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Bone age assessment (BAA) is a widely used clinical practice for the biological development of adolescents. The Tanner Whitehouse (TW) method is a traditionally mainstream method that manually extracts multiple regions of interest (ROIs) related to skeletal maturity to infer bone age. In this paper, we propose a deep learning-based method for fully automatic ROIs localization and BAA. The method consists of two parts: a U-net-based backbone, selected for its strong performance in semantic segmentation, which enables precise and efficient localization without the need for complex pre- or post-processing. This method achieves a localization precision of 99.1% on the public RSNA dataset. Second, an InceptionResNetV2 network is utilized for feature extraction from both the ROIs and the whole image, as it effectively captures both local and global features, making it well-suited for bone age prediction. The BAA neural network combines the advantages of both ROIs-based methods (TW3 method) and global feature-based methods (GP method), providing high interpretability and accuracy. Numerical experiments demonstrate that the method achieves a mean absolute error (MAE) of 0.38 years for males and 0.45 years for females on the public RSNA dataset, and 0.41 years for males and 0.44 years for females on an in-house dataset, validating the accuracy of both localization and prediction.

Abstract Multidrug-resistant (MDR) Acinetobacter baumannii (A. baumannii) is currently recognized not only as a significant nosocomial pathogen but also is an emerging bacterial infection in food-producing animals, posing a critical threat to global health. However, this is a hindrance to detailed bioinformatic studies of MDR A. baumannii of chicken origin due to the lack of its complete genome sequence. Here, we report whole-genome sequencing analysis of MDR A. baumannii Y03 isolated from chickens. The Y03 genome consists of 1 circular chromosome and 4 circular plasmids, The Y03 chromosome harbors 41 antimicrobial resistance genes conferring resistance to major classes of antibiotics, including β-lactams, phenicols, macrolides, lincosamides, aminoglycosides, and nitrofurans, as well as 135 virulence factors involved in effector delivery system, immune modulation, adherence, stress survival, biofilm, exotoxin, and nutritional/metabolic factor. The in vivo infection experiments certificated that Y03 was virulent to chickens. Meanwhile, we used PCR amplification method to detect 10 antimicrobial resistance genes including abeM, adeB, adeH, adeK, bla apmC , bla OXA−90 , catB9, macB, folP, and parE, as well as 14 virulence genes including lpxC, pilO, fimT, ompA, basA, bauA, gspL, csu, pgaC, plc2, tssA, tviB, bap, and vgrG. Whole-genome sequencing analysis revealed that Y03 contained 46 horizontal gene transfer elements, including 11 genomic islands, 30 transposons, and 5 prophages, as well as 518 mutations associated with reduced virulence and 44 mutations resulting in loss of pathogenicity. Furthermore, there were 22 antibiotic targets and 28 lethal mutations on the Y03 chromosome that could be used as potential targets to prevent, control, and treat infections caused by MDR A. baumannii Y03. Therefore, this study contributes to the development of strategies for the prevention, control, and treatment of A. baumannii infections and their spread in chickens.

Abstract Avian pathogenic Escherichia coli (APEC) is a significant pathogen infecting poultry that is responsible for high mortality, morbidity and severe economic losses to the poultry industry globally, posing a substantial risk to the health of poultry. APEC encounters reactive oxygen species (ROS) during the infection process and thus has evolved antioxidant defense mechanisms to protect against oxidative damage. The imbalance of ROS production and antioxidant defenses is known as oxidative stress, which results in oxidative damage to proteins, lipids and DNA, and even bacterial cell death. APEC uses transcription factors (TFs) to handle oxidative stress. While many TFs in E. coli have been well characterized, the mechanism of the YbdO TF on protecting against oxidative damage and regulating the virulence and pathogenicity of APEC has not been clarified. Here we focus on the regulatory mechanism of YbdO on the pathogenicity of APEC. The results from this study showed that YbdO attenuated the pathogenicity of APEC in chicks infection models by inhibiting the expression of virulence genes fepG and ycgV using quantitative real-time reverse transcription PCR (RT-qPCR) experiments. The electrophoretic mobility shift assays (EMSA) confirmed that YbdO specifically bound to the promoters of fepG and ycgV. Additionally, YbdO increases H2O2-induced oxidative damage to APEC via repressing the expression of oxidative stress response genes sodA, soxR, ahpC, ahpF, katG, and oxyR by binding to their promoter regions. The repression effect facilitates host immune response to eliminate APEC and to generate beneficial immune protection to the body, thereby indirectly attenuating the pathogenicity of APEC. These findings might provide further insights into the mechanism of oxidative damage to APEC and offer new perspectives for further studies on the prevention and control of APEC infections.

Porcine circovirus type 2 (PCV2) is an important swine pathogen that has caused considerable economic losses in the global swine industry. During our surveillance of pigs in Shandong, China, from 2018 to 2020, we found that the PCV2 infection rate was 7.89% (86/1090). In addition, we found frequent mixed infections of PCV2 with porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), and porcine herpesvirus (PRV). Thirteen positive clinical samples were selected to amplify the complete genome of PCV2, and were sequenced. Among the 13, we detected two genotypes: PCV2b (1/13) and PCV2d (12/13). This suggests that PCV2d is the dominant genotype circulating in Shandong Province. Additionally, we found three positively selected sites in the ORF2 region, located on the previously reported antigenic epitopes. This investigation will contribute to understanding of the molecular epidemiology and genetic diversity of PCV2 strains in China.

Swine influenza virus (SIV) is a highly contagious pathogen that poses significant economic challenges to the swine industry and carries zoonotic potential, underscoring the need for vigilant surveillance. In this study, we performed a comprehensive genetic and molecular analysis of H3N2 SIV isolates obtained from 372 swine samples collected in Shandong Province, China. Phylogenetic analysis revealed two distinct genotypes. The surface genes of both genotypes clustered with the human-like H3N2 lineage, while the internal genes of one genotype clustered with the 2009 pandemic H1N1 (pdm/09) lineage. In the second genotype, the NS gene clustered with the classical swine (CS) H1N1 lineage, while the remaining internal genes clustered with pdm/09, suggesting stable integration of pdm/09 gene segments into H3N2 SIV. Homology analysis showed over 96% genetic similarity between the isolates and reference strains from China and Brazil, suggesting potential transmission through swine trade or human movement. Molecular characterization identified amino acid substitutions in the HA protein (190D, 226I, and 228S), potentially enhancing the virus’s affinity for human-like receptors, thereby increasing the zoonotic risk. Key mutations in the PB2 (271A, 591R), PA (336M, 356R, 409N), and M2 (S31N) proteins, along with novel drug resistance mutations, indicate the potential for enhanced virulence and drug resistance in these isolates. Moreover, glycosylation site analysis revealed four differences, and antigenic site analysis showed 13 differences between the HA proteins of the isolates and the WHO-recommended vaccine strain A/Cambodia/E0826360/2020 for the 2021–2022 season, which may reduce vaccine efficacy. Serological analysis revealed that 11 out of the tested serum samples were positive for H3N2 antibodies, resulting in an overall positivity rate of 0.42%. These findings emphasize the urgent need for strengthened SIV surveillance in China to monitor the risk of human transmission and ensure better preparedness for future influenza outbreaks.

The facultative anaerobic Gram-positive bacterium Enterococcus faecium is a ubiquitous member of the human gut microbiota. However, it has gradually evolved into a pathogenic and multidrug resistant lineage that causes nosocomial infections. The establishment of high-level intestinal colonization by enterococci represents a critical step of infection. The majority of current research on Enterococcus has been conducted under aerobic conditions, while limited attention has been given to its physiological characteristics in anaerobic environments, which reflects its natural colonization niche in the gut. In this study, a high-density transposon mutant library containing 26,620 distinct insertion sites was constructed. Tn-seq analysis identified six genes that significantly contribute to growth under anaerobic conditions. Under anaerobic conditions, deletion of sufB (encoding Fe-S cluster assembly protein B) results in more extensive and significant impairments on carbohydrate metabolism compared to aerobic conditions. Consistently, the pathways involved in this utilization-restricted carbohydrates were mostly expressed at significantly lower levels in mutant compared to wild-type under anaerobic conditions. Moreover, deletion of sufB or pflA (encoding pyruvate formate lyase-activating protein A) led to failure of gastrointestinal colonization in mice. These findings contribute to our understanding of the mechanisms by which E. faecium maintains proliferation under anaerobic conditions and establishes colonization in the gut.

Streptococcus dysgalactiae infection can cause bovine mastitis and lead to huge economic losses for the dairy industry. The abuse of antibiotics has resulted in growing drug resistance of S. dysgalactiae, which causes hard-to-treat infections. Bacteriophage lysin, as a novel antibacterial agent, has great potential for application against drug-resistant gram-positive bacteria. However, few studies have been conducted on the prophage lysin of S. dysgalactiae. In this study, we mined a novel prophage lysin, named Lys1644, from a clinical S. dysgalactiae isolate by genome sequencing and bioinformatic analysis. Lys1644 was expressed and purified, and the lytic activity, antibacterial spectrum, optimal pH and temperature, lytic activity in milk in vitro, and synergistic bacteriostasis with antibiotics were assessed. The Lys1644 prophage lysin showed high bacteriolysis activity specifically on S. dysgalactiae, which resulted in CFU 100-fold reduction in milk. Moreover, Lys1644 maintained high activity over a wide pH range (pH 5–10) and a wide temperature range (4–42 °C). Synergistic bacteriostatic experiments showed that the combination of low-dose Lys1644 (50 μg/mL) with a subinhibitory concentration of aminoglycoside antibiotics (kanamycin or spectinomycin) can completely inhibit bacterial growth, suggesting that the combination of Lys1644 and antibiotics could be an effective therapeutic strategy against S. dysgalactiae infection.

This study aims to investigate the probiotic properties of various isolated strains of Lactiplantibacillus plantarum. Specifically, the focus is on examining the expression of the glutamic acid decarboxylase (GAD) gene and its role in the production of gamma-aminobutyric acid (GABA), a promising postbiotic metabolite. The investigation includes comprehensive analyses of morphology, genetics, resilience against bile, NaCl, and simulated pancreatin juice (SPJ), carbohydrate fermentation patterns, antibacterial activity, susceptibility to antibiotics, and the presence of β-D-galactosidase and GAD enzymes. Six L. plantarum strains exhibited remarkable resilience against bile, NaCl, and SPJ, as well as susceptibility to antibiotics and antagonistic behavior against pathogens. These strains also showed the presence of β-D-galactosidase. Additionally, five L. plantarum strains were found to harbor the gad gene. Further biochemical analysis of four specific L. plantarum strains revealed promising profiles consisting of antibiotics, vitamins, hormones, and a diverse array of metabolites with potential immunotherapeutic properties. This study highlights the substantial potential of Lactiplantibacillus plantarum in generating beneficial postbiotic metabolites. The identified strains offer exciting avenues for further exploration, with potential applications in functional foods and pharmaceuticals. This research opens up possibilities for harnessing the probiotic and postbiotic potential of L. plantarum to develop novel products with health-promoting properties.

Abstract Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) is one of the most severe swine diseases that affects almost all swine-breeding countries. Nonstructural protein 2 (NSP2) is one of the most important viral proteins in the PRRSV life cycle. Our previous study showed that PRRSV NSP2 could induce the formation of aggresomes. In this study we explored the effects of aggresome formation on cells and found that NSP2 could induce autophagy, which depended on aggresome formation to activate aggrephagy. The transmembrane and tail domains of NSP2 contributed to aggrephagy and the cellular protein 14-3-3ε played an important role in NSP2-induced autophagy by binding the tail domain of NSP2. These findings provide information on the function of the C-terminal domain of NSP2, which will help uncover the function of NSP2 during PRRSV infection.

Chicken skin is considered the most susceptible to bacterial contamination during slaughter. It is rich in bushy feather follicles with complex internal structures that can absorb bacteria via cross-contamination during slaughter. Until now, the microstructural changes and local bacterial composition of feather follicles during slaughter have not been thoroughly investigated. This study used hematoxylin-eosin (HE) staining of the tissue paraffin section to investigate the structure of the feather follicles on chicken skin. In addition, the biopsy sampling method was employed for the high-throughput sequencing of 16S RNA genes to study the composition and source of bacterial contamination during slaughter. The results show that the feather follicles on chicken skin form a closed cavity structure during the slaughtering process. The volume of the irregular follicle cavity was about Ø: 200 μm × D: 1040 μm, which provides a place for the bacteria to absorb and resist the cleaning and disinfection during the slaughtering process. The composition of bacteria in the feather follicle was mainly Acinetobacter (37%), Psychrobacter (8%), Macrococcus (5%), and Comamonas (2%). The heat map obtained via the species abundance analysis of the feather follicle samples as well as the slaughter environment samples suggests that the gastrointestinal feces contaminated the feather follicles on the chicken skin mainly during the evisceration, defeathering, and chilling processes, and the last-stage chilling water also caused severe cross-contamination to the feather follicles during the chilling process.

Abstract The highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) emerged in 2006 in China and caused great economic losses for the swine industry because of the lack of an effective vaccine. 14-3-3 proteins are generating significant interest as potential drug targets by allowing the targeting of specific pathways to elicit therapeutic effects in human diseases. In a previous study, 14-3-3s were identified to interact with non-structural protein 2 (NSP2) of PRRSV. In the present study, the specific subtype 14-3-3ε was confirmed to interact with NSP2 and play a role in the replication of the HP-PRRSV TA-12 strain. Knockdown of 14-3-3ε in Marc-145 cells and porcine alveolar macrophages (PAMs) caused a significant decrease in TA-12 replication, while stable overexpression of 14-3-3ε caused a significant increase in the replication of TA-12 and low pathogenic PRRSV (LP-PRRSV) CH-1R. The 14-3-3 inhibitor difopein also decreased TA-12 and CH-1R replication in Marc-145 cells and PAMs. These findings are consistent with 14-3-3ε acting as a proviral factor and suggest that 14-3-3ε siRNA and difopein are therapeutic candidates against PRRSV infection.

Pigs are considered a “mixing vessel” that can produce new influenza strains through genetic reassortments, which pose a threat to public health and cause economic losses worldwide. The timely surveillance of the epidemiology of the swine influenza virus is of importance for prophylactic action. In this study, 15 H1N1, one H1N2, and four H3N2 strains were isolated from a total of 4080 nasal swabs which were collected from 20 pig farms in three provinces in China between 2016 and 2019. All the isolates were clustered into four genotypes. A new genotype represented by the H1N2 strain was found, whose fragments came from the triple reassortant H1N2 lineage, classical swine influenza virus (cs-H1N1) lineage, and 2009 H1N1 pandemic virus lineage. A/Sw/HB/HG394/2018(H1N1), which was clustered into the cs-H1N1 lineage, showed a close relationship with the 1918 pandemic virus. Mutations determining the host range specificity were found in the hemagglutinin of all isolates, which indicated that all the isolates had the potential for interspecies transmission. To examine pathogenicity, eight isolates were inoculated into 6-week-old female BALB/c mice. The isolates replicated differently, producing different viral loadings in the mice; A/Swine/HB/HG394/2018(H1N1) replicated the most efficiently. This suggested that the cs-H1N1 reappeared, and more attention should be given to the new pandemic to pigs. These results indicated that new reassortments between the different strains occurred, which may increase potential risks to human health. Continuing surveillance is imperative to monitor swine influenza A virus evolution.

Background/Aims: Mycobacterium tuberculosis is an extremely successful intracellular pathogen armed with multiple tactics to subvert host immunity. PPE (Pro-Pro-Glu) family exclusively distributed in mycobacteria might be responsible for the virulence and pathogenicity of M.tuberculosis. The up-regulation of Rv1808 (PPE32) in many conditions prompted us to define its role in host innate immune response. Methods: The Rv1808 encoding gene was expressed in nonpathogenic fast growing Mycobacterium smegmatis, mycobacteria- Escherichia coli shuttle plasmid pNITmyc served as control. RT-PCR and ELISA were used to detect the transcription and translation of host cytokines in culture supernatant from macrophage incubated with purified Rv1808 protein. Pharmacological inhibitors were applied to confirm the specificity of the effector interfering of host signaling. Results: Recombinant Ms_Rv1808 survived better than Ms_pNITmyc within macrophage, accompanied by slightly higher host cell death. Rv1808 protein is associated with the cell wall and exposed on the cell surface. Physical binding of Rv1808 to TLR2 resulted in increase in the secretion of anti-inflammatory cytokine interleukin-10 (IL-10) and pro-inflammatory cytokines tumor necrosis factor (TNF-a) and interleukin-6 (IL-6) possibly via co-activation of NF-κB and MAPK (p38MAPK, JNK and ERK) signalling. Conclusion: Cell wall associated Rv1808 protein manipulated the host cytokines via MAPK and NF-κB signaling pathways.