Effects of a novel Bacillus subtilis GXYX crude lipopeptide against Salmonella enterica serovar Typhimurium infection in mice.

The increased rate of antibiotic resistance strongly limits the resolution of Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. Therefore, new strategies to control bacterial infections are urgently needed. Bacillus subtilis (B. subtilis) and its metabolites are desirable antibacterial agents. Here, we aimed to evaluate the antibacterial activity of the novel B. subtilis strain GXYX (No: PRJNA940956) crude lipopeptide against S. Typhimurium. In vitro, GXYX crude lipopeptides affected S. Typhimurium biofilm formation and swimming and attenuated the adhesion and invasion abilities of S. Typhimurium toward BHK-21 cells; in addition, it inhibited the mRNA expression of the filA, filC, csgA, and csgB genes, which are related to the adhesion and invasion ability of S. Typhimurium. In vivo, pretreatment with GXYX crude lipopeptide via intragastric administration improved the survival rate by 30%, which was related to reductions in organ bacterial loads and clinical signs in mice. Intragastric administration of GXYX crude lipopeptide significantly downregulated the mRNA levels of TNF-α, IL-1ß, IL-12 and IL-6 in response to S. Typhimurium-induced inflammation compared with intraperitoneal injection. Moreover, it significantly improved the intestinal barrier-related gene (ZO-1, claudin-1, occludin-1) mRNA levels in intestinal tissue damaged by S. Typhimurium infection. In conclusion, GXYX crude lipopeptides were effective at reducing S. Typhimurium colonization, laying a foundation for the further development of novel antibacterial agents.

Porcine Epidemic Diarrhea Virus: Etiology, Epidemiology, Antigenicity, and Control Strategies in China.

Porcine epidemic diarrhea virus (PEDV) is a porcine enteric coronavirus, which is one of the main causative agents of porcine epidemic diarrhea (PED), with 100% morbidity and 80-100% mortality in neonatal piglets. Since 2010, large-scale PED caused by highly pathogenic variants of PEDV has occurred successively in China and other countries in the world, posing a great threat to the global pig industry. It has been demonstrated in many investigations that the classic attenuated vaccine strain, PEDV CV777, is insufficient to fully protect against the PEDV variants. Moreover, the maternally derived antibodies elicited by inactivated vaccines also cannot completely protect piglets from infection. In addition, feedback feeding poses a risk of periodic PEDV recurrence in pig farms, making it challenging to successfully limit the spread of PEDV in China. This review focuses on the etiology, epidemiology, antigenicity, and control strategies of PEDV in China and provides information for the formulation of effective control measures.

Characterization of Lung Microbiomes in Pneumonic Hu Sheep Using Culture Technique and 16S rRNA Gene Sequencing.

Hu sheep, a locally bred species in China known for its high productivity, is currently suffering from pneumonia. Here, we combine high-throughput 16SrRNA gene sequencing and bacterial culturing to examine the bacterial community in pneumonic Hu Sheep lungs (p < 0.05). The results showed that the abundance and diversity of lung bacteria in healthy sheep were significantly higher than those in pneumonia sheep (p = 0.139), while there was no significant difference between moderate and severe pneumonia. Furthermore, the composition of the lung microbiota community underwent significant alterations between different levels of pneumonia severity. The application of LEfSe analysis revealed a notable enrichment of Mannheimiae within the lungs of sheep afflicted with moderate pneumonia (p < 0.01), surpassing the levels observed in their healthy counterparts. Additionally, Fusobacterium emerged as the prevailing bacterial group within the lungs of sheep suffering from severe pneumonia. Integrating the results of bacterial isolation and identification, we conclusively determined that Mannheimia haemolytica was the primary pathogenic bacterium within the lungs of sheep afflicted with moderate pneumonia. Furthermore, the exacerbation of pneumonia may be attributed to the synergistic interplay between Fusobacterium spp. and other bacterial species. Our results provide new insights for guiding preventive and therapeutic measures for pneumonia of different severities in sheep.

Isolation and Identification of Aeromonas veronii in Sheep with Fatal Infection in China: A Case Report.

According to the findings of a sheep breeding farm in Shaanxi, China, 2.53% (15/594) of sheep exhibited respiratory (clinical) symptoms such as dyspnoea, nasal discharge, wet cough, fever, and progressive emaciation. Although multi-drug treatment strategies (including ampicillin, tylosin, florfenicol, and ceftiofur) have been attempted to improve clinical outcomes, they have only been met with limited success, with a mortality rate of 40%. Ultimately, Aeromonas veronii (A. veronii) was identified as the causative pathogen for respiratory disease. The rates of symptomatic and asymptomatic sheep positive to A. veronii were 64.28% (95% CI 52.25-76.31%) and 8.02% (95% CI 6.96-9.08%), respectively. Pathogenicity tests demonstrated that the A. veronii is pathogenic to sheep and mice. The results of the antibiotic susceptibility tests revealed that the strain was sensitive to cefotaxime, gentamicin, and enrofloxacin and resistant to ampicillin, ceftiofur, amoxicillin, kanamycin, neomycin, streptomycin, tetracycline, florfenicol, and tylosin. We suggest that the combination of cefotaxime and gentamicin is an effective treatment based on the results of an antimicrobial susceptibility test, which exhibited good therapeutic efficacy. To the best of our knowledge, this is the first report in which pathogenic A. veronii has been documented as the cause of death in sheep in China. We concluded that pathogenic A. veronii poses a potential risk to the industry of sheep husbandry. This study's findings can help guide prevention and treatment plans for A. veronii infection in sheep.

ESBLs-producing Escherichia coli from sheep-origin: Genome-wide virulence genes identification and in vivo virulence assessment in mice and Galleria mellonella.

The worldwide spread of pathogenic Escherichia coli, together with the multidrug resistant linked with extended-spectrum ß-lactamases (blaCTX-M , blaTEM and blaOXA ), not only affect the health of animals and humans but also bring huge economic losses to animal husbandry. Despite the high levels of virulence present in many extended-spectrum beta-lactamases (ESBLs)-producing E. coli isolates, however, few studies have comprehensively assessed the pathogenicity of ESBLs-producing E. coli isolates. Thus, the aim of the present study was to investigate the presence of virulence genes in third-generation cephalosporin-resistant E. coli and to assess their pathogenicity and zoonotic potential. Previously, we identified 67 ESBLs-producing E. coli strains from sheep anal swabs in northwest China. In this study, we genotypically and phenotypically characterized isolates of E. coli that produce ESBLs. According to the VirulenceFinder and virulence factors database, all ESBLs-producing E. coli strains harboured a wide range of virulence genes. The ColV plasmid-related genes (hlyF, ompT, iss, iutA and cvaC) were present in 52 (77.6%) ESBLs-producing E. coli isolates. Surprisingly, quite a number of extraintestinal pathogenic E. coli virulence-related genes were detected in 62 (92.5%) of 67 isolates. A total of 33 serotypes and 37 sequence types (STs) were found in 67 ESBLs-producing isolates. ST10 is the most prevalent ST, which is represented by five strains. The cluster analysis showed that CC10 and CC23 were the common clonal complexes (CCs). Predominant serotypes were O8 (10%) and O9 (9%) followed by 6% each of O89, O101 and O185. Most sheep-origin ESBLs-producing E. coli held the highly pathogenic to human and displayed moderate-to-vigorous-intensity motor capacity. The ESBLs-producing E. coli isolates with numerous virulence-related genes were able to cause multiple infectious diseases in animal models (mice, neonatal rats and Galleria mellonella). To our knowledge, this study represents an important first step for a comprehensive characterization of pathogenicity and zoonotic potential of sheep-origin ESBLs-producing E. coli isolates. These findings may be of significant value for the identification of pathogenicity and zoonotic potential risks associated with sheep-origin ESBLs-producing E. coli.

Characterization of Extended-Spectrum ß-Lactamase-Producing Escherichia coli Isolates That Cause Diarrhea in Sheep in Northwest China.

Development of extended-spectrum-ß-lactamase (ESBL)-producing Escherichia coli is one the greatest threats faced by mankind. Among animals, chickens, pigs, and cattle are reservoirs of these pathogens worldwide. Nevertheless, there is a knowledge gap on ESBL-producing E. coli from small ruminants (i.e., sheep and goats) in China. The aim of this study was to identify and characterize the resistance profiles, resistomes, and sequence features of 67 ESBL-producing E. coli isolates from sheep in northwest China. The findings showed that blaCTX-M and blaTEM were the most prevalent. Interestingly, we found that the resistance gene mcr-1 was widespread in sheep merely from Shaanxi areas, accounting for 19.2% (5/26). The highly prevalent serotypes and FumC-FimH (CH) typing isolates were O8 and C4H32, respectively. High-risk E. coli clones, such as sequence type 10 (ST10), ST23, ST44, and ST58, were also found in China's sheep population. A total of 67 ESBL-producing isolates were divided into five phylogenetic groups, namely, B1 (n = 47, 70.1%), B2 (n = 1, 1.5%), C (n = 14, 20.9%), E (n = 1, 1.5%), and F (n = 1, 1.5%), with the phylogenetic groups for 3 isolates (4.5%) remaining unknown. Moreover, ESBL-producing E. coli isolates were also characterized by the abundance and diversity of biocide/metal resistance genes and insert sequences. We found that in ESBL-producing E. coli isolates, there were two different types of isolates, those containing ESBL genes or not, which led to large discrepancies between resistance phenotypes and resistomes. In summary, our study provides a comprehensive overview of resistance profiles and genome sequence features in ESBL-producing E. coli and highlights the possible role of sheep as antibiotic resistance gene disseminators into humans. IMPORTANCE Antimicrobial resistance (AMR), especially the simultaneous resistance to several antibiotics (multidrug resistance [MDR]), is one of the greatest threats to global public health in the 21st century. Among animals, chickens, pigs, and cattle are reservoirs of these pathogens worldwide. Nevertheless, there is a knowledge gap on ESBL-producing E. coli from small ruminants in China. This study is the largest and most comprehensive analysis of ESBL-producing E. coli isolates from sheep, including antibiotic resistance profiles, phylogenetic groups, serotypes, multilocus sequence types (MLST), insert sequences (IS), antibiotic resistance genes, disinfectant resistance genes, and heavy metal resistance genes. We recommend extending the surveillance of AMR of sheep-origin E. coli to prevent future public health risks.

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.

Polysaccharides from Pinus massoniana pollen improve intestinal mucosal immunity in chickens.

Intestinal mucosa is the largest immune organ in animals, and its immune function is directly related to the resistance against various diseases. Taishan Pinus massoniana pollen polysaccharides (TPPPS) have been recognized as an effective vaccine adjuvant and potential immune enhancer against viral infections. However, little is known about their direct immune-enhancing activity on intestinal mucosa. In this study, we extracted the polysaccharides from Taishan masson pine pollen to investigate its promotive effect on intestinal mucosal immunity. A total of 120 1-day-old chickens were divided into 4 groups and inoculated with PBS or 3 different doses of TPPPS (10 mg/mL, 20 mg/mL, and 40 mg/mL), respectively. Feces, intestinal specimens, and serum samples were collected from the chickens at 7, 14, and 21 d after inoculation. The antibodies in serum, mucosal secretion of IgA, structure of intestinal villi, and expressions of cytokine genes and mucosal immune-related genes in the chickens were all significantly improved by TPPPS treatments. At 21 d after inoculation following the challenge of Newcastle disease virus, the chickens inoculated with 20 and 40 mg/mL TPPPS exhibited decreased weight loss and reduced intestinal pathologic damage and viral loads in the intestine. In summary, our results demonstrate that TPPPS can enhance mucosal immunity and promote intestinal villi development. This study has established the foundation for the development of novel immune-enhancing agent with immune-regulatory effects on intestinal mucosa.

Effects of Pinus massoniana pollen polysaccharides on intestinal microenvironment and colitis in mice.

The stability of the intestinal microenvironment is the basis for maintaining the normal physiological activities of the intestine. On the contrary, disordered dynamic processes lead to chronic inflammation and disease pathology. Pinus massoniana pollen polysaccharide (PPPS), isolated from Taishan Pinus massoniana pollen, has been reported with extensive biological activities, including immune regulation. However, the role of PPPS in the intestinal microenvironment and intestinal diseases is still unknown. In this work, we initiated our investigation by using 16S rRNA high-throughput sequencing technology to assess the effect of PPPS on gut microbiota in mice. The result showed that PPPS regulated the composition of gut microbiota in mice and increased the proportion of probiotics. Subsequently, we established immunosuppressive mice using cyclophosphamide (CTX) and found that PPPS regulated the immunosuppressive state of lymphocytes in Peyer's patches (PPs). Moreover, PPPS also regulated systemic immunity by acting on intestinal PPs. PPPS alleviated lipopolysaccharide (LPS) -induced Caco2 cell damage, indicating that PPPS has the ability to reduce the damage and effectively improve the barrier dysfunction in Caco2 cells. In addition, PPPS alleviated colonic injury and relieved colitis symptoms in dextran sodium sulfate (DSS)-induced colitis mice. Overall, our findings indicate that PPPS shows a practical regulatory effect in the intestinal microenvironment, which provides an essential theoretical basis for us to develop the potential application value of PPPS further.

Screening active fractions from Pinus massoniana pollen for inhibiting ALV-J replication and their structure activity relationship investigation.

The objective was to identify the active fractions of polysaccharide against replication of ALV-J and elucidate their structure activity relationship. The optimal extraction conditions were extracting temperature 90℃, pH 9 and the ratio of liquid to solid 30:1. Under these conditions, extraction yield of total polysaccharide was 6.5 % ± 0.19 %. Total polysaccharide was then purified by DEAE-52 cellulose and Sephadex G-200 gel. Three fractions, PPP-1, PPP-2, and PPP-3, were identified with molecular weight of 463.70, 99.41, and 26.97 kDa, respectively. Three polysaccharide fractions were all composed of 10 monosaccharides in different proportions. Compared with PPP-1, which was mainly composed of glucose, PPP-2 and PPP-3 contained a higher proportion of galactose, glucuronic acid and galacturonic acid. The Congo red assay indicated that the PPP-2 may have a triple helical structure, while PPP-1 and PPP-3 were absent. In vitro assay showed that there was no significant cytotoxicity among the polysaccharide fractions under the concentration of 800 µg mL-1 (P > 0.05). The antiviral test showed that PPP-2 had the strongest activity, indicating PPP-2 was the major antiviral component. The structure-activity relationship showed that the antiviral activities of polysaccharide fractions were affected by their monosaccharide composition, molecular weight, and triple helical structure, which was a result of a combination of multiple molecular structural factors. These results showed that the PPP-2 could be exploited as a valued product for replacing synthetic antiviral drugs, and provided support for future applications of polysaccharide from Pinus massoniana pollen as a useful source for antiviral agent.

Taishan Pinus massoniana pollen polysaccharide inhibits H9N2 subtype influenza virus infection both in vitro and in vivo.

The H9N2 subtype avian influenza virus (AIV) is one of the most prevalent AIV subtypes that can be found throughout most countries. Currently, due to the neglect of low pathogenic avian influenza virus (LPAIV) and monotonous control technique, an expanding H9N2 virus epizootic have been arisen and causes great economic losses in the poultry industry. Therefore, novel anti-influenza drugs are necessary for the prevention and control of H9N2 AIV. Our previous studies have found that Taishan Pinus massoniana pollen polysaccharides (TPPPS) have antiviral effects, but whether they can inhibit the H9N2 AIV remains unclear. Here, we further investigated the effects of TPPPS on the H9N2 virus and its underlying mechanisms of action. We found that TPPPS significantly inhibited the replication of the H9N2 virus in a dose-dependent manner, especially during the period of virus adsorption in vitro. Transmission electron microscopy demonstrated that TPPPS reduce infection by interfering with virus entry into host cells rather than by interacting with the H9N2 virus particles. A fluorescence quantitative PCR (qPCR) assay and an animal experiment were performed to evaluate the anti-viral effect of TPPPS in vivo. As expected, the lungs of chickens treated with TPPPS had fewer lesions and lower virus contents compared with the PBS group. In addition, pre-treatment with TPPPS clearly enhanced host disease resistance and delayed infection by the H9N2 virus. Taken together, our results reveal that TPPPS suppress H9N2 virus replication both in vitro and in vivo and therefore shows promising as an anti-AIV agent.

Immunization of mice against alpha, beta, and epsilon toxins of Clostridium perfringens using recombinant rCpa-b-x expressed by Bacillus subtilis.

The anaerobic pathogen Clostridium perfringens is the most potent cause of intestinal diseases, such as enterotoxemia, hemorrhagic enteritis, and lamb dysentery, in sheep. Three toxinotypes (B, C, and D) are usually the cause of these diseases and are mainly mediated via three important exotoxins: alpha toxin (CPA), beta toxin (CPB), and epsilon toxin (ETX). We have designed a chimeric protein, rCpa-b-x, that contains the C-terminal binding region of CPA, partial sequence of CPB, and ETX (Cpa247-370, Cpb108-305, and EtxH118P, respectively) according to the principle of structural vaccinology. The rCpa-b-x protein was then expressed by pHT43 plasmid in vivo using Bacillus subtilis as a delivery vector (Bs-pHT43-Cpa-b-x). The immunological activity of the rCpa-b-x protein was verified by western blot and its immunological efficacy was evaluated in a murine model. Oral administration with a recombinant agent caused local mucosal and systemic immune responses, and serum lgG and intestinal mucosal secretory IgA (sIgA) antibody titers were significantly increased. Levels of IL-2, IL-4, and IFN-γ were significantly higher in lymphocytes isolated from the Bs-pHT43-Cpa-b-x group compared with levels from the control groups. The percentages of CD4+ and CD8+ T lymphocytes in the Bs-pHT43-Cpa-b-x and inactivated vaccine (IV) groups were in the normal range. Mice of vaccine groups and control groups were challenged with 1x LD100 unit filtrate containing alpha, beta, and epsilon toxins. Mice in the Bs-pHT43-Cpa-b-x group were found to have lower rates of morbidity. The active immunization of mice with Bs-pHT43-Cpa-b-x still maintained 85% to 90% survival at the end of the 10-day observation period, whereas mice of control groups died within two to five days. The results of this study demonstrate the effectiveness of Bs-pHT43-Cpa-b-x in preventing C. perfringens infection in mice, and that Bs-pHT43-Cpa-b-x could be considered a potential vaccine against C. perfringens.

Recombinant Lactococcus Lactis Expressing M1-HA2 Fusion Protein Provides Protective Mucosal Immunity Against H9N2 Avian Influenza Virus in Chickens.

H9N2 subtype low pathogenicity avian influenza virus (LPAIV) is distributed worldwide and causes enormous economic losses in the poultry industry. Despite immunization of almost all chickens with inactivated vaccines, the disease still remains widespread. We speculated that improving mucosal or cellular immune responses could contribute to improved control of H9N2 viruses. In this study, we constructed a novel Lactococcus lactis (L. lactis) strain expressing a recombinant fusion protein consisting of the M1 and HA2 proteins derived from an antigenically conserved endemic H9N2 virus strain. The M1-HA2 fusion protein was cloned downstream of a gene encoding a secretory peptide, and we subsequently confirmed that the fusion protein was secreted from L. lactis by Western blotting. We assessed the immunogenicity and protective effects of this recombinant L. lactis strain. Eighty 1-day-old chickens were divided into four groups, and the experimental groups were orally vaccinated twice with the recombinant L. lactis strain. Fecal and intestinal samples, sera, and bronchoalveolar lavage fluid were collected at 7, 14, and 21 days post-vaccination (dpv). Chickens vaccinated with the recombinant L. lactis strain showed significantly increased levels of serum antibodies, T cell-mediated immune responses, and mucosal secretory IgA (SIgA). Following challenge with H9N2 virus at 21 dpv, chickens vaccinated with the recombinant L. lactis strain showed decreased weight loss, lower viral titers in the lung, and reduced lung pathological damage. In summary, our results demonstrated that a recombinant L. lactis strain expressing an H9N2 M1-HA2 fusion protein could induce protective mucosal and systemic immunity. This oral vaccine is H9N2 virus-specific and represents a significant design improvement compared with previous studies. Our study provides a theoretical basis for improving mucosal immune responses to prevent and control H9N2 virus infection.

Taishan Pinus Massoniana pollen polysaccharide inhibits the replication of acute tumorigenic ALV-J and its associated tumor growth.

Avian leukosis virus subgroup J (ALV-J) has resulted in considerable economic losses in the poultry industry. In recent years, fibrosarcoma induced by ALV-J, which contains the v-fps oncogene, has gained momentum, and this has brought about new challenges to the poultry industry. To study the inhibitory effects of Taishan Pinus Massoniana pollen polysaccharide (TPPPS) on acute ALV-J infection and tumor development, antiviral and antitumor models of the Fu-J (SDAU1005) strain of ALV-J were established in vitro and in vivo. The results of in vitro experiments showed that TPPPS significantly inhibited viral replication in a dose-dependent manner during adsorption and pretreatment stages. The results of in vivo experiments have shown that TPPPS significantly reduced the viral load in the plasma and tumor tissues, as well as inhibited tumor growth. We further examined the difference in transcriptome expression by using RNA-Seq technology. A total of 560 differentially expressed genes were identified that included 329 up-regulated genes and 231 down-regulated genes. The up-regulated genes were mainly immune-related genes, whereas the down-regulated genes were mainly tumor-regulated genes. Gene Ontology (GO) term enrichment included immune system processes, positive regulation of immune system processes, regulation of immune system processes, leukocyte activation, cell activation, and protein binding. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the main immune and tumor-related pathways included T-cell receptor signaling pathway, cytokine-cytokine receptor interactions, natural killer cell-mediated cytotoxicity, PI3K-Akt signaling pathway, JAK-STAT signaling pathway, NF-κB signaling pathway, and Ras signaling pathway. In summary, our results preliminarily point to the antiviral and antitumor mechanism of TPPPS in vivo and in vitro.