Two mutations on S2 subunit were critical for Vero cell tropism expansion of infectious bronchitis virus HV80.

Infectious bronchitis virus (IBV) restricts cell tropism. Except for the Beaudette strain, other IBVs cannot infect mammalian cell lines. The limited cell tropism of other IBVs has hindered IBV vaccine development and research on the mechanisms of IBV infection. A novel Vero cell-adapted strain, HV80, has been previously reported. In this study, we constructed recombinants expressing the chimeric S glycoprotein, S1 or S2 subunit of strain H120 and demonstrated that mutations on S2 subunit are associated with the strain HV80 Vero cell adaptation. R687P or P687R substitution recombinants were constructed with the genome backbone of strains HV80 or H120. We found that the RRRR690/S motif at the S2' cleavage site is crucial to the Vero cell adaptation of strain HV80. Another six amino acid substitutions in the S2 subunit of the recombinants showed that the Q855H mutation induced syncytium formation. A transient transfection assay demonstrated the S glycoprotein with the PRRR690/S motif at the S2' cleavage site induced low-level cell-cell fusion, while H855Q substitution hindered cell-cell fusion and blocked cleavage event with S20 product. This study provides a basis for the construction of IBV recombinants capable of replicating in Vero cells, thus contributing to the advancement in the development of genetically engineered cell-based IBV vaccines.

A One-Pot Convenient RPA-CRISPR-Based Assay for Salmonella enterica Serovar Indiana Detection.

Salmonella enterica serovar Indiana (S. Indiana) is among the most prevalent serovars of Salmonella and is closely associated with foodborne diseases worldwide. In this study, we combined a recombinase polymerase amplification (RPA) technique with clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas) protein Cas12b (CRISPR/Cas12b)-based biosensing in a one-pot platform to develop a novel one-step identification method for S. Indiana infection diagnosis. The entire RPA-CRISPR/Cas12b reaction can be completed at 41 °C within 1 h without the need for specific instruments. The optimal concentrations of Cas12b and single-guide RNA (sgRNA) for the reaction were the same at 250 nM. The single-stranded DNA (ssDNA) reporter 8C-FQ (5'-/6-FAM/CCCCCCCC/BHQ1/-3') presented the best performance in the reaction compared with the other reporters. The limit of detection (LoD) of the RPA-CRISPR/Cas12b assay was 14.4 copies per reaction. As for specificity, we successfully identified four S. Indiana strains among twenty-two Salmonella strains without any false-positive results, presenting 100% accuracy for S. Indiana, and no cross-reactions were observed in eight other pathogens. Moreover, a total of 109 chicken carcasses were classified by the S. Indiana RPA-CRISPR assay and PCR methods from three processing points, including 43 post-shedding, 35 post-evisceration, and 31 post-chilling. There were 17 S. Indiana-positive samples identified during the whole processing step, consisting of nine post-shedding, five post-evisceration, and three post-chilling. The corresponding S. Indiana-positive rates of post-shedding, post-evisceration, and post-chilling were 20.93% (9/43), 14.29% (5/35), and 9.68% (3/31), respectively. Results from the S. Indiana one-step RPA-CRISPR/Cas12b assay were totally in agreement with those obtained using a traditional culture method, demonstrating 100% agreement with no false-positive or false-negative results observed. Altogether, the RPA-CRISPR/Cas12b assay developed in this study represents a promising, accurate, and simple diagnostic tool for S. Indiana detection.

Effects of Bacillus-based inoculum on odor emissions co-regulation, nutrient element transformations and microbial community tropological structures during chicken manure and sawdust composting.

This study aims to evaluate whether different doses of Bacillus-based inoculum inoculated in chicken manure and sawdust composting will provide distinct effects on the co-regulation of ammonia (NH3) and hydrogen sulfide (H2S), nutrient conversions and microbial topological structures. Results indicate that the Bacillus-based inoculum inhibits NH3 emissions mainly by regulating bacterial communities, while promotes H2S emissions by regulating both bacterial and fungal communities. The inoculum only has a little effect on total organic carbon (TOC) and inhibits total sulfur (TS) and total phosphorus (TP) accumulations. Low dose inoculation inhibits total potassium (TK) accumulation, while high dose inoculation promotes TK accumulation and the opposite is true for total nitrogen (TN). The inoculation slightly affects the bacterial compositions, significantly alters the fungal compositions and increases the microbial cooperation, thus influencing the compost substances transformations. The microbial communities promote ammonium nitrogen (NH4+-N), TN, available phosphorus (AP), total potassium (TK) and TS, but inhibit nitrate nitrogen (NO3--N), TP and TK. Additionally, the bacterial communities promote, while the fungal communities inhibit the nitrite nitrogen (NO2--N) production. The core bacterial and fungal genera regulate NH3 and H2S emissions through the secretions of metabolic enzymes and the promoting or inhibiting effects on NH3 and H2S emissions are always opposite. Hence, Bacillus-based inoculum cannot regulate the NH3 and H2S emissions simultaneously.

Genomic Characterization of a Plasmid-Free and Highly Drug-Resistant Salmonella enterica Serovar Indiana Isolate in China.

The emergence of multi-drug resistant (MDR) Salmonella enterica serovar Indiana (S. Indiana) strains in China is commonly associated with the presence of one or more resistance plasmids harboring integrons pivotal in acquiring antimicrobial resistance (AMR). This study aims to elucidate the genetic makeup of this plasmid-free, highly drug-resistant S. Indiana S1467 strain. Genomic sequencing was performed using Illumina HiSeq 2500 sequencer and PacBio RS II System. Prodigal software predicted putative protein-coding sequences while BLASTP analysis was conducted. The S1467 genome comprises a circular 4,998,300 bp chromosome with an average GC content of 51.81%, encompassing 4709 open reading frames (ORFs). Fifty-four AMR genes were identified, conferring resistance across 16 AMR categories, aligning closely with the strain's antibiotic susceptibility profile. Genomic island prediction unveiled an approximately 51 kb genomic island housing a unique YeeVU toxin-antitoxin system (TAS), a rarity in Salmonella species. This suggests that the AMR gene cluster on the S1467 genomic island may stem from the integration of plasmids originating from other Enterobacteriaceae. This study contributes not only to the understanding of the genomic characteristics of a plasmid-free, highly drug-resistant S. Indiana strain but also sheds light on the intricate mechanisms underlying antimicrobial resistance. The implications of our findings extend to the broader context of horizontal gene transfer between bacterial species, emphasizing the need for continued surveillance and research to address the evolving challenges posed by drug-resistant pathogens.

siRNA targeting Atp5a1 gene encoding ATPase α, the ligand of Peg fimbriae, reduced Salmonella Enteritidis adhesion.

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a zoonotic pathogen that can infect both humans and animals. Among the 13 types of fimbrial operons in S. Enteritidis, the highly conserved Peg fimbriae play a crucial role in the adhesion and invasion of S. Enteritidis into host cells but are not well studied. In this study, we identified the ATP synthase subunit alpha (ATPase α) as a ligand of Peg fimbriae using ligand blotting and mass spectrometry techniques. We confirmed the in vitro binding of ATPase α to the purified adhesion protein (PegD). Furthermore, we used siRNA to suppress the expression of ATPase α gene Atp5a1 in Leghorn male hepatoma (LMH) cells, which resulted in a significant reduction in the adhesion rate of S. Enteritidis to the cells (P < 0.05). The findings in this study provide insight into the mechanism of S. Enteritidis infection through Peg fimbriae and highlight the importance of ATPase α in the adhesion process.RESEARCH HIGHLIGHTS Ligand blotting was performed to screen the ligand of S. Enteritidis Peg fimbriae.Binding assay confirmed that ATPase α is the ligand of the Peg fimbriae.siRNA targeting ATPase α gene (Atp5a1) significantly reduced S. Enteritidis adhesion.

Independent and combined effects of antibiotic stress and EM microbial agent on the nitrogen and humus transformation and bacterial community successions during the chicken manure composting.

This study aimed to investigate the independent and combined effects of antibiotic and EM microbial agent on the nitrogen and humus (HS) transformations as well as the bacterial community successions during the chicken manure and rice husk composting. EM microbial agent accelerated the oxytetracycline (OTC) degradation, but slowed down the norfloxacin (NOR) degradation. OTC inhibited the TN retention and promoted the HS accumulation, both NOR and EM microbial agent inhibited the TN retention and HS accumulation, while EM microbial agent showed an antagonistic effect on TN immobilization with antibiotics and reduced the impacts of antibiotics on HS. Obvious bacterial community successions occurred. Firmicutes were related to HS transformation, while Firmicutes, Actinobacteriota and Proteobacteria were associated with nitrogen conversion. NOR promoted the transformations of NH4+-N to NO3--N and FA to HA. The findings provided theoretical data for the recycle of antibiotic-contaminated manure and the efficient production of high-quality compost.

Identification of genes involved in inbreeding depression of reproduction in Langshan chickens.

OBJECTIVE: Inbreeding depression of reproduction is a major concern in the conservation of native chicken genetic resources. Here, based on the successful development of strongly inbred (Sinb) and weakly inbred (Winb) Langshan chickens, we aimed to evaluate inbreeding effects on reproductive traits and identify candidate genes involved in inbreeding depression of reproduction in Langshan chickens. METHODS: A two-sample t-test was performed to estimate the differences in phenotypic values of reproductive traits between Sinb and Winb chicken groups. Three healthy chickens with reproductive trait values around the group mean values were selected from each of the groups. Differences in ovarian and hypothalamus transcriptomes between the two groups of chickens were analyzed by RNA sequencing (RNA-Seq). RESULTS: The Sinb chicken group showed an obvious inbreeding depression in reproduction, especially for traits of age at the first egg and egg number at 300 days (p<0.01). Furthermore, 68 and 618 differentially expressed genes (DEGs) were obtained in the hypothalamus and ovary between the two chicken groups, respectively. In the hypothalamus, DEGs were mainly enriched in the pathways related to vitamin metabolism, signal transduction and development of the reproductive system, such as the riboflavin metabolism, Wnt signaling pathway, extracellular matrix-receptor interaction and focal adhesion pathways, including stimulated by retinoic acid 6, serpin family F member 1, secreted frizzled related protein 2, Wnt family member 6, and frizzled class receptor 4 genes. In the ovary, DEGs were significantly enriched in pathways associated with basic metabolism, including amino acid metabolism, oxidative phosphorylation, and glycosaminoglycan degradation. A series of key DEGs involved in folate biosynthesis (gamma-glutamyl hydrolase, guanosine triphosphate cyclohydrolase 1), oocyte meiosis and ovarian function (cytoplasmic polyadenylation element binding protein 1, structural maintenance of chromosomes 1B, and speedy/RINGO cell cycle regulator family member A), spermatogenesis and male fertility (prostaglandin D2 synthase 21 kDa), Mov10 RISC complex RNA helicase like 1, and deuterosome assembly protein 1) were identified, and these may play important roles in inbreeding depression in reproduction. CONCLUSION: The results improve our understanding of the regulatory mechanisms underlying inbreeding depression in chicken reproduction and provide a theoretical basis for the conservation of species resources.

Serovar-Specific Polymerase Chain Reaction for Detection of Salmonella enterica Serovar Indiana.

Salmonella enterica serovar Indiana (S. Indiana) is a newly emerging pathogen with high levels of drug resistance. It has become one of the most common Salmonella serovars in China with a worldwide distribution, posing significant public health concerns. Detection of S. Indiana by traditional bacteriological methods is time-consuming and laborious, which prevents timely surveillance and effective control of the pathogen. In this study, comparative genomics was used to identify an A7P63_13850 gene that is uniquely present in S. Indiana, but not in other Salmonella serovars or any non-Salmonella bacteria. Then, a polymerase chain reaction (PCR) assay targeting this serovar-specific gene was established for specific detection of S. Indiana. The detection limit of this method is 10 pg per reaction for bacterial genomic DNA, being equivalent to 100 colony-forming units (CFU) per reaction. The established PCR amplifies all S. Indiana strains (n = 56), but none of other Salmonella serovars (n = 146) and non-Salmonella species (n = 14). The assay established in this study was also used to detect clinical samples from poultry, showed a positivity of 14.7% (23/156) for S. Indiana, which were verified by bacteriological methods. The highly sensitive and serovar-specific PCR for S. Indiana established in this study is suitable and convenient for detection of S. Indiana which aids in surveillance and control of the pathogen.

Establishment of a Multiplex Loop-Mediated Isothermal Amplification Method for Rapid Detection of Sulfonamide Resistance Genes (sul1, sul2, sul3) in Clinical Enterobacteriaceae Isolates from Poultry.

Antimicrobial resistance genes play an important role in mediating resistance to sulfonamide in Gram-negative bacteria. While PCR is the current method to detect sulfonamide resistance genes (sul1, sul2, sul3), it is time-consuming and costly and there is an urgent need to develop a more convenient, simpler and rapid test for the sul. In this study, we describe a multiplex loop-mediated isothermal amplification (m-LAMP) assay we developed for the rapid and simultaneous detection of three sul. This m-LAMP assay successfully detected seven reference strains with different sul genotypes, but was negative for nine sul-negative reference strains. The m-LAMP products were verified by HinfI restriction enzyme digestion and the detection limit of the test was 0.5 pg genomic DNA per reaction. Testing 307 sulfonamide-resistant Enterobacteriaceae clinical isolates with the m-LAMP revealed all were positive for the sul with sul2 (79.5%) and sul1 (64.5%) being most prevalent, and sul3 the least (12.1%). Of the Enterobacteriaceae isolates tested, the Salmonella Indiana, a newly emerging serovar resistant to numerous antimicrobials, were most commonly positive with 33% having sul3.

Loop-Mediated Isothermal Amplification of the sefA Gene for Rapid Detection of Salmonella Enteritidis and Salmonella Gallinarum in Chickens.

Salmonella spp. pose a threat to both human and animal health, with more than 2600 serovars having been reported to date. Salmonella serovars are usually identified by slide agglutination tests, which are labor intensive and time consuming. In an attempt to develop a more rapid screening method for the major poultry Salmonella serovars, we developed a loop-mediated isothermal amplification (LAMP) assay, which directly detected the sefA gene, a fimbrial operon gene existing in several specific serovars of Salmonella enterica including the major poultry serovars, namely Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) and Salmonella enterica serovar Gallinarum (Salmonella Gallinarum). With the 177 bacterial strains we tested, positive reactions were only observed with 85 strains of serovar Salmonella Enteritidis and Salmonella Gallinarum. The detection limit of the LAMP assay was 4 CFU/reaction with genomic DNAs of Salmonella Enteritidis (ATCC 13076) from pure culture and 400 CFU/ reaction with DNA extracted from spiked chicken feces. The LAMP assay was more sensitive than conventional culture, especially without enrichment, in detecting Salmonella Enteritidis (CMCC 50041) in the spiked fecal samples. The results show the sefA LAMP method is a rapid, sensitive, specific, and practical method for directly detection of Salmonella Enteritidis and Salmonella Gallinarum in chickens. The sefA LAMP assay can potentially serve as new on-site diagnostics in the poultry industry.

Highly Drug-Resistant Salmonella enterica Serovar Indiana Clinical Isolates Recovered from Broilers and Poultry Workers with Diarrhea in China.

Highly drug-resistant Salmonella enterica serovar Indiana became the most common serovar in broilers with diarrhea in China over the course of this study (15% in 2010 to 70% in 2014). While most S. Indiana isolates (87%, 384/440) were resistant to 13 to 16 of the 16 antibiotics tested, 89% of non-S. Indiana isolates (528/595) were resistant to 0 to 6 antibiotics. Class 1 integrons and IncHI2-type plasmids were detected in all S. Indiana isolates, but only in 39% and 1% of non-S. Indiana isolates.

Characterization of avian pathogenic Escherichia coli isolated in eastern China.

In order to investigate the biological characteristics of avian pathogenic Escherichia coli (APEC) isolated in eastern China, a total of 243 isolates were isolated from diseased poultry on different farms during the period from 2007 to 2014. These isolates were characterized for serogroups (polymerase chain reaction and agglutination), the presence of virulence-associated genes (fimC, iss, ompA, fyuA, stx2f, iroC, iucD, hlyE, tsh, cvaC, irp2, and papC) and class I integrons (polymerase chain reaction), drug susceptibilities (disk diffusion method) and the biofilm-forming abilities (semi-quantitative method). The results showed that the most predominant serogroups were O78 (87 isolates, 35.8%) and O2 (35 isolates, 14.4%). Gene profiling found that fimC and ompA were frequently distributed among the isolates and that 77.4% of the isolates were positive for class 1 integrons. Overall, isolates displayed resistance to tetracycline (97.5%), nalidixic acid (82.3%), ampicillin (81.1%), sulphafurazole (80.7%), streptomycin (79.0%), trimethoprim (78.2%) and cotrimoxazole (78.2%). Multiple-drug resistance was exhibited in 80.3% of the isolates, and the presence of class 1 integrons is associated with multidrug resistance. Finally, 151 isolates had the ability to form biofilms in vitro, and drug resistance seemed relative to biofilm-forming abilities.

Up-regulation of NLRC5 and NF-κB signaling pathway in carrier chickens challenged with Salmonella enterica Serovar Pullorum at different persistence periods.

The immune performance, SNPs and expression levels of candidate genes (IL1-ß, Nramp1, TLR4, MyD88, NF-κB and NLRC5) were analyzed in carrier chickens of a Chinese indigenous breed infected with Salmonella enterica Serovar Pullorum at different persistence periods (12, 19 and 24 weeks of age). Carrier birds at 19 weeks of age presented significant difference in most immune parameters, as compared to carriers at 12 and 24 weeks of age, while no significant difference in most immune parameters was observed between carriers at 12 and 24 weeks of age. The genotype distributions of IL1-ß and TLR4 presented significant differences between carriers and healthy birds. The expression levels of most candidate genes in carriers at 19 weeks of age were significantly higher than that in carriers at 12, 24 weeks of age and healthy birds and reached 1% level of significance between carriers at 19 weeks of age and healthy birds. The expression patterns of all genes, but IL-1fl and NLRC5 between carriers at 12 and 24 weeks of age in all tissues were similar. Compared with carriers at 12 weeks of age, IL1-ß was significantly down-regulated, but NLRC5 was significantly up-regulated in carriers at 24 weeks of age. Our study demonstrated that immune performance of carrier birds was severely impaired at age of sexual maturation and NLRC5 might play as a negative mediator of NF-κB pathway involved in immune response to asymptomatic infection by S. Pullorum. The TLR4/MyD88/NF-κB pathway might be suitable for study on S. Pullorum infection in Chinese indigenous breeds.

Influence of dietary taurine and housing density on oviduct function in laying hens.

Experiments were conducted to study the effects of dietary taurine and housing density on oviduct function in laying hens. Green-shell laying hens were randomly assigned to a free range group and two caged groups, one with low-density and the other with high-density housing. Each group was further divided into control (C) and taurine treatment (T) groups. All hens were fed the same basic diet except that the T groups' diet was supplemented with 0.1% taurine. The experiment lasted 15 d. Survival rates, laying rates, daily feed consumption, and daily weight gain were recorded. Histological changes, inflammatory mediator levels, and oxidation and anti-oxidation levels were determined. The results show that dietary taurine supplementation and reduced housing density significantly attenuated pathophysiological changes in the oviduct. Nuclear factor-κB (NF-κB) DNA binding activity increased significantly in the high-density housing group compared with the two other housing groups and was reduced by taurine supplementation. Tumor necrosis factor-α (TNF-α) mRNA expression in the high-density and low-density C and T groups increased significantly. In the free range and low-density groups, dietary taurine significantly reduced the expression of TNF-α mRNA. Supplementation with taurine decreased interferon-γ (IFN-γ) mRNA expression significantly in the low-density groups. Interleukin 4 (IL-4) mRNA expression was significantly higher in caged hens. IL-10 mRNA expression was higher in the high-density C group than in the free range and low-density C groups. Supplementation with taurine decreased IL-10 mRNA expression significantly in the high-density group and increased superoxide dismutase (SOD) activity in the free range hens. We conclude that taurine has important protective effects against oviduct damage. Reducing housing density also results in less oxidative stress, less inflammatory cell infiltration, and lower levels of inflammatory mediators in the oviduct. Therefore, both dietary taurine and reduced housing density can ameliorate oviduct injury, enhance oviduct health, and promote egg production in laying hens.