Probiotics and vitamins modulate the cecal microbiota of laying hens submitted to induced molting.

Induced molting enables laying hens to relax, restore energy and prolong the laying hen cycle, resolving problems such as poor egg quality and minimizing economic losses caused by rising global feeding costs. However, traditional molting methods may disrupt gut microflora and promote potential pathogens infections. This study used a customized additive with a mixture of probiotics and vitamins to induce molting and examine the cecal microbiota post molting. A total of two hundred 377 day-of-ISA Brown laying hens were randomly assigned to four groups: non-molt with basal diet (C), 12-day feeding restriction (FR) in earlier-molting (B), feed again to 27.12% egg production in middle-molting (A) and reach second peak of egg production over 81.36% in post-molting (D). Sequencing 16S rRNA to analyze cecal microbial composition revealed that there is no significant change in bacterial community abundance post-molting. In contrast to group C, the number of potentially harmful bacteria such as E. coli and Enterococcus was not found to increase in groups B, A, or D. This additive keeps cecal microbiota diversity and community richness steady. In cecal contents, hens in group B had lower Lactobacillus, Lachnospiraceae and Prevotellaceae (vsC, A, and D), no significant differences were found between post-molting and the non-molting. Furthermore, cecal microbiota and other chemicals (antibodies, hormones, and enzymes, etc.) strongly affect immunological function and health. Most biochemical indicators are significantly positively correlated with Prevotellaceae, Ruminococcaceae and Subdoligranulum, while negatively with Phascolarctobacterium and Desulfovibrio. In conclusion, the additive of probiotics and vitamins improved the cecal microbiota composition, no increase in the associated pathogenic microbial community due to traditional molting methods, and enhances hepatic lipid metabolism and adaptive immunological function, supporting their application and induced molting technology in the poultry breeding industry.

Simultaneous Expression of Chicken Granulocyte Monocyte Colony-Stimulating Factor and the Hemagglutinin-Neuraminidase Epitope of the Virulent Newcastle Disease Virus Genotype VII C22 Strain in a Functional Synthetic Recombinant Adenovirus as a Genotype-Matched Vaccine with Potential Antiviral Activity.

When it comes to the prevention of clinical signs and mortality associated with infection of the Newcastle disease virus (NDV), vaccination has been very effective. However, recent evidence has proven that more highly virulent strains are emerging that bypass existing immune protection and pose a serious threat to the global poultry industry. Here, a novel rescued adenovirus 5-coexpressed chicken granulocyte monocyte colony-stimulating factor (ChGM-CSF) bio-adjuvant and C22-hemagglutinin-neuraminidase (HN) boosted chickens' immunological genetic resistance and thus improved the immunological effectiveness of the critical new-generation vaccine in vitro and in vivo. Accordingly, the hemagglutination inhibition (HI) titers (log2) of the recombinant adenovirus (rAdv)-ChGM-CSF-HN-immunized chickens had greater, more persistent, and longer-lasting NDV-specific antibodies than the La Sota and rAdv-HN-inoculated birds. Moreover, humoral and adaptive immunological conditions were shown to be in harmony after rAdv-ChGM-CSF-HN inoculation and uniformly enhanced the expression of alpha interferon (IFN-α), IFN-ß, IFN-γ, interleukin-1ß (IL-1ß), IL-2, IL-16, IL-18, and IL-22. Postchallenge, the control challenge (CC), wild-type adenovirus (wtAdv), and rAdv-ChGM-CSF groups developed unique NDV clinical manifestations, significant viral shedding, high tissue viral loads, gross and microscopic lesions, and 100% mortality within 7 days. The La Sota, rAdv-HN, and rAdv-ChGM-CSF-HN groups were healthy and had 100% survival rates. The rAdv-ChGM-CSF-HN group swiftly regulated and stopped viral shedding and had lower tissue viral loads than all groups at 5 days postchallenge (dpc). Thus, the antiviral activity of ChGM-CSF offered robust immune protection in the face of challenge and reduced viral replication convincingly. Our advance innovation concepts, combining ChGM-CSF with a field-circulating strain epitope, could lead to the development of a safe, genotype-matched, universal transgenic vaccine that could eradicate the disease globally, reducing poverty and food insecurity. IMPORTANCE We studied the biological characterization of the developed functional synthetic recombinant adenoviruses, which showed a high degree of safety, thermostability, and genetic stability for up to 20 passages. It was demonstrated through both in vitro and in vivo testing that the immunogenicity of the proposed vaccine, which uses the T2A peptide from the Thosea asigna virus capsid protein supported by glycine and serine, helps with efficiency to generate a multicistronic vector, enables expression of two functional proteins in rAdv-ChGM-CSF-HN, and is superior to that of comparable vaccines. Additionally, adenovirus can be used to produce vaccines matching the virulent field-circulating strain epitope. Because there is no preexisting human adenoviral immunity detected in animals, the potency of adenoviral vaccines looks promising. Also, it ensures that the living vector does not carry the resistance gene that codes for the kanamycin antibiotic. Accordingly, a human recombinant adenoviral vaccine that has undergone biological improvements is beneficial and important.

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.

A novel Pseudorabies virus vaccine developed using HDR-CRISPR/Cas9 induces strong humoral and cellular immune response in mice.

Outbreaks of Pseudorabies (PR) by numerous highly virulent and antigenic variant Pseudorabies virus (PRV) strains have been causing severe economic losses to the pig industry in China since 2011. However, current commercial vaccines are often unable to induce thorough protective immunity. In this study, a TK/gI/gE deleted recombinant PRV expressing GM-CSF was developed by using the HDR-CRISPR/Cas9 system. Here, a four-sgRNA along with the Cas9D10A targeting system was utilized for TK/gI/gE gene deletion and GM-CSF insertion. Our study showed that the four-sgRNA targeting system appeared to have higher knock-in efficiency for PRVs editing. The replication of the recombinant PRVs were slightly lower than that of the parental strain, but they appeared to have similar properties in terms of growth curves and plaque morphology. The mice vaccinated with the recombinant PRV expressing GM-CSF via intramuscular injection showed no obvious clinical symptoms, milder pathological lesions, and were completely protected against wild-type PRV challenge. When compared to the triple gene-deleted PRV, the gB antibodies and neutralizing antibody titers were improved and the immunized mice appeared to have lower viral load and higher mRNA levels of IL-2, IL-4, IL-6, and IFN-γ in spleens. Our study offers a novel approach for recombinant PRV construction, and the triple gene-deleted PRV expressing GM-CSF could serve as a promising vaccine candidate for PR control.

Comparison of Antimicrobial Resistance, Virulence Genes, Phylogroups, and Biofilm Formation of Escherichia coli Isolated From Intensive Farming and Free-Range Sheep.

Pathogenic E. coli are among the most frequently isolated bacterial pathogens on large-scale sheep farms in China. Antibiotic use in wool sheep production is a risk factor for promoting the emergence of resistant E. coli. To reveal the differences of E. coli populations in sheep from different farming systems the antimicrobial resistance, virulence genes, biofilm formation, and phylogroups of 500 E. coli isolates obtained between September 2019 and December 2020 in northwest China from diarrheic infections of intensive farming and free-range sheep were analyzed. The antimicrobial susceptibility test for 12 classes of antimicrobial agents was determined using the broth microdilution susceptibility method, and PCR was used to detect the differences in virulence genes and phylogroups. Additionally, biofilm formation was determined using microtiter plate and slide agglutination methods. Among the 500 E. coli isolates, the majority of the isolates were multidrug resistant (75.4%) and carried at least one virulence gene (94.8%). We observed that 412 (82.4%), 360 (72.0%), and 266 (53.2%) are found to be resistant to sulfisoxazole, florfenicol, and tetracyclines, respectively. Resistance was also observed to mequindox (46.8%), ampicillin (43.6%), spectinomycin (38.6%), enrofloxacin (34.2%), ceftiofur (21.0%), gentamycin (20.4%), ceftazidime (17.8%), and polymyxin B (7.8%) but no resistance was found to meropenem. These results showed that strains from free-range subjects had fewer antibiotic resistance strains rather than sheep that were intensively farmed (P < 0.05). We observed fifteen virulence genes, of which etrA (n = 401, 80.2%) is the most common. In addition, EAEC (86.4%) is dominant among free-range sheep and EHEC (80.1%) is dominant among intensive farming. Among all virulence genes, the strongest correlation was found between etrA and papC gene (P < 0.001, OR = 455.68). Similarly, the strongest correlation was also found between eltA and sulfisoxazole (P < 0.001, OR = 877). Furthermore, the majority of the E. coli isolates belonged to phylogroup B1 (50.6%), followed by phylogroup C (20.6%), A (7.4%), E (7.4%), D (5.8%), B2 (1.6%), and F (1%). Interestingly, phylogroup B2 and D were all distributed in intensive farms. In addition, 33 (6.6%), 373 (74.6%), and 94 (18.8%) showed moderate, weak, and no connection biofilm formation ability, respectively. These data uncovered that wool sheep serve as a reservoir of pathogenic E. coli harboring multiple resistance phenotypes and virulence genes. The overlapping virulence-associated traits between IPEC and ExPEC indicated the zoonotic potential and safety threats of sheep food products. It is urgent to improve the proper use of antimicrobials in China as well as other countries.

C1QBP inhibits proliferation of porcine circovirus type 2 by restricting nuclear import of the capsid protein.

Complement component 1 Q subcomponent-binding protein (C1QBP) has been shown to interact with the porcine circovirus type 2 (PCV2) Cap protein. Here, using yeast two-hybrid (Y2H) and co-immunoprecipitation assays, as well as laser confocal microscopy, the interaction between C1QBP and Cap was confirmed. Furthermore, overexpression of C1QBP in cells altered the intracellular location of Cap, which was observed using confocal microscopy and verified by detection of Cap in nuclear protein extracts in a Western blot assay. By inhibiting nuclear transport of Cap, overexpression of C1QBP downregulated PCV2 proliferation in PK-15 cells, as determined by quantitative polymerase chain reaction (qPCR). As C1QBP plays a similar role in a fusion of green fluorescent protein (GFP) with the Cap nuclear localisation signal (NLS) sequence, (CapNLS-GFP), we propose that the target site for C1QBP in Cap is possibly located in the NLS region. Considering all the results together, this study demonstrated that C1QBP interacts with the Cap NLS region, resulting in changes in the intracellular localisation of the Cap protein. We confirmed that overexpression of C1QBP inhibits the proliferation of PCV2, and this is possibly related to the function of C1QBP in controlling nuclear transport of Cap.

Newcastle Disease Virus V Protein Inhibits Cell Apoptosis and Promotes Viral Replication by Targeting CacyBP/SIP.

Newcastle disease virus (NDV) has been classified by the World Organization for Animal Health (OIE) as a notable disease-causing virus, and this virus has the ability to infect a wide range of birds. V protein is a non-structural protein of NDV. V protein has been reported to inhibit cell apoptosis (Park et al., 2003a) and promote viral replication (Huang et al., 2003), however, the mechanisms of action of V protein have not been elucidated. In the present study, a yeast two-hybrid screen was performed, and V protein was found to interact with the CacyBP/SIP protein. The results of co-immunoprecipitation and immuno-colocalization assays confirmed the interaction between V protein and CacyBP/SIP. The results of quantitative-PCR and viral plaque assays showed that overexpression of CacyBP/SIP inhibited viral replication in DF-1 cells. Overexpression of CacyBP/SIP in DF-1 cells induced caspase3-dependent apoptosis. The effect of knocking down CacyBP/SIP by siRNA was the opposite of that observed upon overexpression. Moreover, it is known that NDV induces cell apoptosis via multiple caspase-dependent pathways. Furthermore, V protein inhibited cell apoptosis and downregulated CacyBP/SIP expression in DF-1 cells. Taken together, the findings of the current study indicate that V protein interacts with CacyBP/SIP, thereby regulating cell apoptosis and viral replication.

Common microRNA⁻mRNA Interactions in Different Newcastle Disease Virus-Infected Chicken Embryonic Visceral Tissues.

To investigate the roles and explore the altered expression of microRNAs (miRNAs) and mRNAs in chicken embryos in response to Newcastle disease virus (NDV) infection, deep sequencing was performed. Then, a conjoint analysis of small RNA-seq and mRNA-seq was performed to screen interactional miRNA⁻mRNA pairs during NDV infection. In total, 15 and 17 up- and downregulated miRNAs were identified that potentially targeted 4279 and 6080 mRNAs in NDV-infected chicken embryonic tissues, respectively; in addition, 595 upregulated and 480 downregulated mRNAs were identified. The conjoint analysis of the obtained data identified 1069 miRNA⁻mRNA pairs. Among these pairs, 130 pairs were related to immune or inflammatory responses. The relationship between gga-miR-203a and its target transglutaminase 2 (TGM2) was confirmed using a dual-luciferase reporter system and a real time quantitative polymerase chain reaction (RT-qPCR) assay. Overall, the discovery of miRNAs, mRNAs, and their potential pairing relationships, which may be involved in the regulation of NDV infection, will facilitate our understanding of the complex regulatory relationship between the host and the virus.