Both recombinant Bacillus subtilis Expressing PCV2d Cap protein and PCV2d-VLPs can stimulate strong protective immune responses in mice.

Porcine circovirus type 2 (PCV2) is one of the most serious pathogens in pig herds worldwide. The Capsid protein (Cap), a structural protein of PCV2, is involved in the host's immune response; it induces neutralizing-antibody production and has good immunogenicity. The main PCV2 subtype currently prevalent in the Chinese pig herd is PCV2d. In this study, We constructed a recombinant Bacillus subtilis (B. subtilis) capable of secreting Cap protein, named pHT43-Cap/B. subtilis; we concentrated the supernatant of the recombinant bacteria and observed virus-like particles (VLPs) of PCV2d formed by Cap protein under transmission electron microscopy, named PCV2d-VLPs. The immunocompetence of the pHT43-Cap/B. subtilis and PCV2d-VLPs were then assessed by oral administration and by intramuscular injection into mice, respectively. The results showed that the levels of PCV2d-Cap protein-specific IgG in the serum and of PCV2d-Cap protein-specific sIgA in the small intestinal fluid of pHT43-Cap/B. subtilis immunized mice were elevated compared to the control group, both of them highly significant (p < 0.01), and the corresponding serum-specific IgG antibodies were effective in neutralizing PCV2d virulence. The virus load in the liver of the immunized mice was significantly lower than that in the control group (p < 0.01), as was the virus load in the spleen and lungs of the immunized mice (p < 0.05). In addition, the serum levels of PCV2d-Cap-specific IgG in mice immunized with PCV2d-VLPs by intramuscular injection were significantly elevated compared to the control group (p < 0.05), and the viral load in all tissues was significantly lower in immunized mice (p < 0.05). In conclusion, the recombinant bacterium pHT43-Cap/B. subtilis can induce effective mucosal and humoral immunity in mice, PCV2d-VLPs can induce humoral immunity in mice, and both vaccines have good immunogenicity; these results provide a theoretical and material basis for the development of a new vaccine against PCV2d.

Ecological risk under the dual threat of heavy metals and antibiotic resistant Escherichia coli in swine-farming wastewater in Shandong Province, China.

Mineral elements and antibiotic-resistant bacterial pollutants in livestock and poultry farms' wastewater are often sources of ecological and public health problems. To understand the heavy-metal pollution status and the characteristics of drug-resistant Escherichia coli (E. coli) in swine-farm wastewater in Shandong Province and to provide guidance for the rational use of mineral-element additives, common antibiotics, and quaternary ammonium compound disinfectants on swine farms, 10 mineral elements were measured and E. coli isolated from wastewater and its resistance to 29 commonly used antibiotics and resistance genes was determined. Finally, phylogenetic and multi-locus sequence typing (MLST) analyses was performed on E. coli. The results showed serious pollution from iron and zinc, with a comprehensive pollution index of 708.94 and 3.13, respectively. It is worth noting that average iron levels in 75% (12/16) of the districts exceed allowable limits. Multidrug-resistant E. coli were found in every city of the province. The E. coli isolated from swine-farm wastewater were mainly resistant to tetracyclines (95.3%), chloramphenicol (77.8%), and sulfonamides (62.2%), while antibiotic resistance genes for quinolones, tetracyclines, sulfonamides, aminoglycosides, and ß-lactams were all more than 60%. The clonal complex 10 (CC10) was prevalent, and ST10 and ST48 were dominant in E. coli isolates. Multidrug-resistant E. coli were widely distributed, with mainly A genotypes. However, the mechanism of the effect of iron on antibiotic resistance needs more study in this area. Thus, further strengthening the prevention and control of iron and zinc pollution and standardizing the use of antibiotics and mineral element additives in the swine industry are necessary.

Prevalence and antimicrobial susceptibility profiles of ESBL-producing Klebsiella Pneumoniae from broiler chicken farms in Shandong Province, China.

Klebsiella pneumoniae (K. pneumoniae) is a conditionally pathogenic bacterium present in the intestinal or the respiratory tract of animals, and it is a common factor in acquired infections and a major threat to public health. Increased production of extended-spectrum ß-lactamases (ESBLs) has become a serious issue in the treatment of K. pneumoniae infections. In this study, we examined the serotypes and antibiotic resistance profiles of K. pneumoniae isolated from broiler chickens on farms in Shandong Province, China. The K. pneumoniae isolation rate was 4.67% (33/707), and the serotype Capsular K54 (42.42%, 14/33) was the most prevalent serotype in broilers in Shandong. The antimicrobial susceptibility assay revealed that the 33 isolates were resistant to 28 antimicrobial drugs to varying degrees; among these, the highest resistance rate was observed for tetracyclines (90.91%), and the lowest rate of resistance was observed for moxifloxacin and fosfomycin (0%). The multidrug resistance (MDR) rate was 87.88% (29/33). The carrying rate of ß-lactam-resistance genes was as high as 100%, with blaSHV having the highest rate (93.94%). It is worth noting that one carbapenem-resistant K. pneumoniae (CRKP) isolate carrying blaNDM-1 and one colistin-resistant K. pneumoniae (COLR-KP) isolate carrying mcr-3 were found in broiler chickens. This study indicates that ESBL-producing CRKP isolates and COLR-KP isolates have emerged on poultry farms in Shandong and could be a potential threat to food safety and public health.

Research Note: Molecular characterization of antimicrobial resistance and virulence gene analysis of Enterococcus faecalis in poultry in Tai'an, China.

Enterococcus faecalis (E. faecalis) is a zoonotic pathogen that causes severe economic losses in the poultry-breeding industry. In our study, cecal samples from broilers with cecal enlargement at slaughterhouses in Tai'an, China, were analyzed. The results revealed that the 61 E. faecalis strains had drug resistance rates ranging from 96.72 to 8.20% against 11 antibiotics in 5 classes, of which erythromycin (96.72%) and tetracycline (96.72%) had the highest rates and vancomycin (8.20%) the lowest. The highest detection rate of multiple drug-resistant strains in 61 isolates was 72.13%. The results of polymerase chain reaction showed that, of the 12 virulence genes, ccf had the highest detection rate (80.33%), followed by asal and cob (both 78.69%), whereas hyl had the lowest (6.56%). Among 15 drug resistance genes, ermB had the highest detection rate (95.08%), followed by tetM (91.80%) and tetL (90.16%), whereas tetK (0.00%) and vanB (0.00%) remained undetected. Of the 34 sequence types found with multilocus sequence typing, the most predominant were ST631 (13.11%, 8/61) and ST634 (8.2%, 5/61). Our results provide a theoretical basis for guiding the rational use of antibiotics and preventing the spread of drug-resistant bacteria, along with epidemiological data for the risk analysis of food-borne bacteria and antimicrobial resistance in poultry farms in Shandong Province.

Multi-Locus Sequence Typing and Drug Resistance Analysis of Swine Origin Escherichia coli in Shandong of China and Its Potential Risk on Public Health.

The extensive use of antibiotics has caused antimicrobial resistance and multidrug resistance in Escherichia coli and gradual expands it into a worldwide problem. The resistant E. coli could be transmitted to humans through animal products, thereby creating a problem for bacterial treatment in humans and resulting in a public health issue. This study aims to investigate the molecular typing and drug resistance of swine and human origin E. coli within the same prefecture-level cities of Shandong Province and the potential risk of E. coli on public health. The drug sensitivity results indicated that tetracycline (TE) (97.17%) is a major antibiotic with high drug resistance in 106 swine origin E. coli. There was a significant difference in the drug-resistant genotypes between the two sources, of which the blaTEM positive rate was the highest in the genera of ß-lactams (99% in swines and 100% in humans). Among the 146 E. coli isolates, 98 (91.51% swine origin) and 31 (77.5% human origin) isolates were simultaneously resistant to three or more classes of antibiotics, respectively. The multi-locus sequence typing (MLST) results indicate that the 106 swine origin E. coli isolates are divided into 25 STs with ST1258, ST361, and ST10 being the dominant sequence analysis typing strains. There were 19 MLST genotypes in 40 strains of human E. coli from Tai'an, Shandong Province, with ST1193, ST73, ST648, ST131, ST10, and ST1668 being the dominant strains. Moreover, the cluster analysis showed that CCl0 and CC23 were the common clonal complexes (CCs) from the two sources. Our results provide a theoretical basis for guiding the rational use of antibiotics and preventing the spread of drug-resistant bacteria, and also provide epidemiological data for the risk analysis of foodborne bacteria and antimicrobial resistance in swine farms in Shandong Province.

Analysis of In Vivo Transcriptome of Intracellular Bacterial Pathogen Salmonella enterica serovar Typhmurium Isolated from Mouse Spleen.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important intracellular pathogen that poses a health threat to humans. This study tries to clarify the mechanism of Salmonella survival and reproduction in the host. In this study, high-throughput sequencing analysis was performed on RNA extracted from the strains isolated from infected mouse spleens and an S. Typhimurium reference strain (ATCC 14028) based on the BGISEQ-500 platform. A total of 1340 significant differentially expressed genes (DEGs) were screened. Functional annotation revealed DEGs associated with regulation, metabolism, transport and binding, pathogenesis, and motility. Through data mining and literature retrieval, 26 of the 58 upregulated DEGs (FPKM > 10) were not reported to be related to the adaptation to intracellular survival and were classified as candidate key genes (CKGs) for survival and proliferation in vivo. Our data contribute to our understanding of the mechanisms used by Salmonella to regulate virulence gene expression whilst replicating inside mammalian cells.

TruthTrust: Truth Inference-Based Trust Management Mechanism on a Crowdsourcing Platform.

On a crowdsourcing platform, in order to cheat for rewards or sabotage the crowdsourcing processes, spam workers may submit numerous erroneous answers to the tasks published by requesters. This type of behavior extremely reduces the completion rate of tasks and the enthusiasm of honest users, which may lead a crowdsourcing platform to a failure. Defending against malicious attacks is an important issue in crowdsourcing, which has been extensively addressed by existing methods, e.g., verification-based defense mechanisms, data analysis solutions, trust-based defense models, and workers' properties matching mechanisms. However, verification-based defense mechanisms will consume a lot of resources, and data analysis solutions cannot motivate workers to provide high-quality services. Trust-based defense models and workers' properties matching mechanisms cannot guarantee the authenticity of information when collusion requesters publish shadow tasks to help malicious workers get more participation opportunities. To defend such collusion attacks in crowdsourcing platforms, we propose a new defense model named TruthTrust. Firstly, we define a complete life cycle system that from users' interaction to workers' recommendation, and separately define the trust value of each worker and the credence of each requester. Secondly, in order to ensure the authenticity of the information, we establish a trust model based on the CRH framework. The calculated truth value and weight are used to define the global properties of workers and requesters. Moreover, we propose a reverse mechanism to improve the resistance under attacks. Finally, extensive experiments demonstrate that TruthTrust significantly outperforms the state-of-the-art approaches in terms of effective task completion rate.

Longitudinal monitoring of multidrug resistance in Escherichia coli on broiler chicken fattening farms in Shandong, China.

The extensive use of antibiotics has, in recent years, caused antimicrobial resistance and multidrug resistance in Escherichia coli to gradually develop into a worldwide problem. These resistant E. coli could be transmitted to humans through animal products and animal feces in the environment, thereby creating a problem for bacterial treatment for humans and animals and resulting in a public health issue. Monitoring the resistance of E. coli throughout the broiler fattening period is therefore of great significance for both the poultry industry and public health. In this longitudinal study, samples were taken from 6 conventional broiler fattening farms in Shandong Province, China, at 3 different times within 1 fattening period. The overall isolation rate of E. coli was 53.04% (375/707). Antibiotic resistance was very common in the E. coli isolated from these farms, and differed for different antibiotics, with ampicillin having the highest rate (92.86%) and cefoxitin the lowest (10.12%). Multidrug resistance was as high as 91.07%. More importantly, both the resistance rate of E. coli to the different drugs and the detection rate of drug resistance genes increased over time. The mobile colistin resistance (mcr-1) gene was detected in 24.40% of the strains, and these strains often carried other drug resistance genes, such as those conferring aminoglycoside, ß-lactamase, tetracycline, and sulfonamide resistance. Antimicrobial resistance and drug resistance genes in E. coli were least common in the early fattening stage. The individual detection rates of sul1, sul3, aacC4, aphA3, and mcr-1 were significantly lower (P < 0.05) for the early fattening stage than for the middle and late stages. The rational use of antibiotics, in conjunction with the improvement of the breeding environment during the entire broiler fattening cycle, will be helpful in the development of the poultry industry and the protection of public health.

Distribution characteristics and potential risks of heavy metals and antimicrobial resistant Escherichia coli in dairy farm wastewater in Tai'an, China.

Heavy metals and antimicrobial resistant bacteria in livestock and poultry environments can cause declines in production and significant economic losses, leading to potential environmental and public health issues. In this study, the heavy metal pollution status of livestock breeding water bodies in the Dawen river basin of Shandong Province in China was evaluated, and a total of 10 heavy metals were measured. In addition, antimicrobial susceptibility tests were conducted for Escherichia coli strains isolated from the water samples. The results showed that among all the metals, copper, zinc, and iron were detected at each sampling point, followed by nickel (detection rate of 95.74%), arsenic (detection rate of 89.36%), selenium (detection rate of 68.09%), lead (detection rate of 27.66%), and mercury (detection rate of 12.77%). Cadmium and hexavalent chromium were not detected. The contents of nine heavy metals were below the existing water standard values in China, whereas the iron pollution index in the water body in the study area was large and may pose a potential risk. A total of 17 E. coli isolates showed different resistance to ß-lactams, aminoglycosides, tetracyclines, quinolone antibiotics and chloramphenicol, but were mainly resistant to ß-lactams and tetracyclines. The detection rate of the tetA resistance gene was relatively high, indicating the overuse of cephalosporins and tetracyclines. The results of the present study might provide evidence of metal pollution and theoretical basis on the treatment of colibacillosis in the livestock industries.

Application of Bacillus subtilis as a live vaccine vector: A review.

Bacillus subtilis is widely used as a probiotic in various fields as it regulates intestinal flora, improves animal growth performance, enhances body immunity, has short fermentation cycle, and is economic. With the rapid development of DNA recombination technology, B. subtilis has been used as a potential vaccine expression vector for the treatment and prevention of various diseases caused by bacteria, viruses, and parasites as it can effectively trigger an immune response in the body. In this review, we refer to previous literature and provide a comprehensive analysis and overview of the feasibility of using B. subtilis as a vaccine expression vector, with an aim to provide a valuable reference for the establishment of efficient vaccines.

Prevalence of avian-origin mcr-1-positive Escherichia coli with a potential risk to humans in Tai'an, China.

Multidrug-resistant (MDR) Escherichia coli are responsible for difficult-to-treat infections. We sought to determine the prevalence and characteristics of MDR E. coli strains isolated from poultry and clinical patients in the same geographical region. Eighty-seven E. coli strains were isolated from poultry with perihepatitis lesions at different slaughterhouses, and 356 nonrepetitive E. coli strains were isolated from clinical patients. All samples were continuously collected from October to December 2017 in Tai'an, China. The presence of the mcr-1 gene in the strains was assessed by PCR. The genetic relationships of the polymyxin (POL)-resistant E. coli strains were analyzed by pulsed-field gel electrophoresis and multilocus sequence typing. The results indicate that the POL resistance rate for the E. coli isolates from poultry was 31.03% (27 of 87), whereas the human-origin E. coli isolates were 100% sensitive to POL. The mcr-1 gene and extended-spectrum ß-lactamase blaCTX-M-14 genes were identified in all 27 POL-resistant avian-origin E. coli isolates. Our pulsed-field gel electrophoresis analysis suggested that the 27 strains were represented by 14 pulsotypes, among which there were 3 strains each with A, E, I, and K pulsotypes, and 1 to 2 strains represented by the other 10 pulsotypes. Furthermore, multilocus sequence typing molecular typing identified 16 sequence types, including 4 ST156 strains, 3 ST533 strains, and 1 to 2 strains represented by the remaining 14 sequence types. In summary, the E. coli strains isolated in the Tai'an area all showed the MDR phenotype, the rate of which for poultry was higher than that for humans. No POL-resistant human-origin E. coli strains were identified in the clinical patients. Our study reveals that poultry-derived MDR mcr-1-positive E. coli strains may pose a potential risk to humans, and the surveillance findings presented herein will be conducive to our understanding of the prevalence and characteristics of mcr-1-positive E. coli strains in the Tai'an area.

Prevalence and characteristics of multidrug-resistant mcr-1-positive Escherichia coli isolates from broiler chickens in Tai'an, China.

Colibacillosis, caused by Escherichia coli, is one of the most common bacterial diseases of chickens. The high incidence and considerable economic losses associated with colibacillosis make it a significant concern worldwide. In recent years, the efficacy of colistin has been severely impacted by the emergence of plasmid-mediated colistin resistance genes, especially mcr-1. Therefore, monitoring of antibiotic resistance, particularly colistin resistance, amongst E. coli strains is vitally important to the future growth and sustainability of the poultry industry. In this study, a total of 130 E. coli strains were isolated from the livers of chickens displaying symptoms of colibacillosis in Tai'an, China. Isolates were screened for their susceptibility to various antibiotics and for the presence of mobile colistin resistance genes and other antibiotic resistance genes. Overall, 75 (57.7%) isolates showed resistance to colistin and were positive for mcr-1. The mobile colistin resistance genes, mcr-2, -3, and -4, were not detected in this study. Of the 75 mcr-1-positive isolates, all (100%) also carried tetracycline resistance genes, 71 (94.7%) also contained genes associated with ß-lactam resistance, 59 (78.7%) contained aminoglycoside resistance genes, and 57 (76%) contained sulfonamide resistance genes. This high prevalence of multidrug resistance among mcr-1-positive E. coli isolates, including the production of extended-spectrum ß-lactamases, is highly concerning. The surveillance findings presented here will be conducive to our understanding of the prevalence and characteristics of multidrug-resistance in E. coli in the Tai'an area and will provide a better scientific basis for the clinical treatment of colibacillosis in chickens.

Turbot (Scophthalmus maximus) hepcidin-1 and hepcidin-2 possess antimicrobial activity and promote resistance against bacterial and viral infection.

Hepcidin is an antimicrobial peptide and a regulator of iron homeostasis. In turbot (Scophthalmus maximus), two types of hepcidins have been identified, which share approximately 50% sequence identity. In this study, we examined the antimicrobial potentials of the two hepcidins in the form of synthesized peptides, SmHep1P and SmHep2P. We found that SmHep1P and SmHep2P exhibited apparent bactericidal activities against both Gram-positive and Gram-negative bacteria in a dose-dependent manner. The bactericidal effect of SmHep1P was stronger against Gram-positive bacteria, while the bactericidal effect of SmHep2P was stronger against Gram-negative bacteria. Fluorescence and electron microscopy showed that both peptides were able to bind to the target bacterial cells and alter the surface structure of the cells. In vitro studies showed that SmHep1P and SmHep2P reduced bacterial invasion into cultured fish cells. In vivo studies showed that turbot administered with SmHep1P and SmHep2P exhibited significantly enhanced resistance against bacterial and viral infection. In both in vivo and in vitro studies, the antimicrobial activities of SmHep2P were in most cases significantly stronger than those of SmHep1P. Together these results indicate that the two hepcidins of turbot most likely possess antimicrobial properties and play a role in the innate immune defense against bacterial and viral pathogens.

Immunological study of the outer membrane proteins of Vibrio harveyi: insights that link immunoprotectivity to interference with bacterial infection.

Vibrio harveyi is a bacterial pathogen that affects marine vertebrates and invertebrates. In this study, we identified 13 outer membrane proteins (OMPs) from a pathogenic V. harveyi strain and analyzed their immunological properties. In vivo immunogenicity analysis showed that antibodies specific to recombinant proteins of the 13 OMPs were detected in the antiserum of V. harveyi-infected rat. When used as subunit vaccines to immunize Japanese flounder (Paralichthys olivaceus), all OMPs were able to elicit specific serum antibody production in the vaccinated fish; however, only two OMPs (OMP173 and OMP214) induced high levels (>70%) of relative percent survival. Pre-incubation of V. harveyi with the antisera of protective OMPs significantly impaired bacterial infectivity against peripheral blood leukocytes (PBL), whereas the antisera of non-protective OMPs had no apparent effect on infection. OMP173 antibodies could bind whole V. harveyi cells and exhibit bactericidal effect in a complement-dependent manner. Passive immunization showed that fish received OMP173 antiserum before being infected with V. harveyi exhibited significantly reduced mortality rate and lower bacterial loads in liver, spleen, and kidney. Finally, treatment of FG cells with OMP173 prior to V. harveyi infection protected the cells from bacterial invasion to a significant extent. Take together, these results indicate that two of the examined OMPs induce protective immunity through production of specific antibodies that block bacterial invasion, and that one OMP is likely to be involved in host cell interaction during the infection process. Thus, the immunoprotectivity of the OMPs is probably associated with functional participations of the OMPs in bacterial infection.

Characterization of a c-type lysozyme of Scophthalmus maximus: expression, activity, and antibacterial effect.

Lysozyme is a key component of the innate immune system and plays an important role in antibacterial infection. In this study, we analyzed the expression and activity of a chicken-type (c-type) lysozyme (named SmLysC) from turbot (Scophthalmus maximus). SmLysC is composed of 143 residues and shares 67-90% overall sequence identities with the c-type lysozymes of a number of teleost fish. SmLysC possesses a typical c-type lysozyme domain, which contains the conserved residues E50 and D67 that form the putative catalytic site. SmLysC expression was detected, in increasing order, in head kidney, gill, heart, muscle, brain, spleen, blood, and liver. Bacterial infection caused significant inductions of SmLysC expression in head kidney, spleen, and liver in a time-dependent manner. Immunoblot analysis indicated that SmLysC has a subcellular localization in the extracellular milieu. Recombinant SmLysC (rSmLysC) was able to bind to bacterial cells and inhibit bacterial growth. Enzyme assay showed that the optimal temperature and pH of rSmLysC were 37 °C and pH 6.0 respectively. In contrast to rSmLysC, the mutant protein rSmLysCM1, which bears alanine substitutions at E50 and D67, displayed drastically reduced bacteriolytic activity. rSmLysC was able to inhibit the growth of several fish bacterial pathogens in a manner that depended on the dose of the protein; however, Gram-positive bacteria were in general more sensitive to rSmLysC than Gram-negative bacteria. Together these results indicate that SmLysC is a functional lysozyme that is likely to participate in innate immune defense against extracellular bacterial pathogens, in particular those of Gram-positive nature.

C312M: an attenuated Vibrio anguillarum strain that induces immunoprotection as an oral and immersion vaccine.

Vibrio anguillarum, a Gram-negative bacterial pathogen, is the causative agent of vibriosis that affects a wide range of aquatic animals. In this study, we obtained a mutant V. anguillarum, C312M, derived from the pathogenic V. anguillarum C312 by selection of rifampicin resistance. C312M was slower in growth than the wild type C312, particularly under conditions of iron depletion. Compared to C312, C312M was altered in protein production profile and exhibited a dramatically increased median lethal dose. Safety analysis showed that C312M was stable in virulence in the absence of selective pressure. To examine the potential of C312M as a live attenuated vaccine, Japanese flounder Paralichthys olivaceus were vaccinated with C312M via oral, immersion, and oral plus immersion routes. Microbiological analysis showed that C312M was recovered from the gut, liver, kidney, and spleen of the vaccinated fish in 1 to 14 d post-vaccination. When the fish were challenged with C312 at 1 mo post-vaccination, C312M-vaccinated fish exhibited relative percent survival rates of 60 to 84%. Comparable protection was observed when the fish were challenged with a heterologous V. anguillarum strain. Further analysis showed that C312M-vaccinated fish produced specific serum antibodies which enhanced serum bactericidal activity in a manner that is probably complement-dependent. These results indicate that C312M is highly attenuated in virulence but still retains residual infectivity, and that C312M is an effective vaccine when delivered alive via immersion and oral feeding.