Thermostable vacuum foam dried Newcastle disease vaccine: Process optimization and pilot-scale study.

Vacuum foam drying (VFD) has been shown to improve the thermostability and long-term shelf life of Newcastle Disease Virus (NDV). This study optimized the VFD process to improve the shelf life of NDV at laboratory-scale and then tested the optimized conditions at pilot-scale. The optimal NDV to T5 formulation ratio was determined to be 1:1 or 3:2. Using the 1:1 virus to formulation ratio, the optimal filling volumes were determined to be 13-17% of the vial capacity. The optimized VFD process conditions were determined to be at a shelf temperature of 25℃ with a minimum overall drying time of 44 h. The vaccine samples prepared using these optimized conditions at laboratory-scale exhibited virus titer losses of ≤ 1.0 log10 with residual moisture content (RMC) below 3%. Furthermore, these samples were transported for 97 days around China at ambient temperature without significant titer loss, thus demonstrating the thermostability of the NDV-VFD vaccine. Pilot-scale testing of the NDV-VFD vaccine at optimized conditions showed promising results for up-scaling the process as the RMC was below 3%. However, the virus titer loss was slightly above 1.0 log10 (approximately 1.1 log10). Therefore, the NDV-VFD process requires further optimization at pilot scale to obtain a titer loss of ≤ 1.0 log10. Results from this study provide important guidance for possible industrialization of NDV-VFD vaccine in the future. KEY POINTS: • The process optimization and scale-up test of thermostable NDV vaccine prepared through VFD is reported for the first time in this study. • The live attenuated NDV-VFD vaccine maintained thermostability for 97 days during long distance transportation in summer without cold chain conditions. • The optimized NDV-VFD vaccine preparations evaluated at pilot-scale maintained acceptable levels of infectivity after preservation at 37℃ for 90 days, which demonstrated the feasibility of the vaccine for industrialization.

A pyrylium salt-based fluorescent probe for the highly sensitive detection of methylamine vapour.

The amine vapour produced by microorganisms is an important indicator of food spoilage. Amines are also ubiquitous in the chemical industry. The development of molecule-based ion sensors has been a pivotal issue that is currently receiving considerable attention. In this work, a new pyrylium salt-based fluorescent probe MTPY has been developed as a rapid, highly sensitive, and selective sensor for methylamine vapour. MTPY exhibits an obvious fluorescence response from yellow to cyan towards CH3NH2 vapour. The calibration curve of titration analysis shows a linear relationship of the fluorescence intensity at 514 nm versus the methylamine concentration in the range of 0.1-2 ppm. In addition to the linearity (R2 = 0.974) and short response time with a low detection limit (2.6 ppt, 8.4 × 10-8 M), the sensing mechanism was traced using mass spectrometry.

A benzophenoxazine-based NIR fluorescent probe for the detection of hydrogen sulfide and imaging in living cells.

Hydrogen sulfide (H2S) plays a vital role in regulating many important physiological functions. However, H2S-containing industrial wastewater is inevitably pumped into the environment which seriously contaminates water supplies and foodstuffs. So it is crucial to develop a single fluorescent probe for H2S detection with high sensitivity and selectivity. Herein, a colorimetric and turn-on near infrared (NIR) fluorescent probe NRDNP based on benzophenoxazine was designed and synthesized by thiolysis of 2,4-dinitrophenyl (DNP) ether as the specific reaction site strategy to achieve highly specific H2S detection in living systems. The studies demonstrated that the probe NRDNP exhibited excellent sensing performance toward H2S with an about 80-fold NIR fluorescence enhancement, a rapid response within 10 min, excellent sensitivity with a detection limit of 19 nM and good selectivity. Furthermore, the NRDNP is an optical sensor which visually changes in terms of the fluorescence colour/intensity upon sensing gaseous H2S molecules. NRDNP has low cytotoxicity and has been successfully applied in the fluorescence imaging of H2S in living cells, suggesting that it would be an effective tool for H2S detection in living systems.

Vacuum Foam Drying Method Improved the Thermal Stability and Long-Term Shelf Life of a Live Attenuated Newcastle Disease Virus Vaccine.

Vaccines used for managing Newcastle disease virus (NDV) rely heavily on cold chain, and this results in major constraints in their successful application, shipping, and storage. This study was undertaken to improve the thermotolerance properties of live attenuated NDV vaccines using vacuum foam drying (VFD) technology. The live attenuated NDV vaccine formulated in 15% trehalose, 2.5% gelatin, 0.05% pluronic, and 25 mmol/L potassium phosphate buffer (T5) and dried using VFD showed improved heat tolerance in comparison to the vaccine formulated in T5 as well, but dried using freeze-drying (FD) method. The T5-formulated VFD vaccine was stored at 37°C for 120 days, 45°C for 7 days, and 60°C for 3 days; the virus titer loss decreased by no more than 1.0 Log10. In contrast, the FD vaccine prepared in T5 could only be stored at 37°C for 7-10 days. Furthermore, the T5-formulated NDV-VFD vaccine remained infectious when heated at 100°C for 30 min. Shelf-life studies confirmed the improved thermal tolerance of the T5-formulated NDV-VFD vaccine since it could be stably stored at 2-8°C for 42 months and 25°C for 15 months. Moreover, immunization of 1-month-old specific pathogen-free (SPF) chickens with the T5-formulated NDV-VFD vaccine stored at 25 and 37°C could produce hemagglutination inhibition (HI) antibody levels comparable to those of commercial NDV-FD vaccines, which require strict adherence to the cold chain. In conclusion, not only did the VFD technology improve the thermostability and long-term shelf life of the vaccine, it also maintained its immunogenicity.

Role of long polar fimbriae type 1 and 2 in pathogenesis of mammary pathogenic Escherichia coli.

Escherichia coli is a leading cause of bovine mastitis worldwide. The bacteria can rapidly grow in milk and elicit a strong lipopolysaccharide (LPS)/toll-like receptor-4 (TLR4)-dependent inflammatory response. Recently, the long polar fimbriae (LPF) were identified as a promising virulence factor candidate widely distributed in mammary pathogenic E. coli (MPEC) strains. Mammary pathogenic E. coli possess 2 lpf loci encoding LPF1 and LPF2, respectively. By deleting the major fimbrial subunit gene, lpfA, we found that both LPF1 and LPF2 contribute to MPEC adhesion, invasion, and biofilm formation in vitro. The lpf1A and lpf2A mutants showed reduced cytotoxicity in our in vitro cell infection model. Furthermore, we observed that LPF2 induced a mild TLR4-independent proinflammatory response. The median lethal dose (LD50) of both ∆lpf2A and ∆lpf1A∆lpf2A mutants to BALB/c mice increased by 0.38 and 0.15 logs, respectively, whereas that of wild-type strain MPJS13 was 8.69 logs. In contrast, LPF1 deficiency significantly enhanced the LPS/TLR4-mediated inflammatory response in mammary epithelial cells, and the LD50 of the mutant decreased to 8.18 logs. In conclusion, our data suggested that LPF are important in MPEC colonization of mammary cells and may provide a benefit to bacterial intracellular survival that induces persistent bovine mastitis.

Engineering T7 bacteriophage as a potential DNA vaccine targeting delivery vector.

BACKGROUND: DNA delivery with bacteriophage by surface-displayed mammalian cell penetrating peptides has been reported. Although, various phages have been used to facilitate DNA transfer by surface displaying the protein transduction domain of human immunodeficiency virus type 1 Tat protein (Tat peptide), no similar study has been conducted using T7 phage. METHODS: In this study, we engineeredT7 phage as a DNA targeting delivery vector to facilitate cellular internalization. We constructed recombinant T7 phages that displayed Tat peptide on their surface and carried eukaryotic expression box (EEB) as a part of their genomes (T7-EEB-Tat). RESULTS: We demonstrated that T7 phage harboring foreign gene insertion had packaged into infective progeny phage particles. Moreover, when mammalian cells that were briefly exposed to T7-EEB-Tat, expressed a significant higher level of the marker gene with the control cells infected with the wide type phage without displaying Tat peptides. CONCLUSION: These data suggested that the potential of T7 phage as an effective delivery vector for DNA vaccine transfer.

Development of a novel oil-in-water emulsion and evaluation of its potential adjuvant function in a swine influenza vaccine in mice.

BACKGROUND: Vaccination is the principal strategy for prevention and control of diseases, and adjuvant use is an effective strategy to enhance vaccine efficacy. Traditional mineral oil-based adjuvants have been reported with post-immunization reactions. Developing new adjuvant formulations with improved potency and safety will be of great value. RESULTS: In the study reported herein, a novel oil-in-water (O/W) Emulsion Adjuvant containing Squalane (termed EAS) was developed, characterized and investigated for swine influenza virus immunization. The data show that EAS is a homogeneous nanoemulsion with small particle size (~ 105 nm), low viscosity (2.04 ± 0.24 cP at 20 °C), excellent stability (at least 24 months at 4 °C) and low toxicity. EAS-adjuvanted H3N2 swine influenza vaccine was administrated in mice subcutaneously to assess the adjuvant potency of EAS. The results demonstrated that in mice EAS-adjuvanted vaccine induced significantly higher titers of hemagglutination inhibition (HI) and IgG antibodies than water-in-oil (W/O) vaccines or antigen alone, respectively, at day 42 post vaccination (dpv) (P < 0.05). EAS-adjuvanted vaccine elicited significantly stronger IgG1 and IgG2a antibodies and higher concentrations of Th1 (IFN-γ and IL-2) cytokines compared to the W/O vaccine or antigen alone. Mice immunized with EAS-adjuvanted influenza vaccine conferred potent protection after homologous challenge. CONCLUSION: The O/W emulsion EAS developed in the present work induced potent humoral and cellular immune responses against inactivated swine influenza virus, conferred effective protection after homologous virus challenge and showed low toxicity in mice, indicating that EAS is as good as the commercial adjuvant MF59. The superiority of EAS to the conventional W/O formulation in adjuvant activity, safety and stability will make it a potential veterinary adjuvant.

Expression and functional role of Cdx2 in intestinal metaplasia of cystitis glandularis.

PURPOSE: Cdx2 is an essential transcription factor in intestinal epithelial cell differentiation and proliferation. However, to our knowledge the expression and role of Cdx2 in the development of intestinal cystitis glandularis, a metaplastic lesion induced by chronic inflammation, remained to be explored. MATERIALS AND METHODS: Real-time polymerase chain reaction was used to examine Cdx2, LI-cadherin and villin expression in typical and intestinal cystitis glandularis, and normal bladder tissue. Cdx2 cDNA was subcloned to the retroviral vector pLNCX2 for subsequent transfection into human bladder urothelium cells and rat bladder urothelium. Cdx2 mRNA and protein levels, and cell morphology and proliferation were assessed after transfection using real-time polymerase chain reaction, phase contrast microscopy, transmission electron microscopy and MTT assay, respectively. RESULTS: Higher mRNA levels of Cdx2, villin and LI-cadherin were detected in intestinal cystitis glandularis compared to normal bladder and typical cystitis glandularis. Only Cdx2 groups attained statistical significance (p <0.001). Retroviral over expression of Cdx2 resulted in increased mRNA and protein expression of Cdx2 as well as villin and LI-cadherin levels, and increased cell proliferation. A distinct change in cellular morphology, in which cells resembled intestinal-like cells, was also observed in vitro and in vivo. CONCLUSIONS: Cdx2 may have a critical role in regulating intestinal metaplasia in cystitis glandularis. Further studies are planned to assess the potential of using Cdx2 as a marker and therapeutic target for cystitis glandularis.

Interleukin-6 levels in female rats with protamine sulfate-induced chronic cystitis treated with hyaluronic acid.

OBJECTIVES: To measure interleukin-6 levels in a protamine sulfate-induced chronic cystitis rat model treated with hyaluronic acid, and to study the correlation among interleukin-6, bladder inflammatory degree and voiding frequency. METHODS: A chronic cystitis model was created in female rats by using long-term intermittent intravesical protamine sulfate (0.5 mL, 30 mg/mL). Then, hyaluronic acid (0.5 mL, 0.8 mg/mL) was also instilled intravesically in the rats. Interleukin-6 levels were analyzed with immunohistochemistry, real-time reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Hematoxylin-eosin staining was carried out to examine bladder inflammatory degree based on a four-point scoring system (from 0 - none to 3 - severe). Voiding patterns were investigated by cystometrography. RESULTS: According to cystometrography, protamine sulfate-induced rats had significantly shorter intercontraction intervals and less bladder capacity (P < 0.001). The bladder tissue of the rats showed severe chronic inflammation. Immunohistochemistry, reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay showed significantly higher expression of interleukin-6 (P < 0.001). After intravesical administration of hyaluronic acid, both intercontraction intervals and bladder capacity increased significantly (P < 0.001), whereas both bladder inflammatory degree and interleukin-6 levels decreased significantly (P < 0.001). Furthermore, there was a strong correlation between interleukin-6 levels and inflammatory degree (r = 0.727, P < 0.001), and also between interleukin-6 levels and voiding frequency (r = -0.761, P < 0.001). CONCLUSIONS: Intravesical administration of hyaluronic acid decreases interleukin-6 levels, as well as the severity of bladder inflammation and voiding frequency in a rat model of chronic cystitis. Interleukin-6 levels closely correlate with the inflammatory degree and voiding frequency. Thus, they can be regarded as an assessment measure of therapeutic impact.

Formulation and Evaluation of Lipidized Imiquimod as an Effective Adjuvant.

Adjuvants are essential for the induction of robust immune responses against vaccine antigens. Small-molecule TLR7 agonists hold high potential for this purpose. In this communication, imiquimod (IMQ) bearing a cholesterol lipid moiety derivative, IMQ-Chol, was designed and synthesized as a vaccine adjuvant, which could release parent IMQ molecules in aqueous conditions via amide bond hydrolysis. We performed a series of immunological evaluations by cooperating with the inactivated foot-and-mouth disease virus (FMDV). All of the results confirmed that IMQ-Chol could stimulate the body for a prolonged time to produce strong humoral and cellular immunity with a balanced Th1/Th2 immune response through a TLR7-related MAPK pathway. In addition, the results of the proof-of-concept vaccine indicated IMQ-Chol had a good effect on preventing and treating FMD in pigs.

Biodegradable Imiquimod-Loaded Mesoporous Organosilica as a Nanocarrier and Adjuvant for Enhanced and Prolonged Immunity against Foot-and-Mouth Disease Virus in Mice.

Foot-and-mouth disease (FMD), a serious, fast-spreading, and virulent disease, has led to huge economic losses to people all over the world. Vaccines are the most effective way to control FMD. However, the weak immunogenicity of inactivated FMD virus (FMDV) requires the addition of adjuvants to enhance the immune effectiveness of the vaccines. Herein, we formulated and fabricated biodegradable dendritic mesoporous tetrasulfide-doped organosilica nanoparticles SOMSN with imiquimod complex (SOMSN-IMQ) and used it as a platform for FMD vaccine delivery and as an adjuvant. SOMSN-IMQ demonstrated excellent stability for 6 months when stored in PBS, while it could be completely degraded within 42 days in SBF at room temperature. Biosafety experiments such as cell toxicity, hemolysis, and histology indicated that the as-prepared SOMSN-IMQ showed nontoxicity and good biocompatibility. Furthermore, SOMSN-IMQ exhibited a maximum adsorption capacity of 1000 µg/mg for inactivated FMDV antigens. Our results showed that SOMSN-IMQ can be effectively engulfed by RAW264.7 cells in a dose-dependent manner. After immunization, SOMSN-IMQ@FMDV can elicit persistent higher antibody levels, higher IgG2a/IgG1 ratio, and cytokine expression, which indicated that SOMSN-IMQ@FMDV triggered superior humoral and cellular immune responses. Moreover, SOMSN-IMQ could provoke maturation and activation of dendritic cells in lymph nodes (LDCs) as well as the proliferation of lymphocytes in vivo. Thus, SOMSN-IMQ could promote effective and potent immunity and provide a promising adjuvant platform for FMDV vaccination with acceptable safety.

A single dose of Astragalus saponins adjuvanted inactivated vaccine for pseudorabies virus protected mice against lethal challenge.

Pseudorabies (PR) is an important infectious disease of swine that causes enormous economic losses to the swine industry in China. Immunization with vaccines is a routine practice to control this disease. PRV inactivated vaccines usually require a booster vaccination to provide complete immune protection. Therefore, Astragalus saponins (AST) have been added as an immunopotentiator to improve the immune efficacy and reduce the immunization times for the PRV inactivated vaccine. The results in mice have shown that a single dose of AST-adjuvanted PRV inactivated vaccine promoted higher production of gB-specific IgG, IgG1, and IgG2a and neutralizing antibody, secretion of Th1-type (IFN-γ) and Th2-type (IL-4) cytokines, and lymphocyte proliferation than mice immunized without AST. Compared to mice immunized without AST, a single dose of the AST-adjuvanted PRV inactivated vaccine improved the survival percentage of mice and reduced the PRV viral loads in the lungs and brains after lethal challenge. In summary, AST was an effective immunopotentiator to improve the immune efficacy of a single dose PRV inactivated vaccine.

NADPH oxidase-derived reactive oxygen species production activates the ERK1/2 pathway in neutrophil extracellular traps formation by Streptococcus agalactiae isolated from clinical mastitis bovine.

Streptococcus agalactiae (S. agalactiae) continues to be challenging for milk quality in some countries and leads to huge economic losses. A large number of neutrophils are recruited into inflammatory foci when S. agalactiae infection occurs, and most studies have focused on the interaction between neutrophil extracellular traps (NETs) and this bacterium in the context of human pathogenicity. However, there is little information on the NET formation mechanism induced by S. agalactiae in the context of bovine mastitis. Here, neutrophils isolated from BALB/c mice were infected with S. agalactiae SAG-FX17, and NET formation was evaluated. SAG-FX17 could induce NADPH oxidase-derived reactive oxygen species (NOX-ROS)-dependent NET formation, and 21.8% of bacteria could be eliminated by NETs via NET DNA and associated proteins. SAG-FX17 could induce the phosphorylation of p38 MAPK, ERK1/2 MAPK, and JNK/SAPK in neutrophils. However, only ERK1/2 MAPK was shown to play an important role in SAG-FX17-induced NET formation. Importantly, NOX-ROS production occurs upstream of ERK1/2 MAPK activation and then induces NET release. ERK1/2 MAPK phosphorylation can, in turn, enhance NOX-ROS generation, which further contributes to NET release and bacterial elimination. This study provides evidence of the molecular mechanism underlying serotype Ia S. agalactiae SAG-FX17-induced NET formation and the interaction between bacteria and NETs, and these findings will increase our knowledge about bacterial mastitis in dairy cattle and contribute to the prevention and clinical treatment of bovine mastitis.

Evaluation of dendritic cell-targeting T7 phages as a vehicle to deliver avian influenza virus H5 DNA vaccine in SPF chickens.

Introduction: There is a growing demand for effective technologies for the delivery of antigen to antigen-presenting cells (APCs) and their immune-activation for the success of DNA vaccines. Therefore, dendritic cell (DC)-targeting T7 phages were used as a vehicle to deliver DNA vaccine. Methods: In this study, a eukaryotic expression plasmid pEGFP-C1-HA2-AS containing the HA2 gene derived from the avian H5N1 virus and an anchor sequence (AS) gene required for the T7 phage packaging process was developed. To verify the feasibility of phage delivery, the plasmid encapsulated in DC-targeting phage capsid through the recognition of AS was evaluated both in vitro and in vivo. The pEGFP-C1-HA2-AS plasmid could evade digestion by DNase I by becoming encapsulated into the phage particles and efficiently expressed the HA2 antigen in DCs with the benefit of DC-targeting phages. Results: For chickens immunized with the DC-targeting phage 74 delivered DNA vaccine, the levels of IgY and IgA antibodies, the concentration of IFN-γ and IL-12 cytokines in serum, the proliferation of lymphocytes, and the percentage of CD4+/CD8+ T lymphocytes isolated from peripheral blood were significantly higher than chickens which were immunized with DNA vaccine that was delivered by non-DC-targeting phage or placebo (p<0.05). Phage 74 delivered one-fiftieth the amount of pEGFP-C1-HA2-AS plasmid compared to Lipofectin, however, a comparable humoral and cellular immune response was achieved. Although, the HA2 DNA vaccine delivered by the DC-targeting phage induced enhanced immune responses, the protection rate of virus challenge was not evaluated. Conclusion: This study provides a strategy for development of a novel avian influenza DNA vaccine and demonstrates the potential of DC-targeting phage as a DNA vaccine delivery vehicle.

Construction of a T7 phage display nanobody library for bio-panning and identification of chicken dendritic cell-specific binding nanobodies.

Dendritic cells (DCs) are the antigen-presenting cells that initiate and direct adaptive immune responses, and thus are critically important in vaccine design. Although DC-targeting vaccines have attracted attention, relevant studies on chicken are rare. A high diversity T7 phage display nanobody library was constructed for bio-panning of intact chicken bone marrow DCs to find DC-specific binding nanobodies. After three rounds of screening, 46 unique sequence phage clones were identified from 125 randomly selected phage clones. Several DC-binding phage clones were selected using the specificity assay. Phage-54, -74, -16 and -121 bound not only with chicken DCs, but also with duck and goose DCs. In vitro, confocal microscopy observation demonstrated that phage-54 and phage-74 efficiently adsorbed onto DCs within 15 min compared to T7-wt. The pull-down assay, however, did not detect any of the previously reported proteins for chicken DCs that could have interacted with the nanobodies displayed on phage-54 and phage-74. Nonetheless, Specified pathogen-free chickens immunized with phage-54 and phage-74 displayed higher levels of anti-p10 antibody than the T7-wt, indicating enhanced antibody production by nanobody mediated-DC targeting. Therefore, this study identified two avian (chicken, duck and goose) DC-specific binding nanobodies, which may be used for the development of DC-targeting vaccines.

Dermal Microvascular Units in Domestic Pigs (Sus scrofa domestica): Role as Transdermal Passive Immune Channels.

The dermal microvascular unit (DMU) is a perivascular functional unit in the dermis. It is composed of microvascular and capillary lymphatics surrounded by immune cells. In this study, jet needle-free injection system was used to injected biocompatible carbon nanoparticles into the cervical skin of domestic pigs (Sus scrofa domestica) and assessed the morphological distribution of DMUs by hematoxylin erythrosine staining, immunohistochemistry (IHC), and transmission electron microscopy (TEM), and TEM was also used to observe the ultrastructural changes of DMUs after jet needle-free injection. Following our study, we identified DMUs in the dermis stratum papillare and similar structures in the dermis stratum reticulare, but the aggregation of CD68+ and CD1a+ cells in the dermis stratum papillare of DMUs by IHC confirmed that DMUs act as reservoirs of dermal immune cells, while similar structures in the dermis stratum reticulare should not be considered as DMUs. Ultrastructure of DMUs was revealed by TEM. Marvelous changes were found following xenobiotics attack, including the rearrangement of endothelial cells and pericytes, and the reactivity of immune cells. Novel interstitial cell telocyte (TC) was also identified around the microvasculature, which may have been previously known as the veil cell. Our results successfully identified the distribution of DMUs in the skin of domestic pigs, which might act as reservoirs of immune cells in the skin and play a role in immune surveillance and immune defense.

Effect of multiplicity of infection on the evasion of neutrophil killing by Streptococcus agalactiae isolated from clinical mastitis bovine.

Streptococcus agalactiae (S. agalactiae) causes intramammary infection in dairy cows. Increased neutrophils and a high bacterial load are important characteristics of bovine bacterial mastitis. We hypothesized that the multiplicity of infection (MOI) of S. agalactiae in bovine mastitis plays an important role in bacterial pathogenicity by modulating the neutrophil response to promote bacterial survival. Neutrophils from BALB/c mice were infected with the bovine mastitis isolate of S. agalactiae SAG-FX17 at various MOIs, and neutrophil responses were investigated. Infecting neutrophils with SAG-FX17 at an MOI of 1 induced reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) formation. Bacteria at an MOI of 10 suppressed neutrophil responses, including ROS bursts, NET formation, and cell necrosis, which are conducive to bacterial multiplication within 30 min postinfection. In addition, neutrophils are destroyed by SAG-FX17 at an MOI of 100 or greater. This study identified the MOIs related to the ROS and NET suppression caused by SAG-FX17, and the findings suggested that interventions to decrease bacterial loads before the MOI of 10 could be necessary and effective to harness the power of innate immune response to eliminate pathogens.

Optimization of Heat-Resistance Technology for a Duck Hepatitis Lyophilized Live Vaccine.

In this study, to improve the quality of a live attenuated vaccine for duck viral hepatitis (DHV), the lyophilization of a heat-resistant duck hepatitis virus vaccine was optimized. The optimized heat protectors were made of 10% sucrose, 1.2% pullulan, 0.5% PVP, and 1% arginine, etc., with a titer freeze-drying loss of ≤0.50 Lg. The vaccine product's valence measurements demonstrated the following: the vaccine could be stored at 2-8 °C for 18 months with a virus titer loss ≤0.91 Lg; at 37 °C for 10 days with a virus valence loss ≤0.89 Lg; and at 45 °C for 3 days with a virus titer loss ≤0.90 Lg. Regarding safety, no deaths occurred in two-day-old ducklings immunized with a 10 times dose vaccine; their energy, diet, and weight gain were all normal, demonstrating that the DHV heat-resistant vaccines were safe for ducklings and did not cause any immune side effects. Duck viral hepatitis freeze-dried vaccine began to produce antibodies at 7 d after immunization, reached above 5.0 on 14 d, and reached above 7.0 on 21 d, showing a continuous upward trend. This indicates that duck viral hepatitis vaccine has a good immunogen level. The optimization of the freeze-drying process saves costs and also improves the quality of the freeze-drying products, which provides important theoretical and technical support for the further study of vaccine products.

Biological Characterization and Evolution of Bacteriophage T7-△holin During the Serial Passage Process.

Bacteriophage T7 gene 17.5 coding for the only known holin is one of the components of its lysis system, but the holin activity in T7 is more complex than a single gene, and evidence points to the existence of additional T7 genes with holin activity. In this study, a T7 phage with a gene 17.5 deletion (T7-△holin) was rescued and its biological characteristics and effect on cell lysis were determined. Furthermore, the genomic evolution of mutant phage T7-△holin during serial passage was assessed by whole-genome sequencing analysis. It was observed that deletion of gene 17.5 from phage T7 delays lysis time and enlarges the phage burst size; however, this biological characteristic recovered to normal lysis levels during serial passage. Scanning electron microscopy showed that the two opposite ends of E. coli BL21 cells swell post-T7-△holin infection rather than drilling holes on cell membrane when compared with T7 wild-type infection. No visible progeny phage particle accumulation was observed inside the E. coli BL21 cells by transmission electron microscopy. Following serial passage of T7-△holin from the 1st to 20th generations, the mRNA levels of gene 3.5 and gene 19.5 were upregulated and several mutation sites were discovered, especially two missense mutations in gene 19.5, which indicate a potential contribution to the evolution of the T7-△holin. Although the burst size of T7-△holin increased, high titer cultivation of T7-△holin was not achieved by optimizing the culture process. Accordingly, these results suggest that gene 19.5 is a potential lysis-related component that needs to be studied further and that the T7-△holin strain with its gene 17.5 deletion is not adequate to establish the high-titer phage cultivation process.

Cellular evidence of autophagy in Sertoli cells during spermatogenesis in goats.

Sertoli cells (SCs) play their nursing role as structural and functional supporting cells during spermatogenesis to ensure the production of highly specialized mature spermatozoa. Besides that, the role of SCs in autophagy during active (adult) and inactive (young) spermatogenesis in the caprine testis is still largely unknown. In this study, we investigated autophagy in goat SCs by light microscopy, immunohistochemistry (IHC), double immunofluorescence (double-IF), and transmission electron microscopy. Light microscopy showed active seminiferous tubules with SCs and layers of developing germ cells in the adult goat testis. In young goats, layer of germ cells and SCs was viewed on the basal membrane in the seminiferous tubule. IHC of autophagy-related 7 (ATG7) showed moderate expressions in the cytoplasmic extensions of SCs during inactive spermatogenesis, and strong expression was observed during active spermatogenesis in the testis of goat. Co-immunolabeling of p62 or light chain 3 (LC3) with vimentin showed increasing expression from the basal to the luminal compartment of the seminiferous tubule and stronger expression during active than inactive spermatogenesis in the testis of goat. Ultrastructure assessment of the cytoplasm in SCs showed phagophores, generated from the endoplasmic reticulum during active spermatocytogenesis. Numerous autophagosomes and autolysosomes were noted in the SCs cytoplasm, which surrounds the spermatogenic cells in the basal compartment of the seminiferous tubules. At a later stage, SCs showed autophagosomes and autolysosomes, together with multivesicular bodies (MVB), during spermiogenesis at different phases of the acrosome formation. Numerous embedded elongated spermatozoa were found in the cytoplasm of SCs, surrounded by autophagic components and MVB. Under TEM, the mean diameter of autophagosomes was 952.35 nm and that of autolysosomes was 504.38 nm. Collectively, these results suggest that autophagy is active in SCs during caprine spermatogenesis and that the level of autophagy becomes more evident as spermatogenesis advances from the basal to the luminal compartment of SC.