mm×100 mm, 1.8 μm) by gradient elution using methanol and 5 mmol/L ammonium acetate containing 0.1% formic acid solution as mobile phase. The secondary mass information of alkaloid was collected in positive electrospray ionization and targeted-selected ion monitor-ddMS2 mode. The mass spectrometry data were applied to the automatic screening of actual samples. Results The mass spectrometry database of 72 alkaloids were established containing the theoretical accurate molecular mass, retention time collision energy and fragment ions. The multiple alkaloids in actual samples were locked rapidly by automatic screening with the mass spectra database. Conclusion This method is simple, fast, accurate and suitable for the rapid automatic screening of alkaloids in poisoned food sample.

The active vibration damping actuator uses dual-motors to drive two sets of eccentric wheels to achieve active vibration reduction, but the backlash nonlinearity and eccentric load will cause the load speed and load position to be out of synchronization, which will influence the system vibration reduction effect. The traditional parallel control strategy controls the dual motors separately without the consideration of internal coupling effect, leading to a poor synchronization performance. In order to solve these problems, a load position difference cross-coupled control strategy based on multiple-input and multiple-output (MIMO) is proposed in this paper. The minimum singular value of return difference matrix is used to analyze the stability margin of active vibration damping actuator system under the traditional parallel control strategy and the cross-coupled control strategy, and the analysis procedure has considered the influences of load disturbance and backlash nonlinearity. At the same time, the sensitivity comparison of the two strategies is conducted, and the results indicate that the system under cross-coupled control strategy has stronger relative stability and robustness. On this basis, a method for designing synchronization controller parameters is given. The stability region of parameters satisfying the requirement of stability margin is determined with the help of minimum singular value of return difference matrix. Then, the optimal value is found in the obtained parameter stability region according to the dynamic and steady-state performance. Finally, an experimental platform is set up to verify the effectiveness of cross-coupled control strategy and the rationality of parameter tuning.

Abstract 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 K D 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.

Abstract Background Air-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. Results In 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. Conclusions Ali-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.

Background Mycoplasma hyopneumoniae (Mhp) is the main pathogen causing respiratory disease in the swine industry. Mhp infection rates differ across pig breeds, with Chinese native pig breeds that exhibit high fecundity (e.g., Jiangquhai, Meishan, Erhualian) more sensitive than Duroc, Landrace, and other imported pig breeds. However, the genetic basis of the immune response to Mhp infection in different pig breeds is largely unknown. Aims The aims of this study were to determine the relative Mhp susceptibility of the Chinese native Jiangquhai breed compared to the Duroc breed, and identify molecular mechanisms of differentially expressed genes (DEGs) using an RNA-sequencing (RNA-seq) approach. Methods Jiangquhai and Duroc pigs were artificially infected with the same Mhp dose. The entire experiment lasted 28 days. Daily weight gain, Mhp-specific antibody levels, and lung lesion scores were measured to evaluate the Mhp infection susceptibility of different breeds. Experimental pigs were slaughtered on the 28th day. Lung tissues were collected for total RNA extraction. RNA-seq was performed to identify DEGs, which were enriched by gene ontology (GO) and the Kyoto Encyclopedia annotation of Genes and Genomes (KEGG) databases. DEGs were validated with real-time quantitative polymerase chain reaction (RT-qPCR). Results Infection with the same Mhp dose produced a more serious condition in Jiangquhai pigs than in Duroc pigs. Jiangquhai pigs showed poorer growth, higher Mhp antibody levels, and more serious lung lesions compared with Duroc pigs. RNA-seq identified 2,250 and 3,526 DEGs in lung tissue from Jiangquhai and Duroc pigs, respectively. The two breeds shared 1,669 DEGs, which were involved in immune-relevant pathways including cytokine-cytokine receptor interaction, PI3K-Akt signaling pathway, and chemokine signaling pathway. Compared to Jiangquhai pigs, more chemokines, interferon response factors, and interleukins were specifically activated in Duroc pigs; CXCL10, CCL4, IL6 and IFNG genes were significantly up-regulated, which may help Duroc pigs enhance immune response and reduce Mhp susceptibility. Conclusion This study demonstrated differential immune-related DEGs in lung tissue from the two breeds, and revealed an important role of genetics in the immune response to Mhp infection. The biological functions of these important DEGs should be further confirmed and maybe applied as molecular markers that improve pig health.