Massive extrapancreatic solid pseudopapillary neoplasm misdiagnosed as hepatic tumor: a case report and literature review.

Background: Solid pseudopapillary neoplasms (SPNs) of the pancreas are uncommon, low-malignancy neoplasms. Moreover, the occurrence of extrapancreatic SPNs is rarely encountered. Case summary: A 45-year-old female presented with a right upper abdominal mass and abdominal pain for 3 and 1 months as chief complaints, respectively. Initially, the patient was misdiagnosed with hepatocellular carcinoma based on her symptoms and results of physical and imaging examinations. Following multidisciplinary discussion and ruling out surgical contraindications, a decision was taken to proceed with surgical intervention. Interestingly, the tumor was found to originate from the retroperitoneum and had invaded the right half of the liver and the right wall of the inferior vena cava. The operation was uneventful, and the pathological findings confirmed the tumor as an extrapancreatic SPN. The patient remained asymptomatic after 15 months of follow-up. Conclusion: Surgical treatment remains the preferred option for extrapancreatic SPN. The preoperative misdiagnosis also highlights the importance of accurate diagnosis and the development of appropriate treatment strategies for liver masses.

Bacterial dynamic of flue-cured tobacco leaf surface caused by change of environmental conditions.

Microorganisms present on the surface of tobacco leaves play a significant role in shaping the composition of the tobacco microbial ecosystem, which undergoes continuous changes throughout the curing process. In the present study, a total of four distinct tobacco curing periods were selected for sampling, namely the fresh, yellowing, leaf-drying, and stem-drying stages. The bacterial 16S rRNA gene sequences of the collected samples were subsequently analyzed to identify operational taxonomic units (OTUs). The findings indicated that the complete dataset of leaf microbial samples was clustered, resulting in the identification of 1,783 operational taxonomic units (OTUs). Furthermore, the analysis of diversity revealed a pattern of initially increasing and subsequently decreasing community diversity. Redundancy Analysis (RDA) and weighted gene correlation networks for analysis (WGCNA) were employed in conjunction with environmental factors to assign OTUs to 22 modules for functional analysis. Additionally, a classification model utilizing the random forest algorithm was utilized to identify seven marker microorganisms (Escherichia coli, Faecalibacterium prausnitzii, Faecalibacterium, Escherichia-Shigella, Peptostreptococcaceae, Peptostreptococcales-Tissierellales, and Proteobacteria) that exhibited discriminative characteristics across different time periods. This study aimed to investigate the dynamic changes in the bacterial community throughout the curing process and their impact on the community's function. Additionally, certain bacteria were identified as potential markers for detecting changes in the curing stage. These findings offer a novel opportunity to accurately regulate the curing environment, thereby enhancing the overall quality of tobacco leaf curing.

Epidemiological investigation and drug resistance characteristics of Riemerella anatipestifer strains from large-scale duck farms in Shandong Province, China from March 2020 to March 2022.

Infectious serositis is a common disease caused by Riemerella anatipestifer (R. anatipestifer) in ducks, characterized by respiratory distress, septicemia, and neurological symptoms. In this study, 1,020 samples (brain and liver) were collected from ducks with suspected R. anatipestifer infection from March 2020 to March 2022 in Shandong Province, of which 171 R. anatipestifer strains were identified by PCR and isolation culture. The serotype of all strains was analyzed, and 74 strains were subjected to drug sensitivity tests and drug resistance genes detection. The results showed that the overall prevalence rate of R. anatipestifer in Shandong Province was 16.7% (171/1,020), with most strains coming from brain samples of ducklings under 3-mo old collected from September to December each year. Histopathological examination showed that heart vessels of the diseased duck were highly dilated and filled with red blood cells, with obvious fibrin exudates outside the pericardium, and fatty degeneration of liver cells. There were 45 strains of serotype 1, 45 strains of serotype 2, 2 strains of serotype 4, 33 strains of serotype 6, 44 strains of serotype 7, and 2 strains of serotype 10. The minimum inhibitory concentration (MIC) of 10 common antibiotics against 74 representative strains was determined by the agar dilution method. It was found that 74 strains had the most severe resistance to gentamicin (77%) and fully susceptible to ceftriaxone, but the 81.1% isolated strains were multidrug resistant. Resistance genes testing of 74 R. anatipestifers showed that tetracycline resistance gene tet X had the highest detection rate of 95.9%, followed by macrolide resistance gene ermF with 77%, and the rate of ß-lactam resistance gene blaTEM is the lowest (10.8%). The animal experiment of 4 R. anatipestifer strains with different serotypes showed that they had strong pathogenicity to 7-day-old ducklings, which could cause nervous symptoms, and the mortality rate was 58% to 70%. The autopsy showed obvious pathological changes. These findings of this study on R. anatipestifer will help us to understand the latest prevalence, drug resistance characteristics, and pathogenicity of R. anatipestifer in Shandong, China, and provide a scientific guide for the treatment and control of the disease.

Clinical value and potential mechanisms of BUB1B up-regulation in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) has insidious onset, late clinical diagnosis and high recurrence rate, which leads to poor quality of patient life. Therefore, it is necessary to further explore the pathogenesis and therapy targets of NPC. BUB1 mitotic checkpoint serine/threonine kinase B (BUB1B) was found to be up-regulated in a variety of cancers, but only two previous study showed that BUB1B was overexpressed in NPC and the sample size was small. The clinical role of BUB1B expression and its underlying mechanism in NPC require more in-depth research. Immunohistochemical samples and public RNA-seq data indicated that BUB1B protein and mRNA expression levels were up-regulated in NPC, and summary receiver operating characteristic curve indicated that BUB1B expression level had a strong ability to distinguish NPC tissues from non-NPC tissues. Gene ontology and Kyoto Encyclopedia of genes and genomes were performed and revealed that BUB1B and its related genes were mainly involved in cell cycle and DNA replication. Protein- Protein Interaction were built to interpret the BUB1B molecular mechanism. Histone deacetylase 2 (HDAC2) could be the upstream regulation factor of BUB1B, which was verified by Chromatin Immunoprecipitation Sequencing samples. In summary, BUB1B was highly expressed in NPC, and HDAC2 may affect cell cycle by regulating BUB1B to promote cancer progression.

Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus strains circulating in China from 2020 to 2021.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV), an enteric coronavirus, has become the major causative agent of acute gastroenteritis in piglets since 2010 in China. RESULTS: In the current study, 91 complete spike (S) gene sequences were obtained from PEDV positive samples collected from 17 provinces in China from March 2020 to March 2021. A phylogenetic analysis showed that 92.3% (84 out of 91) of the identified strains belonged to GII subtype, while 7.7% (7 out of 91) were categorized as S-INDEL like strains and grouped within GI-c clade. Based on a recombination analysis, six of S-INDEL like strains were recombinant strains originated from S-INDEL strain FR/001/2014 and virulent strain AJ1102. In addition, PEDV variant strains (CH/GDMM/202012, CH/GXDX/202010 et al) carrying novel insertions (360QGRKS364 and 1278VDVF1281) in the S protein were observed. Furthermore, the deduced amino acid sequences for the S protein showed that multiple amino acid substitutions in the antigenic epitopes in comparison with the vaccine strains. CONCLUSIONS: In conclusion, these data provide novel molecular evidence on the epidemiology and molecular diversity of PEDV in 2020-2021. This information may help design a strategy for controlling and preventing the prevalence of PEDV variant strains in China.

Potential Molecular Mechanism of Upregulated Aryl Hydrocarbon Receptor Nuclear Translocator 2 in Nasopharyngeal Carcinoma.

Background: Currently, the benefits of nasopharyngeal carcinoma (NPC) therapy are limited, and it is necessary to further explore possible therapeutic targets. Aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) has been extensively studied in other cancer species, but little has been explored in NPC. The aim of this study was to verify the expression level of ARNT2 and its underlying mechanism in NPC. Methods: Datasets containing ARNT2 mRNA expression levels were retrieved and collected from various databases to explore the expression status of ARNT2 in NPC. ARNT2-related coexpressed genes, differential expressed genes, and target genes were obtained for functional enrichment analysis. The potential target gene of ARNT2 and their regulatory relationship were studied through ChIP-seq data. CIBERSORTx was used to assess the immune infiltration of NPC, and the association with ARNT2 expression was calculated through correlation analysis. Results: ARNT2 was upregulated and possessed an excellent discriminatory capability in NPC samples. ARNT2 positively correlated target genes were clustered in pathways in cancer, while negatively correlated target genes were enriched in immune-related pathway. The ChIP-seq information of ARNT2 and histone showed that prostaglandin-endoperoxide synthase 2 (PTGS2) was a potential target gene of ARNT2. CIBERSORTx revealed the immunity status in NPC, and ARNT2 expression was correlated with infiltration of five immune cells. Conclusions: ARNT2 is overexpressed in NPC and may regulate PTGS2 to participate in the cancer process. ARNT2 serves as a key oncogenic target in NPC patients.

The Function of BBX Gene Family under Multiple Stresses in Nicotiana tabacum.

B-box (BBX) is a zinc finger transcription factor, which is involved in regulating the growth and development of plants and resisting various stresses. In this study, 43 NtBBX genes were identified and divided into five subgroups in tobacco. The members in each subgroup had similar characteristics. The promoter region of NtBBX genes had cis-acting elements related to light response, hormone regulation and stress response. Transcriptome analysis showed that NtBBX30 was significantly up-regulated, and NtBBX12, NtBBX13, NtBBX16 and NtBBX17 were significantly down-regulated under abiotic stresses. The NtBBX genes also responded to the infection of Ralstonia solanacearum. NtBBX9, NtBBX1, NtBBX15 and NtBBX17 showed the greatest response under stresses. The NtBBX genes are expressed in various degrees under different tissues. This research will provide a solid foundation for further study of the biological function of NtBBX genes in tobacco.

Downregulated miR-150-5p in the Tissue of Nasopharyngeal Carcinoma.

The clinical significance and potential targets of miR-150-5p have not been elucidated in nasopharyngeal carcinoma (NPC). The pooled analysis based on 539 NPC samples and 75 non-NPC nasopharyngeal samples demonstrated that the expression of miR-150-5p was down-regulated in NPC, with the area under the curve being 0.89 and the standardized mean difference being -0.66. Subsequently, we further screened the differentially expressed genes (DEGs) of 14 datasets, including 312 NPC samples and 70 non-NPC nasopharyngeal samples. After the DEGs were narrowed down with the predicted targets from the miRWalk database, 1316 prospective target genes of miR-150-5p were identified. The enrichment analysis suggested that "pathways in cancer" was the most significant pathway. Finally, six hub genes of "pathways in cancer", including EGFR, TP53, HRAS, CCND1, CDH1, and FGF2, were screened out through the STRING database. In conclusion, the down-regulation of miR-150-5p modulates the tumorigenesis and progression of NPC.

Genome-wide identification and expression analyses of late embryogenesis abundant (LEA) gene family in tobacco (Nicotiana tabacum L.) reveal their function in abiotic stress responses.

Late embryogenesis abundant (LEA) proteins play an important role in plant growth and response to abiotic stresses. However the late embryogenesis abundant (LEA) gene family in Nicotiana tabacum has not been systematically studied. In this study, 123 NtLEA genes were identified in Nicotiana tabacum, and divided into 8 groups, including LEA_1, LEA_2, LEA_3, LEA_4, LEA_5, LEA_6, DHN (dehydratin) and SMP (Seed Maturation Protein). The LEA_2 group is the most abundant of the NtLEA family. The gene structure, conserved motifs, subcellular localization and physicochemical properties of the NtLEA genes were analyzed. RNA-seq and qPCR analyses showed that the NtLEA genes were significantly induced under two different abiotic stresses and showed different expression patterns. The expression patterns of 35 NtLEA genes responding to ABA and 3 NtLEA genes responding to NaCl abiotic stress, respectively, were characterized. The protein-protein interaction network revealed that most NtLEA proteins (>78%) had the potential function to enhance tobacco resistance to abiotic stress. The transcriptional regulatory network showed that 21 transcription factor families were involved in regulating the expression of the NtLEA genes. These results are beneficial for future studies of the function of the NtLEA genes.

Florfenicol/Chlortetracycline Effect on Pharmacodynamic Indices for Mutant Selection of Riemerella anatipestifer in Ducks.

Riemerella anatipestifer can cause septicemia and death in ducks and geese, leading to significant economic losses to animal farms. The emergence of resistance of R. anatipestifer to commonly used antibiotics increases the difficulty of treating R. anatipestifer infection. The aim of this study was to evaluate the utility of antibiotic combination to restrict mutant selection of multidrug-resistant (MDR) R. anatipestifer isolates. Pharmacokinetics of florfenicol and chlortetracycline in Pekin ducks were evaluated using both noncompartmental analysis and population pharmacokinetic models. The areas under the curve of florfenicol and chlortetracycline after single 20 and 10 mg/kg oral administration were 49.3 and 6.84 mg*h/L, respectively. Chlortetracycline exhibited high apparent clearance and low systemic exposure. Minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) values of the two antibiotics were determined in 10 and 2 MDR R. anatipestifer isolates, respectively, to derive fTMSW (the fraction of time over 24 hours wherein the free drug concentration was within the mutant selection window [MSW]) and fT>MPC (the fraction of time that the free drug concentration was above the MPC). Both fTMSW and fT>MPC were estimated from simulated concentration-time profiles relative to MIC and MPC. Florfenicol and chlortetracycline combination have additive activities against R. anatipestifer in majority of isolates and could significantly decrease monotherapy MPC of florfenicol and chlortetracycline, as well as optimize both fTMSW and fT>MPC parameters, provided that the bioavailability of chlortetracycline is improved. The application of pharmacokinetic/pharmacodynamic analyses to MPC concepts to restrict selection of mutant bacterial strains can help improve short- and long-term outcomes of antibiotic treatment in animal farms.

Serological Surveillance of the H1N1 and H3N2 Swine Influenza A Virus in Chinese Swine between 2016 and 2021.

Background: Swine influenza A virus (IAV-S) is a common cause of respiratory disease in pigs and poses a major public health threat. However, little attention and funding have been given to such studies. The aim of this study was to assess the prevalence of the Eurasian avian-like H1N1 (EA H1N1), 2009 pandemic H1N1 (pdm/09 H1N1), and H3N2 subtype antibodies in unvaccinated swine populations through serological investigations. Such data are helpful in understanding the prevalence of the IAV-S. Methods: A total of 40,343 serum samples from 17 regions in China were examined using hemagglutination inhibition (HI) tests against EA H1N1, pdm/09 H1N1, and H3N2 IAV-S from 2016 to 2021. The results were analyzed based on a reginal distribution, seasonal distribution, and in different breeding stages. Results: A total of 19,682 serum samples out of the 40,343 were positive for IAV-S (48.79%). The positivity rates to the EA H1N1 subtype, pdm/09 H1N1 subtype, and H3N2 subtype were 24.75% (9,986/40,343), 7.94% (3,205/40,343), and 0.06% (24/40,343), respectively. The occurrences of coinfections from two or more subtypes were also detected. In general, the positivity rates of serum samples were related to the regional distribution and feeding stages. Conclusions: The results of this study showed that the anti-EA H1N1 subtype and pdm/09 H1N1 subtype antibodies were readily detected in swine serum samples. The EA H1N1 subtype has become dominant in the pig population. The occurrences of coinfections from two or more subtypes afforded opportunities for their reassortment to produce new viruses. Our findings emphasized the need for continuous surveillance of influenza viruses.

Downregulation of MicroRNA-1 and Its Potential Molecular Mechanism in Nasopharyngeal Cancer: An Investigation Combined with In Silico and In-House Immunohistochemistry Validation.

BACKGROUND: This study was aimed at elucidating the molecular biological mechanisms of microRNA-1 (miR-1) in nasopharyngeal carcinoma (NPC). METHOD: In this study, we performed a pooled analysis of miR-1 expression data derived from public databases, such as GEO, ArrayExpress, TCGA, and GTEx. The miRWalk 2.0 database, combined with the mRNA microarray datasets, was used to screen the target genes, and the genes were then subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis using the DAVID 6.8 database. We then used the STRING 11.0 database and Cytoscape 3.80 software to construct a protein-protein interaction (PPI) network for screening hub genes. Immunohistochemistry (IHC) was further used to validate the expression of hub genes. Finally, potential therapeutic agents for NPC were screened by the Connectivity Map (cMap) database. RESULTS: Pooled analysis showed that miR-1 expression was significantly decreased in NPC (SMD = -0.57; P < 0.05). The summary receiver operating characteristic curve suggested that miR-1 had a good ability to distinguish cancerous tissues from noncancerous tissues (AUC = 0.78). The results of GO analysis focused on mitotic nuclear division, DNA replication, cell division, cell adhesion, extracellular space, kinesin complex, and extracellular matrix (ECM) structural constituent. The KEGG analysis suggested that the target genes played a role in key signaling pathways, such as cell cycle, focal adhesion, cytokine-cytokine receptor interaction, ECM-receptor interaction, and PI3K/Akt signaling pathway. The PPI network suggested that cyclin-dependent kinase 1 (CDK1) was the hub gene, and the CDK1 protein was subsequently confirmed to be significantly upregulated in NPC tissues by IHC. Finally, potential therapeutic drugs, such as masitinib, were obtained by the cMap database. CONCLUSION: miR-1 may play a vital part in NPC tumorigenesis and progression by regulating focal adhesion kinase to participate in cell mitosis, regulating ECM degradation, and affecting the PI3K/Akt signaling pathway. miR-1 has the potential to be a therapeutic target for NPC.

Down-Regulation of Activating Transcription Factor 3 (ATF3) in Hepatoblastoma and Its Relationship with Ferroptosis.

PURPOSE: The molecular mechanisms and signal pathways of ferroptosis in hepatoblastoma (HB) have not yet been clarified. In previous studies, activating transcription factor 3 (ATF3) was reported to be correlated with several tumors, but the clinical significance of ATF3 has never been determined. Herein, we investigated the clinicopathological value and mechanisms of ATF3 in regulating ferroptosis in HB. METHODS: The mRNA microarray and RNA-sequencing data of 402 samples from our hospital and public databases were used to estimate ATF3 expression and assess its clinical role in HB. The standard mean difference (SMD) and summary receiver operating characteristic curves were utilized to judge the discrimination ability of ATF3 between HB and non-HB liver tissues. We examined the expression variation of ATF3 in HB cells after the treatment with erastin. We also predicted the target genes of ATF3 as a transcriptional factor from public Chromatin Immunoprecipitation-sequencing data and selected the ferroptosis-related genes for a signaling pathway analysis. RESULTS: In ten series, the pooled SMD for ATF3 was -0.91, demonstrating that ATF3 expression was predominantly lower in HB than in non-HB liver tissues. ATF3 down-regulation showed moderate potential to distinguish HB from non-HB liver tissues (area under curves = 0.83, 95% confidence interval = 0.79-0.86). Altogether, 4855 putative targets of ATF3 as a transcriptional factor were collected, among which, 60 genes were ferroptosis-related. CONCLUSION: The down-regulated ATF3 expression may play a vital role in the occurrence of HB possible partially by regulating ferroptosis.

Molecular Characterization of Blastocystis from Beef Cattle in Northeastern China.

Blastocystis is a common unicellular protist that lives in the intestines of humans and animals. Blastocystis infection and subtypes in cattle have been reported in several regions. However, the information of Blastocystis infection in cattle in China is still largely scant. To assess the prevalence and subtype distribution of Blastocystis in beef cattle in China, 803 fecal samples were collected from beef cattle farms in four cities of Northeast China, and were subjected to an analysis based on small subunit rRNA gene fragment. The overall prevalence of Blastocystis in beef cattle was 2.11% (17/803), with 2.15% in preweaning calves, 1.9% in postweaning calves, and 3.85% in breeding cattle, but absence in adult cattle (p > 0.05). Moreover, five Blastocystis subtypes were identified (ST10, ST21, ST23, ST25, and ST26), among which ST10 and ST26 subtypes were dominant subtypes in beef cattle. Mixed infections were detected in three specimens (ST10/ST25, ST10/ST23/ST25, and ST10/ST26). This is the first report showing Blastocystis infection in beef cattle in Northeast China. In addition, a variety of Blastocystis subtypes are reported in cattle in China for the first time. These results will benefit for better understanding the epidemiology and public health implications of Blastocystis.

Incomplete thermal ablation-induced up-regulation of transcription factor nuclear receptor subfamily 2, group F, member 6 (NR2F6) contributes to the rapid progression of residual liver tumor in hepatoblastoma.

Hepatoblastoma is a kind of extreme malignancy frequently diagnosed in children. Although surgical resection is considered as the first-line treatment for hepatoblastoma, a relatively large population of patients have lost the preferred opportunity for surgery. Administration of locoregional ablation enables local tumor control but with the deficiency of insufficient ablation, residual tumor, and rapid progression. In this study, we integrated 219 hepatoblastoma and 121 non-cancer liver tissues to evaluate the expression of NR2F6, from which a higher NR2F6 level was found in hepatoblastoma compared with non-cancer livers with a standard mean difference (SMD) of 1.04 (95% CI: 0.79, 1.29). The overexpression of NR2F6 also appeared to be an efficient indicator in distinguishing hepatoblastoma tissues from non-cancer liver tissues from the indication of a summarized AUC of 0.90, with a pooled sensitivity of 0.76 and a pooled specificity of 0.89. Interestingly, nude mouse xenografts provided direct evidence that overexpressed NR2F6 was also detected in residual tumor compared to untreated hepatoblastoma. Chromatin immunoprecipitation-binding data in HepG2 cells and transcriptome analysis of HepG2 xenografts were combined to identify target genes regulated by NR2F6. We finally selected 150 novel target genes of NR2F6 in residual tumor of incomplete ablation, and these genes appeared to be associated with the biological regulation of lipid metabolism-related pathway. Accordingly, targeting NR2F6 holds a therapeutic promise in treating residual recurrent hepatoblastoma after incomplete ablation.

Characterization of hydrophobic interaction of galactomannan in aqueous solutions using fluorescence-based technique.

Fluorescence probing was used to study hydrophobic interactions of galactomannan (GM) obtained from fenugreek gum (FG), guar gum (GG), and locust bean gum (LBG) at different M/G ratios. The I1/I3 ratio of pyrene changed from 1.73 to 1.29, 1.22, and 1.29 for FG, GG and LBG, respectively, as the concentration of GM increased from 0.01 to 8.0 g/L at 30 °C. The critical aggregation concentration of FG, GG, and LBG increased from 1.04 to 3.84 g/L, 1.15 to 3.73 g/L, and 0.94 to 3.63 g/L, respectively, as temperature increased from 10 to 70 °C. Addition of Na2SO4 and NaSCN increased the I1/I3 ratio in dilute solution, but reduced it in semi-dilute solution, whereas adding urea reduced I1/I3 in dilute solution but increased it in semi-dilute solution. These results indicated that the CAC of GM, polarity and number of hydrophobic microdomains were highly dependent on the M/G ratio and galactose distribution.

The role of 14-3-3 proteins in cell signalling pathways and virus infection.

14-3-3 proteins are highly conserved in species ranging from yeast to mammals and regulate numerous signalling pathways via direct interactions with proteins carrying phosphorylated 14-3-3-binding motifs. Recent studies have shown that 14-3-3 proteins can also play a role in viral infections. This review summarizes the biological functions of 14-3-3 proteins in protein trafficking, cell-cycle control, apoptosis, autophagy and other cell signal transduction pathways, as well as the associated mechanisms. Recent findings regarding the role of 14-3-3 proteins in viral infection and innate immunity are also reviewed.

Intranasal delivery of plasmids expressing bovine herpesvirus 1 gB/gC/gD proteins by polyethyleneimine magnetic beads activates long-term immune responses in mice.

BACKGROUND: DNA vaccine is one of the research hotspots in veterinary vaccine development. Several advantages, such as cost-effectiveness, ease of design and production, good biocompatibility of plasmid DNA, attractive biosafety, and DNA stability, are found in DNA vaccines. METHODS: In this study, the plasmids expressing bovine herpesvirus 1 (BoHV-1) gB, gC, and gD proteins were mixed at the same mass ratio and adsorbed polyethyleneimine (PEI) magnetic beads with a diameter of 50 nm. Further, the plasmid and PEI magnetic bead polymers were packaged into double carboxyl polyethylene glycol (PEG) 600 to use as a DNA vaccine. The prepared DNA vaccine was employed to vaccinate mice via the intranasal route. The immune responses were evaluated in mice after vaccination. RESULTS: The expression of viral proteins could be largely detected in the lung and rarely in the spleen of mice subjected to a vaccination. The examination of biochemical indicators, anal temperature, and histology indicated that the DNA vaccine was safe in vivo. However, short-time toxicity was observed. The total antibody detected with ELISA in vaccinated mice showed a higher level than PBS, DNA, PEI + DNA, and PBS groups. The antibody level was significantly elevated at the 15th week and started to decrease since the 17th week. The neutralizing antibody titer was significantly higher in DNA vaccine than naked DNA vaccinated animals. The total IgA level was much greater in the DNA vaccine group compared to other component vaccinated groups. The examination of cellular cytokines and the percentage of CD4/CD8 indicated that the prepared DNA vaccine induced a strong cellular immunity. CONCLUSION: The mixed application of plasmids expressing BoHV-1 gB/gC/gD proteins by nano-carrier through intranasal route could effectively activate long-term humoral, cellular, and mucosal immune responses at high levels in mice. These data indicate PEI magnetic beads combining with PEG600 are an efficient vector for plasmid DNA to deliver intranasally as a DNA vaccine candidate.

Establishment and evaluation of an indirect ELISA for detection of antibodies to goat Klebsiella pneumonia.

BACKGROUND: Klebsiella pneumonia, a Gram-negative bacterium belonging to the genus Enterobacter, causes many human and livestock diseases. Notably, infected goats may develop pneumonia, septicemia, which can lead to occasional death, resulting in great economic losses in goat-farming industry. However, there are little systematic methods for detection of goat Klebsiella pneumoniae in livestock production. RESULTS: In this study, we developed a Klebsiella pneumoniae goat polyclonal antibody and established an indirect ELISA method to detect the Klebsiella pneumoniae. After screening and optimizing the conditions for detection, we determined the optimal working dilutions of the coated-bacterial antigen, the polyclonal antibody, and the enzyme-labeled secondary antibody that were 1:800 (2.99 × 107 CFU/ml), 1:6400, and 1:5000, respectively. The optimal condition of coating and blocking were both 4 °C for 12 h. The optimal dilution buffers of bacterial antigen, the antibodies, and the blocking buffer were 0.05 mol/L carbonate buffer, 1% BSA phosphate buffer, and 1.5% BSA carbonate buffer, respectively. The cut-off value was determined to be 0.28, and the analytical sensitivity was 1:800 (dilution of a positive sample). Furthermore, there was no cross-reaction between the coated antigen and goat serum positive for antibodies against other bacteria, indicating that indirect ELISA could detect Klebsiella pneumoniae specifically in most cases. The average coefficients of variation of intra-assay and inter-assay were 4.37 and 5.17% indicating favorable reproducibility of indirect ELISA. In the detection of clinical veterinary samples, the positive rate of indirect ELISA was 6.74%, higher than that of conventional agglutination assays. CONCLUSIONS: Taken together, we successfully established an indirect ELISA method for detecting antibodies against Klebsiella pneumoniae in goats, which can be applied in production.

High Prevalence and Diversity Characteristics of blaNDM, mcr, and blaESBLs Harboring Multidrug-Resistant Escherichia coli From Chicken, Pig, and Cattle in China.

The objective of this study was to understand the diversity characteristics of ESBL-producing Escherichia coli (ESBL-EC) in chicken, pig, and cattle. A high prevalence of ESBL-EC (260/344) was observed in all food animals with prevalence rates of 78.6% (110/140) for chicken, 70.7% (58/82) for cattle, and 75.4% (92/122) for swine. However, the resistance rates presented significant differences in different animal origin ESBL-EC, where resistance to CTX, GEN, IMP, NEO, and OFL was the highest in chicken ESBL-EC, then in cattle, and the lowest in swine. Seriously, most ESBL-EC harbor multidrug resistance to antibiotics (MDR, ≥3 antibiotic categories), and the MDR rates of ESBL-EC were the highest in chicken (98.18%), followed by swine (93.48%), and the lowest in cow (58.62%), while the same trend also was observed in MDR of ≥5 antibiotic categories. This high prevalence and resistance can be partly interpreted by the high carriage rates of the ß-lactamases CTX-M (n = 89), OXA (n = 59), SHV (n = 7), and TEM (n = 259). A significant difference of ß-lactamase genes also presented in different animal species isolates, where the chicken origin ESBL-EC possessed higher carriage rates of almost all genes tested than cattle and swine. Notably, eight chicken origin ESBL-EC carried transferable plasmid-mediated blaNDM-1 or blaNDM-5, especially, of which four ESBL-EC also contained the colistin resistance gene mcr-1, as confirmed by genomic analysis. More interestingly, two deletion events with a 500-bp deletion in ΔISAba125 and a 180-bp deletion in dsbC were observed in three blaNDM-5 IncX3 plasmids, which, as far as we know, is the first discovery. This showed the instability and horizontal transfer of blaNDM genetic context, suggesting that blaNDM is evolving to "pack light" to facilitate rapid and stable horizontal transfer. Sequence types (STs) and PFGE showed diversity patterns. The most prevalent STs were ST48 (n = 5), ST189 (n = 5), ST206 (n = 4), ST6396 (n = 3), ST10 (n = 3), and ST155 (n = 3), where ST48 ESBL-EC originated from three food animal species. The STs of all blaNDM-positive ESBL-EC were attributed to three STs, namely, ST6396 (n = 2), ST206 (n = 2), and ST189 (n = 4), where ST189 was also the unique type for four mcr-1-carrying ESBL-EC. In conclusion, we suggest that the three animal species ESBL-EC show similar high prevalence, diversity in isolate lineages, and significant discrepancies in antibiotic resistance and resistance genes. This suggests that monitoring and anti-infection of different food animal origin ESBL-EC need different designs, which deserves more attention and further surveillance.