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Accurate detection is a prerequisite for effective prevention and control of Mycoplasma synoviae infection. ELISA is the most popular method for the clinical detection of M. synoviae because of its convenience, low cost, and high detection rate. However, the cross-reactivity of commercially available ELISA kits with other avian pathogen-positive sera needs to be addressed. The aim of this study was to establish an ELISA method with high specificity for the detection of anti-M. synoviae antibodies in chicken serum to evaluate the M. synoviae infection status on poultry farms. The recombinant MS087 (rMS087) protein was expressed in Escherichia coli BL21 (DE3) and purified by Ni2+ affinity chromatography. An antibody against rMS087 was generated by immunizing BALB/c mice. Bioinformatic analysis revealed that MS087 was conserved among M. synoviae strains. Western blotting and indirect immunofluorescence results indicated that MS087 was not only localized in the cytoplasm and on the membrane but also secreted by the organism. For the established ELISA method based on rMS087, the optimal antigen concentration, blocking buffer, blocking duration, serum dilution, serum incubation duration, secondary antibody dilution, secondary antibody incubation duration and colorimetric reaction duration were 2 μg/mL, 1% BSA, 3 h, 1:500, 1.5 h, 1:20,000, 2 h and 5 min, respectively. Validation of the rMS087-based ELISA revealed a cut-off value of 0.5. The coefficients of variation of both the intra-batch and inter-batch methods were less than 9%. The assay was able to differentiate positive serum against M. synoviae from antisera against nine other avian pathogens and was able to recognize M. synoviae-positive sera at a dilution of 1:1,000. Compared with the commercial ELISA method, the rMS087-based ELISA has the potential to recognize more positive sera against M. synoviae. Collectively, the rMS087-based ELISA is a reproducible, specific, and sensitive serological method for detecting antibodies against M. synoviae in chicken serum and has robust potential for large-scale serological epidemiology of M. synoviae infection on poultry farms.

ABSTRACT A total of 334 Salmonella isolates were recovered from 6,223 pet rectal samples collected at 50 pet clinics, 42 pet shops, 7 residential areas, and 4 plazas. Forty serovars were identified that included all strains except for one isolate that did not cluster via self-agglutination, with Salmonella Typhimurium monophasic variant, Salmonella Kentucky, Salmonella Enteritidis, Salmonella Pomona, and Salmonella Give being the predominant serovars. Fifty-one sequence types were identified among the isolates, and ST198, ST11, ST19, ST451, ST34, and ST155 were the most common. The top four dominant antimicrobials to which isolates were resistant were sulfisoxazole, ampicillin, doxycycline, and tetracycline, and 217 isolates exhibited multidrug resistance. The prevalence of β-lactamase genes in Salmonella isolates was 59.6%, and among these isolates, 185 harbored blaTEM, followed by blaCTX-M (66) and blaOXA (10). Moreover, six PMQR genes, namely, including qnrA (4.8%), qnrB (4.2%), qnrD (0.9%), qnrS (18.9%), aac(6')-Ib-cr (16.5%), and oqxB (1.5%), were detected. QRDR mutations (76.6%) were very common in Salmonella isolates, with the most frequent mutation in parC (T57S) (47.3%). Furthermore, we detected six tetracycline resistance genes in 176 isolates, namely, tet(A) (39.5%), tet(B) (8.1%), tet(M) (7.7%), tet(D) (5.4%), tet(J) (3.3%), and tet(C) (1.8%), and three sulfonamide resistance genes in 303 isolates, namely, sul1 (84.4%), sul2 (31.1%), and sul3 (4.2%). Finally, we found 86 isolates simultaneously harboring four types of resistance genes that cotransferred 2–7 resistance genes to recipient bacteria. The frequent occurrence of antimicrobial resistance, particularly in dogs and cats, suggests that antibiotic misuse may be driving multidrug-resistant Salmonella among pets.IMPORTANCEPet-associated human salmonellosis has been reported for many years, and antimicrobial resistance in pet-associated Salmonella has become a serious public health problem and has attracted increasing attention. There are no reports of Salmonella from pets and their antimicrobial resistance in Chongqing, China. In this study, we investigated the prevalence, serovar diversity, sequence types, and antimicrobial resistance of Salmonella strains isolated from pet fecal samples in Chongqing. In addition, β-lactamase, QRDR, PMQR, tetracycline and sulfonamide resistance genes, and mutations in QRDRs in Salmonella isolates were examined. Our findings demonstrated the diversity of serovars and sequence types of Salmonella isolates. The isolates were widely resistant to antimicrobials, notably with a high proportion of multidrug-resistant strains, which highlights the potential direct or indirect transmission of multidrug-resistant Salmonella from pets to humans. Furthermore, resistance genes were widely prevalent in the isolates, and most of the resistance genes were spread horizontally between strains.

Abstract Background Mycoplasma hyopneumoniae, the primary pathogen responsible for porcine enzootic pneumonia, reduces average daily weight gain and causes substantial economic losses to the pig industry worldwide. Vaccination is the most common strategy to control this disease but offers partial protection. Therefore, developing next‐generation vaccines by screening protective antigens is crucial. Objectives The aim of this study was to evaluate the antibody response to 33 recombinant proteins in pigs naturally infected with M. hyopneumoniae. Methods The genes encoding 33 (hypothetical) membrane proteins or secretory proteins were ligated into pGEX‐6P‐1, pGEX‐6P‐2, pGEX‐5X‐3 or pGEX‐4T‐3 vectors and transformed into Escherichia coli BL21(DE3) or E. coli XL‐1 Blue to construct recombinant bacteria and to express the recombinant proteins. The recombinant bacteria expressing the target proteins reacted with porcine convalescent sera and negative sera to screen immunodominant proteins by ELISA. Then, recombinant bacteria expressing immunodominant proteins were used to identify the discriminating immunodominant proteins that were recognised by convalescent sera nut not hyperimmune sera. Results All recombinant bacteria could express the target recombinant proteins in soluble form. Twenty‐one proteins were shown to present immunodominant antigens, and four proteins were not recognised by convalescent sera. Moreover, six proteins were considered discriminating and reacted with convalescent sera but not with hyperimmune sera. Conclusions The identified immunodominant proteins were antigenic and expressed during bacterial infection, suggesting that these proteins, especially those capable of discriminating between sera, can be used to identify protective antigens with the view to develop more effective vaccines against M. hyopneumoniae infection.

Abstract Background Staphylococcus aureus infection of livestock animals and humans is a major public health issue. There are reports of antimicrobial resistance and multiple staphylococcal superantigen genes in many countries and several provinces of China, but the status in Chongqing, China is uncertain. Objectives The aim of this study was to determine the prevalence, antimicrobial susceptibility, and other molecular characteristics of S. aureus isolates from livestock animals in Chongqing. Methods Staphylococcus aureus was isolated and identified by selective enrichment and amplification of the nuc gene from 1371 samples collected at farms in Chongqing. The agar dilution method was used to determine the resistant phenotype, and extended spectrum β‐lactamase genes were amplified by PCR. Methicillin‐resistant S. aureus was verified by the presence of the mecA gene, and the presence or absence of SE, SEl, and TSST‐1 genes was detected in the isolates. Results We cultured 89 S. aureus isolates from 1371 samples between March 2014 and December 2017. These isolates were from pigs, cattle, goats, rabbits, and chickens. There were four methicillin‐resistant S. aureus strains (three from pigs and one from a chicken). The 89 isolates had high resistance to penicillin (93.3%) and ampicillin (92.1%), but most were susceptible to amikacin and ofloxacin, with resistance rates below 10%. A total of 62.9% of the isolates had varying degrees of multidrug resistance. Almost all strains, except for three isolates from chickens, were positive for blaTEM‐1a. There were 19 of 20 tested staphylococcal SE/SEl/TSST‐1 genes present (all except for seq), and the predominant genes were sei (58.4%), tst‐1 (56.2%), and seg (51.7%). Conclusions The high antimicrobial resistance and prevalence of blaTEM‐1a reinforce the need to reduce the usage of antimicrobials in livestock. The universal existence of staphylococcal toxin genes implies a potential threat to public health by animal‐to‐human transmission via the food chain.

Abstract Autophagy is an important conserved homeostatic process related to nutrient and energy deficiency and organelle damage in diverse eukaryotic cells and has been reported to play an important role in cellular responses to pathogens and bacterial replication. The respiratory bacterium Mycoplasma hyopneumoniae has been identified to enter porcine alveolar macrophages, which are considered important immune cells. However, little is known about the role of autophagy in the pathogenesis of M. hyopneumoniae infection of porcine alveolar macrophages. Our experiments demonstrated that M. hyopneumoniae infection enhanced the formation of autophagosomes in porcine alveolar macrophages but prevented the fusion of autophagosomes with lysosomes, thereby blocking autophagic flux and preventing the acidification and destruction of M. hyopneumoniae in low-pH surroundings. In addition, using different autophagy regulators to intervene in the autophagy process, we found that incomplete autophagy promoted the intracellular proliferation of M. hyopneumoniae. We also found that blocking the phosphorylation of JNK and Akt downregulated the autophagy induced by M. hyopneumoniae, but pathways related to two mitogen-activated protein kinases (Erk1/2 and p38) did not affect the process. Collectively, M. hyopneumoniae induced incomplete autophagy in porcine alveolar macrophages through the JNK and Akt signalling pathways; conversely, incomplete autophagy prevented M. hyopneumoniae from entering and degrading lysosomes to realize the proliferation of M. hyopneumoniae in porcine alveolar macrophages. These findings raise the possibility that targeting the autophagic pathway may be effective for the prevention or treatment of M. hyopneumoniae infection.

Salmonella is one of the most important foodborne zoonotic pathogens, causing global morbidity and mortality in both humans and animals. Due to the extensive use of antimicrobials in food-producing animals, the antimicrobial resistance of Salmonella has attracted increasing attention globally. There have been many reports concerning the antimicrobial resistance of Salmonella from food-producing animals, meats and the environment. However, few studies on Salmonella from food-producing animals have been reported in Chongqing municipality, China. The aim of the present study was to determine the prevalence, serovar diversity, sequence types, and antimicrobial resistance of Salmonella isolated from livestock and poultry in Chongqing. Meanwhile, we also want to know the presence of β-lactamase genes, plasmid-mediated quinolone resistance (PMQR) genes and quinolone resistance-determining region (QRDR) mutations of Salmonella isolates. A total of 129 Salmonella strains were recovered from 2,500 fecal samples at 41 farms from pigs, goats, beef cattle, rabbits, chickens, and ducks. Fourteen serovars were identified, with S. Agona and S. Derby being the dominant serovars. The 129 isolates had high resistance to doxycycline (87.6%), ampicillin (80.6%), tetracycline (79.8%), trimethoprim (77.5%), florfenicol (76.7%) chloramphenicol (72.9%), and trimethoprim-sulfamethoxazole (71.3%), but were susceptible to cefepime. A total of 114 (88.4%) isolates showed multidrug resistant phenotypes. The prevalence of β-lactamase genes in Salmonella isolates was 89.9% (116/129), and among these isolates, 107 (82.9%) harbored blaTEM, followed by blaOXA (26, 20.2%), blaCTX-M (8, 6.2%), and blaCMY (3, 2.3%). In addition, qnrB, qnrD, qnrS, oqxA, oqxB, and aac(6′)-Ib-cr were detected in 11, 2, 34, 34, 43, and 72 PMQR-producing isolates, respectively. Moreover, QRDR mutations were very common in PMQR-positive Salmonella isolates (97.2%, 70/72) with mutation(s) in parC or combinative mutations in gyrA and parC. More significantly, 32 extended spectrum beta-lactamase (ESBL)-producing isolates were identified, and 62.5% of them were found to harbor one to four PMQR genes. Furthermore, 11 sequence types were identified from the isolates, and most of ESBL-producing isolates were attributed to ST34 (15.6%) and ST40 (62.5%). The coexistence of PMQR genes with β-lactamase genes and the extensive mutations in QRDR present in Salmonella isolates from food-producing animals suggest a potential threat to public health. Reasonable utilization and strict control strategies for antimicrobials in animal husbandry and animal treatment are necessary to reduce the emergence and dissemination of drug-resistant Salmonella isolates.

Abstract Background Evaluating inflammatory severity using imaging is essential for Crohn’s disease, but it is limited by potential interobserver variation and subjectivity. We compared the efficiency of magnetic resonance index of activity (MaRIA) collected by radiologists and a radiomics model in assessing the inflammatory severity of terminal ileum (TI). Methods 121 patients were collected from two centers. Patients were divided into ulcerative group and mucosal remission group based on the TI Crohn’s disease Endoscopic Severity Index. The consistency of bowel wall thickness (BWT), relative contrast enhancement (RCE), edema, ulcer, MaRIA and features of the region of interest between radiologists were described by weighted Kappa test and intraclass correlation coefficient (ICC), and developed receiver operating curve of MaRIA. The radiomics model was established using reproducible features of logistic regression based on arterial staging of T1WI sequences. Delong test was used to compare radiomics with MaRIA. Results The consistency between radiologists were moderate in BWT (ICC = 0.638), fair in edema (κ = 0.541), RCE (ICC = 0.461), MaRIA (ICC = 0.579) and poor in ulcer (κ = 0.271). Radiomics model was developed by 6 reproducible features (ICC = 0.93–0.96) and equivalent to MaRIA which evaluated by the senior radiologist (0.872 vs 0.883 in training group, 0.824 vs 0.783 in validation group, P = 0.847, 0.471), both of which were significantly higher than MaRIA evaluated by junior radiologist (AUC: 0.621 in training group, 0.557 in validation group, all, P

Abstract Background Determining the prognosis of lung adenocarcinoma (LUAD) is challenging. The present study aimed to identify prognostic ferroptosis-related long noncoding RNAs (FRLs) and construct a prognostic model. Moreover, differential analysis of immune and N6-methyladenosine (m6A)-related genes was systematically conducted. Methods A total of 504 patients selected from a dataset from The Cancer Genome Atlas were included. The patients with LUAD were randomly divided into a training group and a test group at a ratio of 1:1. Pearson correlation analysis and univariate Cox regression analysis were used to identify the prognostic FRLs. Then, a prognostic model was constructed from the optimized subset of prognostic FRLs based on the least absolute shrinkage and selection operator (LASSO) algorithm. Subsequently, the receiver operating characteristic (ROC) curve and survival analysis were used to evaluate the performance of the model. The risk score based on the prognostic model was analyzed using Cox regression analysis. Moreover, gene set enrichment analysis and differential analysis of immune- and m6A-related genes were conducted. Results After univariate Cox regression analysis and LASSO algorithm analysis, a total of 19 prognostic FRLs were selected to construct the final model to obtain the risk score. The area under the ROC curve of the prognostic model for 1-year, 3-year, and 5-year overall survival (OS) was 0.763, 0.745, and 0.778 in the training set and 0.716, 0.724, and 0.736 in the validation set, respectively. Moreover, the OS of the high-risk group was significantly worse than that of the low-risk group in the training group (P

Salmonella is one of the most important foodborne zoonotic pathogens, causing global morbidity and mortality in both humans and animals. Due to the extensive use of antimicrobials in food-producing animals, the antimicrobial resistance of Salmonella has attracted increasing attention globally. There have been many reports concerning the antimicrobial resistance of Salmonella from food-producing animals, meats and the environment. However, few studies on Salmonella from food-producing animals have been reported in Chongqing municipality, China. The aim of the present study was to determine the prevalence, serovar diversity, sequence types, and antimicrobial resistance of Salmonella isolated from livestock and poultry in Chongqing. Meanwhile, we also want to know the presence of β-lactamase genes, plasmid-mediated quinolone resistance (PMQR) genes and quinolone resistance-determining region (QRDR) mutations of Salmonella isolates. A total of 129 Salmonella strains were recovered from 2,500 fecal samples at 41 farms from pigs, goats, beef cattle, rabbits, chickens, and ducks. Fourteen serovars were identified, with S. Agona and S. Derby being the dominant serovars. The 129 isolates had high resistance to doxycycline (87.6%), ampicillin (80.6%), tetracycline (79.8%), trimethoprim (77.5%), florfenicol (76.7%) chloramphenicol (72.9%), and trimethoprim-sulfamethoxazole (71.3%), but were susceptible to cefepime. A total of 114 (88.4%) isolates showed multidrug resistant phenotypes. The prevalence of β-lactamase genes in Salmonella isolates was 89.9% (116/129), and among these isolates, 107 (82.9%) harbored blaTEM, followed by blaOXA (26, 20.2%), blaCTX-M (8, 6.2%), and blaCMY (3, 2.3%). In addition, qnrB, qnrD, qnrS, oqxA, oqxB, and aac(6′)-Ib-cr were detected in 11, 2, 34, 34, 43, and 72 PMQR-producing isolates, respectively. Moreover, QRDR mutations were very common in PMQR-positive Salmonella isolates (97.2%, 70/72) with mutation(s) in parC or combinative mutations in gyrA and parC. More significantly, 32 extended spectrum beta-lactamase (ESBL)-producing isolates were identified, and 62.5% of them were found to harbor one to four PMQR genes. Furthermore, 11 sequence types were identified from the isolates, and most of ESBL-producing isolates were attributed to ST34 (15.6%) and ST40 (62.5%). The coexistence of PMQR genes with β-lactamase genes and the extensive mutations in QRDR present in Salmonella isolates from food-producing animals suggest a potential threat to public health. Reasonable utilization and strict control strategies for antimicrobials in animal husbandry and animal treatment are necessary to reduce the emergence and dissemination of drug-resistant Salmonella isolates. [ABSTRACT FROM AUTHOR]