Phage Tail Fiber Protein as a Specific Probe for Recognition of Shiga Toxin-Producing Escherichia coli O91, O103, and O111.

The ability to quickly identify specific serotypes of Shiga toxin-producing Escherichia coli (STEC) could facilitate the monitoring and control of STEC pathogens. In this study, we identified the receptors and receptor-binding proteins (RBPs) of three novel phages (pO91, pO103, and pO111) isolated from hospital wastewater. Recombinant versions of these RBPs (pO91-ORF43, pO103-ORF42, and pO111-ORF8) fused to a fluorescent reporter protein were then constructed. Both fluorescence microscopy and transmission electron microscopy showed that all three recombinant RBPs were bound to the bacterial surface. Indirect enzyme-linked immunosorbent assay was used to verify that each recombinant RBP bound specifically to E. coli O91, O103, or O111, but not to any of the 83 strains of E. coli with different O-antigens, nor to 10 other bacterial species that were tested. The recombinant RBPs adsorbed to their respective host bacteria within 10 min of incubation. The minimum concentration of bacteria required for detection by the recombinant RBPs was 33 colony-forming units (CFU)/mL (range: 3.3 × 10 to 3.3 × 108 CFU/mL). Furthermore, each recombinant RBP was also able to detect bacteria in lettuce, chicken breast meat, and infected mice, indicating that their usage will facilitate the detection of STEC and may help to reduce the spread of STEC-related infections and diseases.

Attenuated African swine fever virus through serial passaging of viruses in cell culture: a brief review on the knowledge gathered during 60 years of research.

African swine fever virus (ASFV) is a highly pathogenic double-stranded DNA virus. It affects various breeds of pigs, causing serious economic losses and health threats because of its rapid spread and high pathogenicity and infectivity. This situation is not helped by the lack of a validated vaccine or effective therapies. Since the 1960s, different strains of ASFV have been subjected to serial passage in a variety of cell lines. The attenuated ASFV strains obtained through serial passage are not only candidates for ASF vaccine research, but also are useful to study the molecular genetic characteristics and pathogenic mechanism of the virus. This review summarizes related studies on the attenuated strains of ASFV acquired through cell passage over the last 60 years, with the aim of providing inspiration for the rational design of vaccines in future.

Fungal aerosols in rabbit breeding environment: Metagenetic insight into PM2.5 based on third-generation sequencing technology.

Fungal aerosols are a vital environmental hazard factor impeding the development of the rabbit breeding industry and threatening public health. This study aimed to determine fungal abundance, diversity, composition, diffusion, and variability in aerosols in rabbit breeding environments. Twenty PM2.5 filter samples were collected from five sampling sites (i.e. En5, In, Ex5, Ex15, and Ex45) in a modern rabbit farm in Linyi City, China. Fungal component diversity at the species level was analyzed in all samples using third-generation sequencing technology. Results revealed that fungal diversity and community composition in PM2.5 significantly differed across different sampling sites, and different pollution levels. The highest concentrations of PM2.5 and fungal aerosols (i.e., 102.5 µg/m3 and 18.8 × 103 CFU/m3, respectively) were found at Ex5, and these concentrations were found to decrease as the distance from the exit increased. However, no significant correlation was observed between the internal transcribed spacer (ITS) gene abundance and overall PM2.5 levels, except for Aspergillus ruber and Alternaria eichhorniae. Although most fungi are not pathogenic to humans, zoonotic pathogenic microorganisms that cause pulmonary aspergillosis (e.g., Aspergillus ruber) and invasive fusariosis (e.g., Fusarium pseudensiforme) were observed. The relative abundance of A. ruber was higher at Ex5 than that at In, Ex15, and Ex45 (p < 0.01), and the relative abundance of the fungal species decreased with an increase in distance from the rabbit houses. Moreover, four potential novel strains of Aspergillus ruber were discovered, with 82.9%-90.3% of the nucleotide and amino acid sequences similar to those of reference strains. This study highlights the importance of rabbit environments as a source in shaping fungal aerosol microbial communities. To the best of our knowledge, this is the first study to uncover the initial characteristics of fungal biodiversity and dispersion of PM2.5 in rabbit breeding environments, contributing to infectious disease control and prevention in rabbits.

Recombinant cucurmosin-based immunotoxin targeting HER-2 with potent in vitro anti-cancer cytotoxicity.

Trastuzumab is a humanized monoclonal antibody against HER2 approved by FDA for breast and gastric cancer therapy. However, only a quarter of patients have the potential to benefit from it, and most of them develop resistance within therapy. The main purpose of this study is to broaden trastuzumab's therapeutic window by conjugating trastuzumab with recombinant cucurmosin to form an immunotoxin called T-CUS245C. T-CUS245C was chemically conjugated and the purification of T-CUS245C was evaluated by SDS-PAGE. SRB tests showed a remarkable cytotoxicity of T-CUS245C with IC50 values in picomolar range on HER2 positive cancer cells without significantly proliferation inhibition on HER2 negative cells (P < 0.01). Confocal microscopy verified the time-dependent internalization effects of T-CUS245C and revealed that the lethal efficacy can be increased by provoking the internalization. These results indicate the therapeutic potential of T-CUS245C for the HER-2 targeted therapy.

Downregulation of ß-catenin blocks fibrosis via Wnt2 signaling in human keloid fibroblasts.

Keloid is a disorder of fibroproliferative diseases that occurs in wounds, characterized by an exaggerated response to injury. The key factor responsible for the disease process has not been identified. This study sought to elucidate the role of ß-catenin in the regulation of keloid phenotypes and signaling. Expression of ß-catenin in keloid and normal non-keloid samples was measured by real-time polymerase chain reaction. Knockdown of ß-catenin was achieved by delivering small interfering RNA to target ß-catenin. Cell proliferation, cell cycle progression, and apoptosis of keloid cells were measured by functional assays in vitro. The proteins related to keloid fibrosis were measured by Western blotting. ß-catenin expression was significantly upregulated in keloid tissue samples compared with the normal non-keloid age-adjusted skin sample counterparts. Functionally, targeting ß-catenin with lipofection-delivered small interfering RNA oligonucleotide inhibited the proliferation and cell cycle arrest in G0/G1 phase and increased apoptosis of fibroblast cells, accompanied by downregulation of Wnt2 and cyclin D1 as well as the phosphorylation level of glycogen synthase kinase 3 beta in the keloid fibrosis. Our study supports a crucial role of ß-catenin in the regulation of fibroproliferation and extracellular matrix deposition. Targeting ß-catenin using small interfering RNA oligonucleotide may be a promising approach for preventing excessive fibroproliferative development after wound healing and may lead to the development of novel strategies for restoring keloid diseases.

Amino acid substitutions in the neuraminidase protein of an H9N2 avian influenza virus affect its airborne transmission in chickens.

Cases of H9N2 avian influenza virus (AIV) in poultry are increasing throughout many Eurasian countries, and co-infections with other pathogens have resulted in high morbidity and mortality in poultry. Few studies have investigated the genetic factors of virus airborne transmission which determine the scope of this epidemic. In this study, we used specific-pathogen-free chickens housed in isolators to investigate the airborne transmissibility of five recombinant H9N2 AIV rescued by reverse genetic technology. The results show that airborne transmission of A/Chicken/Shandong/01/2008 (SD01) virus was related to the neuraminidase (NA) gene, and four amino acid mutations (D368E, S370L, E313K and G381D) within the head region of the SD01 NA, reduced virus replication in the respiratory tract of chickens, reduced virus NA activity, and resulted in a loss of airborne transmission ability in chickens. Similarly, reverse mutations of these four amino acids in the NA protein of r01/NASS virus, conferred an airborne transmission ability to the recombinant virus. We conclude that these four NA residues may be significant genetic markers for evaluating potential disease outbreak of H9N2 AIV, and propose that immediate attention should be paid to the airborne transmission of this virus.

Asp141 and the Hydrogen-Bond Chain Asp141-Asn109-Asp33 Are Respectively Essential for GT80 Sialyltransferase Activity and Structural Stability.

Sialyltransferases are key enzymes involved in the biosynthesis of biologically and pathologically important sialic acid-containing molecules in nature. In this study, the activity of a putative sialyltransferase (Pm0160) harboring an inherent mutation D141Y in the conserved DDG motif, which has been identified in GT52 and GT80 families, was restored by reverse mutation. More interestingly, a hydrogen-bond chain was found to form between three conserved residues (Asp141, Asn109, and Asp33) of GT80 sialyltransferases based on recently determined crystal structures. Our mutagenesis experiments demonstrated that the hydrogen-bond chain connecting the general base Asp141 with Nß4, Nß1, and Nα1 plays an essential role in maintaining protein structural stability other than keeping the general base Asp141 in a productive orientation for sialic acid transfer.

Genome sequence of Shiga toxin-producing Escherichia coli jumbo bacteriophage vB_EcoM_JNE01.

Bacteriophage vB_EcoM_JNE01 was isolated from chicken farm sewage using Escherichia coli O157:H7 as the host bacteria. The total length of the vB_EcoM_JNE01 genome is 355,583 bp, with 584 open reading frames and 36% G+C content. It shares an 80% nucleotide identity with 59% query coverage with the bacteriophage PBECO4 (NC_027364).

Toxin gene detection and antibiotic resistance of Clostridium perfringens from aquatic sources.

Clostridium perfringens is a zoonotic opportunistic pathogen that produces toxins that can cause necrotic enteritis and even "sudden death disease". This bacterium is widely distributed in the intestines of livestock and human, but there are few reports of distribution in aquatic animals (Hafeez et al., 2022). In order to explore the isolation rate of C. perfringens and the toxin genes they carry, 141 aquatic samples, including clams (Ruditapes philippinarum), oysters (Ostreidae), and mud snails (Bullacta exerata Philippi), were collected from the coastal areas of Shandong Province, China. C. perfringens strains were tested for cpa, cpb, etx, iap, cpb2, cpe, netB, and tpeL genes. 45 clam samples were boiled at 100 °C for 5 min before bacteria isolation. 80 strains were isolated from 141 samples with the positive rate being 57 %.And the positive rates of cooked clams was 87 % which was higher than the average. In detection of 8 toxin genes, all strains tested cpa positive, 3 strains netB positive, and 2 cpb and cpe, respectively. 64 strains were selected to analyze the antibiotic resistance phenotype of 10 antibiotics. The average antibiotic resistance rates of the strains to tetracycline, clindamycin, and ampicillin were 45 %, 20 %, and 16 % respectively, and the MIC of 4 strains to clindamycin was ≥128 µg/mL. A high isolation rate of C. perfringens from aquatic animals was shown, and it was isolated from boiled clams for the first time, in which cpe and netB toxin genes were detected for the first time too. The toxin encoded by cpe gene can cause food poisoning of human, thus the discoveries of this study have certain guiding significance for food safety. Antibiotics resistant C. perfringens of aquatic origin may arise from transmission in the terrestrial environment or from antibiotic contamination of the aquaculture environment and is of public health significance.

Characterization, genome analysis, and therapeutic evaluation of a novel Salmonella phage vB_SalS_JNS02: a candidate bacteriophage for phage therapy.

Phage therapy is gaining momentum as an alternative to antibiotics in the treatment of salmonellosis caused by Salmonella. In this study, a novel Salmonella phage, vB_SalS_JNS02, was isolated successfully from poultry farms in Shandong, China. The biological characteristics of vB_SalS_JNS02 were analysed, which revealed a short latent period of approximately 10 min and a burst size of 110 PFU/cell. Moreover, vB_SalS_JNS02 exhibited remarkable stability across a wide pH range (pH 3-12) and temperatures ranging from 30 to 80°C. Genome sequencing analysis provided valuable insights into the genetic composition of vB_SalS_JNS02, which consists of a double-stranded DNA genome that spans 42,450 base pairs and has a G + C content of 49.4%. Of significant importance, the genomic sequence of vB_SalS_JNS02 did not contain any genes related to lysogenicity, virulence, or antibiotic resistance. The phage's efficacy was evaluated in a larval challenge study. Treatment with the phage resulted in increased survival of Galleria mellonella larvae (100, 70, and 85%) (MOI 0.1) in the prophylactic treatment, co-infection treatment, and remedial treatment experiments, respectively. Another in vivo experiment investigated the potential application of the phage in broiler chickens and revealed that a single oral dose of vB_SalS_JNS02 (108 PFU/mL, 100 µL/chick) administered 3 h after S. enteritidis oral administration provided effective protection. The introduction of bacteriophage not only enhances the production of secretory immunoglobulin A (sIgA), but also induces alterations in the composition of the gut microbial community. Phage therapy increases the relative abundance of beneficial bacteria, which helps to maintain intestinal barrier homeostasis. However, it is unable to fully restore the disrupted intestinal microbiome caused by S. enteritidis infection. Importantly, no significant adverse effects were observed in the animal subjects following oral administration of the phage, and our findings highlight vB_SalS_JNS02 is a hopeful candidate as a promising tool to target Salmonella infections in poultry.

Development of the CRISPR-Cas12a system for editing of Pseudomonas aeruginosa phages.

Pseudomonas aeruginosa is a common opportunistic pathogen. The potential efficacy of phage therapy has attracted the attention of researchers, but efficient gene-editing tools are lacking, limiting the study of their biological properties. Here, we designed a type V CRISPR-Cas12a system for the gene editing of P. aeruginosa phages. We first evaluated the active cutting function of the CRISPR-Cas12a system in vitro and discovered that it had a higher gene-cutting efficiency than the type II CRISPR-Cas9 system in three different P. aeruginosa phages. We also demonstrated the system's ability to precisely edit genes in Escherichia coli phages, Salmonella phages, and P. aeruginosa phages. Using the aforementioned strategies, non-essential P. aeruginosa phage genes can be efficiently deleted, resulting in a reduction of up to 5,215 bp (7.05%). Our study has provided a rapid, efficient, and time-saving tool that accelerates progress in phage engineering.

The alternate-form reliability study of six variants of the Brief Visual-Spatial Memory Test-Revised and the Hopkins Verbal Learning Test-Revised.

Introduction: The revised Hopkins Verbal Learning Test-Revised (HVLT-R) and the Brief Visual-Spatial Memory Test-Revised (BVMT-R) are two widely used test involving verbal and visual learning and memory. In the two tests, six different versions are assembled, respectively, to prevent learning effects. Currently, no researchers have compared the six versions of the two tests. Thus, their usefulness in clinical studies requiring multiple follow-ups is limited. In this work, we confirm the equivalence of six HVLT-R and BVMT-R versions. Methods: 20 people completed all six HVLT-R and BVMT-R versions, while 120 people were randomly assigned to complete one of the six versions of each test. The Intelligence Quotient (IQ) level is measured using the short version of the Wechsler Adult Intelligence test. R4.2.0 is used for statistical analysis. The K-Related sample test (a non-parametric test) is used to observe the differences in test scores among the 20 subjects. The one-way Analysis of Variance (ANOVA) test is utilized to analyze the differences in test scores among the 120 subjects. The scores on different versions are compared using two similar sample tests. The HVLT-R Total Learning, the HVLT-R Delayed Recall, the BVMT-R Total Learning, and the BVMT-R Delayed Recall are indexes for comparison. Version and test scores are used as research factors, while different versions are used as research levels. Results: The results suggest that HVLT-R and BVMT-R versions 3, 5 and 6 are equally difficult, and relatively easy compared to versions 1, 2 and 4. HVLT-R versions 3, 5, and 6 show good reliability and can be used interchangeably when testing word learning ability or short-term memory; BVMT-R Versions 3, 5, and 6 show acceptable reliability and can be can be used interchangeably. Discussion: In the study of multiple follow-ups, it is a must to avoid discrepant versions and choose other equivalent versions. The results from this study could be used as a guide for upcoming studies and clinical applications in China.

Effects of cognitive behavioral therapy on improving depressive symptoms and increasing adherence to antiretroviral medication in people with HIV.

The incidence of depression is higher in PLWH (people living with HIV) than in the general population. It is of clinical significance to explore effective measures to improve depression in patients. But the available evidence is still quite limited. CBT (cognitive behavioral therapy) is considered to be one of the effective methods to improve depression, medication adherence and quality of life in PLWH. Therefore, this study aimed to systematically evaluate the effect of cognitive behavioral therapy on improving depressive symptoms and increasing adherence to antiretroviral therapy (ART) in people living with HIV (Human Immunodeficiency Virus). The Cochrane Library, Embase, PubMed, and Web of Science databases were searched by computer to collect randomized controlled trials on the effects of cognitive behavioral therapy on improving depression and increasing ART medication adherence in PLWH, and the retrieval time was from the inception of each database to January 10, 2022. Meta-analysis was performed by two researchers using Stata 15.0 software after screening the literature, extracting data and evaluating quality according to inclusion and exclusion criteria. A total of 16 studies with 1,998 patients were included. Meta-analysis results showed that CBT improved depressive symptoms in PLWH (SMD = -0.09, 95% CI [-0.13 to -0.04], P < 0.001) with better long-term (<6 months) depression improvement (SMD = -0.09, 95% CI [-0.15 to -0.02], P = 0.006) than short-term (0-6 months); the difference in improved ART medication adherence in the CBT group compared to the control group was not statistically significant (SMD = 0.04, 95% CI [-0.06 to 0.13], P = 0.490). There may be publication bias due to incomplete inclusion of literature as only published literature was searched. Cognitive behavioral therapy is effective in improving depressive symptoms in people living with HIV, with better long-term (>6 months) results than short-term (0-6 months).

The dominant influencing factors of desertification changes in the source region of Yellow River: Climate change or human activity?

Due to the combined effects of global warming and human activities, the ecological environment of the Yellow River source area has undergone profound changes and desertification has become increasingly prominent. In this study, an optimal desertification monitoring index based on feature space was proposed for the Yellow River source area, and constructed using Landsat images. Then, the spatial and temporal variation of desertification in the Yellow River source area and its driving mechanism were studied using Geodetector. The main conclusions are as follows: (1) The newly proposed feature space-based desertification monitoring index has good applicability in the study area. The best inversion accuracy of the point-to-point Albedo-NDVI feature space model was 88.4%. (2) Desertification in the eastern and southern regions of the Yellow River source area has a tendency to increase, while the desertification situation in the central region is relatively stable. (3) From 1995 to 2015, there was a significant improvement in desertification in the study area, as evidenced by a decrease in desertification intensity. (4) As the intensity of human disturbance increases, the influence of natural factors on desertification gradually diminishes. The interaction of natural and anthropogenic factors has greater explanatory power for desertification than that of individual natural or anthropogenic factors. The research results can be used as a reference for decision-making on desertification control in the Three-River Source Region.

Concurrent chemoradiotherapy using gemcitabine and nedaplatin in recurrent or locally advanced head and neck squamous cell carcinoma.

BACKGROUND: Patients with recurrent or locally advanced head and neck squamous cell carcinoma (HNSCC) typically have limited treatment options and poor prognosis. AIM: To evaluate the efficacy and safety of two drugs with potent radio-sensitization properties including gemcitabine and nedaplatin as concurrent chemoradiotherapy regimens in treating HNSCC. METHODS: This single-arm prospective study enrolled patients with HNSCC to receive gemcitabine on days 1 and 8 and nedaplatin on days 1 to 3 for 21 days. Intensity-modulated radiation therapy with a conventional fraction was delivered 5 days per week. Objective response rate (ORR), disease control rate, and toxicity were observed as primary endpoints. Overall survival (OS) and progression free survival were recorded and analyzed as secondary endpoints. RESULTS: A total of 24 patients with HNSCC were enrolled. During the median 22.4-mo follow-up, both ORR and disease control rate were 100%. The one-year OS was 75%, and one-year progression-free survival (PFS) was 66.7% (median PFS was 15.1 mo). Recurrent HNSCC patients had a poorer prognosis than the treatment-naïve patients, and patients who achieved complete response had better survival than those in the PR group (all P < 0.05). The most common grade 1-4 (100%) or grade 3-4 toxicities (75%) were hematological, and the most common grade 3-4 non-hematological toxicity was mucositis in 17 (71%) patients. CONCLUSION: Gemcitabine plus nedaplatin with concurrent chemoradiotherapy is a therapeutic option for HNSCC with predictable tolerability. Considering the high adverse event rate, the optimized dose and schedule must be further explored.

Resilience Improves the Sleep Quality in Disabled Elders: The Role of Perceived Stress.

The current research aims to prove the impact of resilience on sleep quality and to investigate the mediating function of perceived stress in the paths from resilience to sleep quality among disabled Chinese elders. The participants were 196 elders with visual and physical disability who resided in the nursing houses in Shanghai, including 102 males and 94 females whose mean age was 74.5 years old (standard deviation = 6.81). All the elders were measured with the 10-item Connor-Davidson Resilience Scale, Pittsburgh Sleep Quality Index (PSQI), and Perceived Stress Scale. The results showed that all the demographic variables were significantly related to perceived stress and PSQI scores. High levels of resilience could significantly enhance the quality of sleep in disabled Chinese elders. In addition, the two-step mediation models also confirmed the impact of resilience on sleep quality as mediated through perceived stress in this special aged population. Better knowledge on the mechanisms of sleep quality among older adults could benefit the prevention and treatment of some geriatric diseases.

[Clinical observation on wheat grain moxibustion for chronic superficial gastritis of spleen-stomach deficiency and cold].

OBJECTIVE: To compare the clinical effect of wheat grain moxibustion and warming acupuncture on chronic superficial gastritis of spleen-stomach deficiency and cold. METHODS: A total of 200 patients with chronic superficial gastritis of spleen-stomach deficiency and cold were randomly divided into a wheat grain moxibustion group (100 cases, 13 cases dropped off ) and a warming acupuncture group (100 cases, 16 cases dropped off ). The acupoints of Zhongwan (CV 12), Tianshu (ST 25), Qihai (CV 6), Liangqiu (ST 34) and Zusanli (ST 36) were selected in both groups. The patients in the wheat grain moxibustion group were treated with wheat grain moxibustion, and 5 cone were given on every acupoint each time. Moxibustion was performed after acupuncture in the warming acupuncture group, and 3 cone each acupoint. Both groups were treated every other day for 15 times. The scores of clinical symptoms and signs, scores of physical component summary (PCS) and mental component summary (MCS) of quality of life in the two groups were recorded before and after treatment and during follow-up 1 month after treatment, and the clinical effects of the two groups were evaluated after treatment and during follow-up. RESULTS: After treatment and during follow-up, the scores of clinical symptoms and signs of the two groups were lower than before treatment (P<0.05), and the scores of PCS and MCS of quality of life were higher than before treatment (P<0.05). At follow-up, the score of clinical symptoms and signs in the wheat grain moxibustion group was lower than that in the warming acupuncture group (P<0.01), and the scores of PCS and MCS of quality of life were higher than those in the warming acupuncture group (P<0.001). The total effective rate of the wheat grain moxibustion group was 93.1% (81/87), which was higher than 82.1% (69/84) of the warming acupuncture group at follow-up (P<0.001). CONCLUSION: The wheat grain moxibustion can effectively improve the clinical symptoms, signs and quality of life in patients with chronic superficial gastritis of spleen-stomach deficiency and cold, and its long-term effect is better than warming acupuncture.

Novel EGFR­bispecific recombinant immunotoxin based on cucurmosin shows potent anti­tumor efficiency in vitro.

Epidermal growth factor receptor (EGFR) is overexpressed in various tumors and is associated with cancer initiation, progression, and poor prognosis. Despite the achievements made by tyrosine kinase inhibitors and monoclonal antibodies in certain cases, many patients have not benefited from such treatment due to resistance. Immunotoxins (ITs) are antibody­cytotoxin chimeric molecules with specific cell killing ability, which have achieved different degrees of success in the treatment of a wide range of cancers in clinical trials. The aim of the current study was to examine a novel targeting EGFR recombinant immunotoxin Bs/cucurmosin (CUS) generated by fusing CUS to the EGFR­specific nanobody 7D12­9G8. Bs/CUS was successfully expressed in Escherichia coli strain BL21 (DE3) in a soluble form. Furthermore, it retained binding capacity and specificity with EGFR and was superior to rE/CUS, a monospecific IT we reported previously. In vitro results showed that Bs/CUS could be internalized into the cytoplasm and selectively kill cells in the picomolar range. Flow cytometry showed that Bs/CUS killed the cells mediated by the apoptosis pathway. Taken together, results of the current study indicated that Bs/CUS is a promising candidate that should be further evaluated as a cancer therapeutic for the treatment of EGFR­positive tumors.

Characteristics of Brucella abortus vaccine strain A19 reveals its potential mechanism of attenuated virulence.

Brucella vaccination is one of the most important strategies for controlling brucellosis in livestock. The A19 strain was the effective vaccine used to control brucellosis in China. However, the characteristics of physiological and attenuated virulence of the A19 strain are not investigated in detail. In this study, we compared the phenotypic characteristics of the A19 to the wild-type strain S2308. Virulence test showed that the A19 was significantly attenuated at chronic infection stage in infected mouse model. In growth analysis, the A19 exhibited a quick growth at exponential phase and premature at stationary phase. The inflammatory response of macrophages infected by the A19 was detected using TaqMan qPCR assay, indicating that the inflammatory level of the A19-infected macrophages was higher than that of the S2308 infection. Cell death analysis showed that the A19 was not cytotoxic for macrophages. Cell infection showed that the A19 reduced its ability to invade, survive and traffic within host cells, and the intracellular A19 hardly excludes lysosome-associated marker LAMP-1, suggesting that the A19 can't escape the lysosome degradation within host cells. In further study, the sensitivity test exhibited that the A19 is more sensitive to stress and bactericidal factors than the S2308 strain, Western blot and silver staining analysis exhibited that the A19 has a different expression pattern of OMPs and reduces LPS O-antigen expression relative to the S2308 strain. Those data give us a more detailed understanding about the A19 vaccine strain, which will be beneficial for improvement of current Brucella vaccine and overcoming its defects.

Pathogenicity and Immune Responses of Aspergillus fumigatus Infection in Chickens.

Aspergillus fumigatus is a ubiquitous pathogen in poultry farms, causing aspergillosis in chickens. To study the pathogenicity of A. fumigatus, 14-days-old chickens were infected with fungal conidia (2 × 107 CFU/mL) via thoracic intra-air sacs inoculation. The clinical symptoms, gross and histopathological lesions, and fungal load in the lungs were examined. Additionally, the mRNAs of Toll like receptors (TLR) and pro-inflammatory cytokines were evaluated by quantitative PCR to explore the immune responses induced by A. fumigatus. The results showed that overt depression, ruffled feathers, and dyspnea were observed in the infected chickens as early as 3 days post infection (dpi). Eleven out of 25 infected chickens died from 5 to 9 dpi, and A. fumigatus could also be reisolated from the infected lung. Histopathological examination revealed obvious airsacculitis and pneumonia, characterized by inflammatory cell infiltration (heterophils and macrophages), and granulomatous lesions in the lung. The mRNA expressions of TLR1 and TLR2 were upregulated in the lung and spleen, and most pro-inflammatory cytokines including IL-1ß, Cxcl-8, TNF-α, IL-12, and IFN-γ were increased in both the lung and spleen during the tested period, suggesting that the innate immune responses were triggered by A. fumigatus infection, and these cytokines participated in the inflammatory responses against A. fumigatus. These results indicate that A. fumigatus infection by thoracic intra-air sacs inoculation can cause severe respiratory damage in chickens, activate TLR1 and TLR2 mediated immune responses, and elicit large expression of pro-inflammatory cytokines such as IL-1ß, Cxcl-8, and IFN-γ. These data will help further understanding of the pathogenesis and immune responses of A. fumigatus infection in the chicken.