Comparison of mouse models of microbial experience reveals differences in microbial diversity and response to vaccination.

Specific pathogen-free (SPF) laboratory mice dominate preclinical studies for immunology and vaccinology. Unfortunately, SPF mice often fail to accurately model human responses to vaccination and other immunological perturbations. Several groups have taken different approaches to introduce additional microbial experience to SPF mice to better model human immune experience. How these different models compare is unknown. Here, we directly compare three models: housing SPF mice in a microbe-rich barn-like environment (feralizing), adding wild-caught mice to the barn-like environment (fer-cohoused), or cohousing SPF mice with pet store mice in a barrier facility (pet-cohoused); the two latter representing different murine sources of microbial transmission. Pet-cohousing mice resulted in the greatest microbial exposure. Feralizing alone did not result in the transmission of any pathogens tested, while fer-cohousing resulted in the transmission of several picornaviruses. Murine astrovirus 2, the most common pathogen from pet store mice, was absent from the other two model systems. Previously, we had shown that pet-cohousing reduced the antibody response to vaccination compared with SPF mice. This was not recapitulated in either the feralized or fer-cohoused mice. These data indicate that not all dirty mouse models are equivalent in either microbial experience or immune responses to vaccination. These disparities suggest that more cross model comparisons are needed but also represent opportunities to uncover microbe combination-specific phenotypes and develop more refined experimental models. Given the breadth of microbes encountered by humans across the globe, multiple model systems may be needed to accurately recapitulate heterogenous human immune responses.IMPORTANCEAnimal models are an essential tool for evaluating clinical interventions. Unfortunately, they can often fail to accurately predict outcomes when translated into humans. This failure is due in part to a lack of natural infections experienced by most laboratory animals. To improve the mouse model, we and others have exposed laboratory mice to microbes they would experience in the wild. Although these models have been growing in popularity, these different models have not been specifically compared. Here, we directly compare how three different models of microbial experience impact the immune response to influenza vaccination. We find that these models are not the same and that the degree of microbial exposure affects the magnitude of the response to vaccination. These results provide an opportunity for the field to continue comparing and contrasting these systems to determine which models best recapitulate different aspects of the human condition.

The influence of Spirulina extract on pathogenicity, immune response, and vaccine efficacy against H9N2 avian influenza virus in specific pathogen free chickens.

Avian influenza (AI) viruses pose a risk to the worldwide poultry industry. Ultimately, improving the efficiency of the H9N2 vaccine is necessary to better control low-pathogenic avian influenza-H9N2 by using natural immunostimulant. Therefore, the goal of the present study was to examine varying doses of the cyanobacterium Spirulina extract on the effectiveness of H9N2 vaccine. Thus, a total of 150 specific pathogen-free (SPF) chickens were allocated into 6 groups, 25 birds each, as follow: G1, G2, and G6 were supplemented with 200, 400, and 400 mg Spirulina extract/kg feed, respectively, whilst the feed in G3, G4, and G5 were not supplemented with Spirulina extract. At 21-days-old, only the chickens in G1, G2, and G3 were vaccinated with the H9N2 AI vaccine. After 4 wk postvaccination, the chickens in G1, G2, G3, G4, and G6 were challenged with H9N2 AI Egyptian strain. The challenged virus was selected from a recent circulating Egyptian strain during 2022, and it was related to A/quail/Hong Kong/G1/97-like virus lineage and clustered with G1-B sub-lineage EGY-2 group. It had a high amino acids identity percentage of 92.6% with the A/chicken/Iran/av1221/1998 (Boehringer Ingelheim) vaccine. The results of real-time reverse-transcriptase polymerase-chain-reaction (rRT-PCR) revealed that no shedding of the virus was reported in G1, G2, G3, and G5. The supplementation of Spirulina extract in low (200 mg/kg of feed) and high (400 mg/kg of feed) concentration with the birds vaccinated with H9N2 AI vaccine (G1 and G2) induced prominent immuno-stimulatory effect in a dose dependent manner where it strongly enhanced the phagocytic activities of broilers' peripheral blood monocytes, and lysozyme at all days postvaccination (dpv) and days postchallenge (dpc) compared to other groups with significant differences at all day of experiment and 21st dpv, 28th dpv, 7th dpc, and 14th dpc, respectively. The supplementation with Spirulina extract in G1 and G2 induced the highest hemagglutination inhibition antibody titer in a dose-dependent manner at all-time intervals. The antibody titer postvaccination was significantly increased in G1 and G2 at 14th, and 21st dpv, in comparison with G3. Furthermore, G1 and G2 showed higher significant antibody titers at 7th and 14th dpc, compared to other groups. Furthermore, Spirulina extract (200 and 400 mg/kg feed) in G1 and G2 showed anti-inflammatory effect in a dose dependant manner by downregulating nitric oxide levels at all times postchallenge with a significant difference at 3 to 7 dpc compared to G3, G4, and G6, with improved histopathological alterations in the trachea, lung, kidney, spleen, and bursa of Fabricius.  G6 supplied with 400 mg/kg Spirulina extract feed only without vaccination had a similar effect as vaccinated groups on innate immunity. However, it delayed the production of antibodies and did not prevent viral shedding as in vaccinated groups. In conclusion, vaccination in conjunction with either dose of Spirulina extract (G1, and G2) prevents viral shedding, increases the immune response, and reduces inflammation and histopathological change caused by H9N2 AI infection in a dose dependent manner. We recommend the use of 400 mg Spirulina extract/kg feed as a natural immunostimulant in conjunction with the H9N2 vaccine to achieve the highest possible level of protection against H9N2 AI infection.

Development and evaluation of a bivalent vaccine based on recombinant newcastle disease virus expressing infectious bursal disease virus VP2L-CH3-CH4 in SPF chickens.

Newcastle disease (ND) and infectious bursal disease (IBD) are two viral infectious diseases that are extremely damaging to the poultry industry and are widespread throughout the world. It is necessary to develop a safe and effective vaccine against IBD and ND because vaccination is an effective preventive measure. It has been discovered that recombinant proteins expressed by an expression system in which a fragment of mammalian Immunoglobulin G (IgG) Fragment crystallizable (Fc) is linked to a segment of a gene have antibody-like properties that increase the exogenous protein's serum half-life. Heavy chain constant region 3 and heavy chain constant region 4 (CH3-CH4) of Avian Immunoglobulin Y (IgY) is structurally very similar to mammalian Ig G Fc. In this study, a bivalent vaccine rClone30-VP2L-CH3-CH4-GMCSF was developed by using NDV rClone30-chGM-CSF vector to produce VP2L-CH3-CH4 fusion protein. The vaccine has been given to 14-day-old specific pathogen free (SPF) free chickens to test whether it has the potential to prevent IBD and ND. Anti-IBDV and anti-NDV antibody levels in serum were evaluated using ELISA and HI, respectively, and the contents of CD4+ T, CD8+ T, and B cells in leukocytes were determined via flow cytometry. The contents and mRNA transcription levels of four inflammatory factors, IL-1ß, IL-4, IFN-γ and chGM-CSF, were detected by ELISA and real-time PCR respectively. The results showed that after vaccination with the rClone30-VP2L-CH3-CH4-GMCSF vaccine, the levels of anti NDV and anti IBDV antibodies in chickens were significantly higher than those of the rClone30 vaccine and commercial vaccines. Meanwhile, the contents and transcription levels of inflammatory factors in chickens inoculated with rClone30-VP2L-CH3-CH4-GMCSF were significantly increased, and the proliferation response of B cells, CD4+ and CD8+ T cells was also stronger. However, the rClone30-VP2L-CH3-CH4-GMCSF vaccine had no significant advantage over the rClone30-VP2L-GMCSF vaccine in any of the above-mentioned features. In summary, rClone30-VP2L-CH3-CH4-GMCSF can stimulate the body to produce a stronger immune response, showing its potential to be considered as vaccine against IBD and ND, but the addition of CH3-CH4 did not improve the vaccine's immune effect as expected. The research lays the foundation for developing vaccines for other infectious viral diseases and avoids a unrealistic vaccine optimization method.

Study of coinfection with local strains of infectious bursal disease virus and infectious bronchitis virus in specific pathogen-free chickens.

Immunosuppressive diseases cause great losses in the poultry industry, increasing the susceptibility to infections by other pathogens and promoting a suboptimal response to vaccination. Among them, infectious bursal disease virus (IBDV) arises as one of the most important around the world. IBDV infects immature B lymphocytes, affecting the immune status of birds and facilitating infections by other pathogens such as avian infectious bronchitis virus (IBV). Although it has been reported that the interaction between these viruses increases IBV clinical signs, there are no actual studies about the interaction between regional circulating isolates that validate this statement. In this context, the objective of our work was to evaluate the effect of the interaction between local isolates of IBDV (belonging to genogroup 4) and IBV (lineage GI-16) in chickens. Thus, specific pathogen-free chickens were orally inoculated with IBDV genogroup (G) 4 or with PBS at 5 d of age. At 14-days postinoculation (dpi) the animals were intratracheally inoculated with a GI-16 IBV or with PBS. At multiple time points, groups of birds were euthanized and different parameters such as histological damage, viral load, lymphocyte populations and specific antibodies were evaluated. The success of IBDV infection was confirmed by the severity of bursal atrophy, viral detection, and presence of anti-IBDV antibodies. In IBV-infected animals, the presence of viral genome was detected in both kidney and bursa. The coinfected animals showed higher degree of lymphocyte infiltration in kidney, higher rate of animals with IBV viral genome in bursa at 28 dpi, and a clear decrease in antibody response against IBV at 28, 35, and 40 dpi. The results indicate that the infection with the local isolate of IBDV affects the immune status of the chickens, causing major severe damage, in response to IBV infection, which could consequently severely affect the local poultry industry.

Efficacy of a plant-produced infectious bronchitis virus-like particle vaccine in specific pathogen-free chickens.

Infectious bronchitis (IB) Gammacoronavirus causes a highly contagious respiratory disease in chickens that is listed by the World Organisation for Animal Health (WOAH). Its high mutation ability has resulted in numerous variants against which the commercially available live or recombinant vaccines singly offer limited protection. Agrobacterium-mediated transient expression in Nicotiana benthamiana (tobacco) plants was used here to produce a virus-like particle (VLP) vaccine expressing a modified full-length IBV spike (S) protein of a QX-like IB variant. In a challenge study with the homologous live IB QX-like virus, VLP-vaccinated birds produced S protein-specific antibodies comparable to those produced by live-vaccinated birds seroconverting with mean geometric titers of 6.8 and 7.2 log2, respectively. The VLP-vaccinated birds had reduced oropharyngeal and cloacal viral shedding compared to an unvaccinated challenged control and were more protected against tracheal ciliostasis than the live-vaccinated birds. While the results appeared similar, plant-produced IB VLPs are safer, more affordable, easier to produce and update to antigenically match any emerging IB variant, making them a more suitable alternative to IBV control than live-attenuated vaccines.

Trastuzumab-induced cardiomyopathy via ferroptosis-mediated mitochondrial dysfunction.

Trastuzumab (TRZ) is a first-line chemotherapeutic agent for HER-2 (ErbB2)-positive breast cancer. Unfortunately, its clinical use is limited due to its cardiotoxicity, referred to as TRZ-induced cardiotoxicity (TIC). However, the exact molecular mechanisms underlying the development of TIC remain unclear. Iron and lipid metabolism and redox reactions participate in the development of ferroptosis. Here, we show that ferroptosis-mediated mitochondrial dysfunction is involved in TIC in vivo and in vitro. We first established TIC models with BALB/c mice or neonatal rat cardiomyocytes and confirmed cardiomyopathy with echocardiography and inhibition of cell viability with a cell counting kit-8 examination, respectively. We showed that TRZ downregulated glutathione peroxidase 4 (GPx4) and elevated lipid peroxidation by-products, 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), by inactivating the ErbB2/PI3K/AKT/Nrf2 signalling pathway. Additionally, upregulated mitochondrial 4-HNE binds to voltage-dependent anion channel 1 (VDAC1), increases VDAC1 oligomerization, and subsequently induces mitochondrial dysfunction, as evidenced by mitochondrial permeability transition pore (mPTP) opening and decreased mitochondrial membrane potential (MMP) and ATP levels. Concomitantly, TRZ affected the mitochondrial levels of GSH/GSSG and iron ions and the stability of mitoGPx4. Ferroptosis inhibitors, such as ferrostatin-1 (Fer-1) or the iron chelator deferoxamine (DFO), ameliorate TRZ-induced cardiomyopathy. Overexpression of mitoGPx4 also suppressed mitochondrial lipid peroxidation and prevented TRZ-induced ferroptosis. Our study strongly suggests that targeting ferroptosis-mediated mitochondrial dysfunction is a potential cardioprotective strategy.

A genetic system for Akkermansia muciniphila reveals a role for mucin foraging in gut colonization and host sterol biosynthesis gene expression.

Akkermansia muciniphila, a mucophilic member of the gut microbiota, protects its host against metabolic disorders. Because it is genetically intractable, the mechanisms underlying mucin metabolism, gut colonization and its impact on host physiology are not well understood. Here we developed and applied transposon mutagenesis to identify genes important for intestinal colonization and for the use of mucin. An analysis of transposon mutants indicated that de novo biosynthesis of amino acids was required for A. muciniphila growth on mucin medium and that many glycoside hydrolases are redundant. We observed that mucin degradation products accumulate in internal compartments within bacteria in a process that requires genes encoding pili and a periplasmic protein complex, which we term mucin utilization locus (MUL) genes. We determined that MUL genes were required for intestinal colonization in mice but only when competing with other microbes. In germ-free mice, MUL genes were required for A. muciniphila to repress genes important for cholesterol biosynthesis in the colon. Our genetic system for A. muciniphila provides an important tool with which to uncover molecular links between the metabolism of mucins, regulation of lipid homeostasis and potential probiotic activities.

Pathogenicity and innate immune responses induced by fowl adenovirus serotype 8b in specific pathogen-free chicken.

Fowl adenovirus serotype 8b (FAdV-8b), as causative agent of inclusion body hepatitis (IBH), poses a great threat to the poultry industry. Considering the importance of innate immune response in host against viral infections, we investigated pathogenicity of a FAdV-8b strain HLJ/151129 in 1-mo-old specific pathogen-free (SPF) chickens and immune responses of host to FAdV-8b infection in this study. The results demonstrated that no obvious clinical signs were observed in infected birds. Neither mobility nor mortality was observed in both FAdV-8b infected and control chickens, as well. However, hepatic necrosis and a small amount of inflammatory cell infiltration were observed by pathological analysis. Viral load was detected in bursa of Fabricius, cecal tonsils, liver, heart, spleen, Harderian glands, and thymus. Virus shedding and viremia generated as early as 3 days postinfection (dpi) (9/10) and reached the peak at 7 dpi (10/10). In addition, the infected birds had developed FAdV-specific antibodies at 7 dpi, and the antibody titers reached the peak at 14 dpi. Furthermore, the results demonstrated that the mRNA expression levels of most of toll-like receptors (TLRs), most of avian ß-defensins (AvBDs), and cytokines [interleukin (IL)-2, IL-6, and interferon (IFN)-γ], were significantly upregulated in most tissues at early phases of FAdV-8b infection, especially in liver and spleen. In contrast, FAdV-8b infection results in downregulation of TLR4, TLR5, and TLR21 expressions in some tissues of infected chickens. In addition, FAdV-8b infection upregulated myeloid differentiation factor 88 (MyD88), nuclear factor-kappa B (NF-κB) p65, and TIR-domain-containing adapter inducing interferon-ß (TRIF) expression in some tissues, while decreased NF-κBp65 and TRIF in spleen at both 72 hpi and 21 dpi. Taken together, these results confirmed that FAdV-8b could replicate in all investigated tissues of infected birds, and then, result in production of FAdV-specific antibody titers. Meanwhile, the FAdV-8b infection induces strong innate immune responses at early stage in chickens, which may associate with the viral pathogenesis.

The role of protein arginine deiminase 4-dependent neutrophil extracellular traps formation in ulcerative colitis.

Background: Neutrophil extracellular traps (NETs) play an important role in the development and progression of ulcerative colitis (UC). Peptidyl arginine deiminase 4 (PAD4) is essential for the formation of NETs via catalyzing histone citrullination. This study mainly to explore the role of PAD4-mediated NETs in intestinal inflammation of dextran sulfate sodium (DSS)-induced UC. Methods: Acute and chronic colitis mouse models were established by supplementing DSS in drinking water. Colon tissues from colitis mice were analyzed for the level of PAD4 expression, citrullinated histone H3(Cit-H3), intestinal histopathology, and inflammatory cytokines secretion. Serum samples were tested for systemic neutrophil activation biomarkers. Colitis mice administered with Cl-amidine, a PAD4 inhibitor, and PAD4 knockout mice were investigated to detect NETs formation, intestinal inflammation, and barrier function. Result: We found the formation of NETs significantly increased in DSS-induced colitis mice and was correlated with disease markers. Blocking NETs formation by Cl-amidine or PAD4 genetic knockout could alleviate clinical colitis index, intestinal inflammation, and barrier dysfunction. Conclusion: This study provided a research basis for the role of PAD4-mediated NETs formation in the pathogenesis of UC and suggested that inhibition of PAD4 activity and the formation of NETs may be helpful for the prevention and treatment of UC.

Effect of oral vitamin A supplementation on host immune response to infectious bronchitis virus infection in specific pathogen-free chicken.

Vitamin A is a fat-soluble vitamin that is a crucial mediator of the immune system. In this study, we evaluated the effect of oral vitamin A supplementation on host immune responses to infectious bronchitis virus (IBV) infection in chickens. Forty 1-day-old specific pathogen-free (SPF) chickens were fed a basal diet and randomly divided into 2 groups (n = 20 birds per group). Chickens in the experimental group were treated orally with vitamin A (dissolved in 0.1 mL soybean oil, at a dose of 8,000 IU per kg diet) daily. Birds in the control group were orally administered 0.1 mL soybean oil without vitamin A until 21 d of age. On d 21 after birth, all chickens were infected with 0.1 mL of 106.5 50% median embryo infectious dose of a pathogenic IBV strain (CK/CH/LDL/091022) by intraocular and intranasal routes. The results demonstrated that oral vitamin A supplementation did not affect the clinical course of disease and growth performance of SPF chickens. However, vitamin A supplementation increased the IBV-specific IgG serum levels and decreased the viral load in some tissues of IBV-infected chickens. In addition, the results demonstrated that vitamin A supplementation decreased the expression levels of most immune-related molecules in some tissues of IBV-infected chickens. Vitamin A supplementation decreased the mRNA expression levels of some avian ß-defensins (AvBD2, 3, 6, 7, 11, and 13) and increased the expression levels of AvBD9 and AvBD12 in some tissues of IBV-infected chickens. Similarly, vitamin A supplementation decreased the mRNA expression levels of some cytokines (interferon-γ, interleukin-1ß [IL-1ß], and IL-6) and increased the mRNA expression levels of IL-2 in some tissues of IBV-infected chickens. Furthermore, vitamin A supplementation decreased the mRNA expression levels of myeloid differentiation primary response protein 88, nuclear factor-κB p65, toll-like receptor 3, toll-like receptor 7, and CD4. In summary, the present study suggests that vitamin A supplementation enhances the immune function of SPF chickens against IBV infection by inhibiting viral replication, increasing the IBV-specific antibody titer, and suppressing the excessive inflammatory responses to IBV infection.

Diet-mediated constitutive induction of novel IL-4+ ILC2 cells maintains intestinal homeostasis in mice.

Group 2 innate lymphoid cells (ILC2s) expressing IL-5 and IL-13 are localized at various mucosal tissues and play critical roles in the induction of type 2 inflammation, response to helminth infection, and tissue repair. Here, we reveal a unique ILC2 subset in the mouse intestine that constitutively expresses IL-4 together with GATA3, ST2, KLRG1, IL-17RB, and IL-5. In this subset, IL-4 expression is regulated by mechanisms similar to but distinct from those observed in T cells and is partly affected by IL-25 signaling. Although the absence of the microbiota had marginal effects, feeding mice with a vitamin B1-deficient diet compromised the number of intestinal IL-4+ ILC2s. The decrease in the number of IL-4+ ILC2s caused by the vitamin B1 deficiency was accompanied by a reduction in IL-25-producing tuft cells. Our findings reveal that dietary vitamin B1 plays a critical role in maintaining interaction between tuft cells and IL-4+ ILC2s, a previously uncharacterized immune cell population that may contribute to maintaining intestinal homeostasis.

Functional neuronal circuits promote disease progression in cancer.

The molecular and functional contributions of intratumoral nerves to disease remain largely unknown. We localized synaptic markers within tumors suggesting that these nerves form functional connections. Consistent with this, electrophysiological analysis shows that malignancies harbor significantly higher electrical activity than benign disease or normal tissues. We also demonstrate pharmacologic silencing of tumoral electrical activity. Tumors implanted in transgenic animals lacking nociceptor neurons show reduced electrical activity. These data suggest that intratumoral nerves remain functional at the tumor bed. Immunohistochemical staining demonstrates the presence of the neuropeptide, Substance P (SP), within the tumor space. We show that tumor cells express the SP receptor, NK1R, and that ligand/receptor engagement promotes cellular proliferation and migration. Our findings identify a mechanism whereby intratumoral nerves promote cancer progression.

Microbiota alters the metabolome in an age- and sex- dependent manner in mice.

Commensal bacteria are major contributors to mammalian metabolism. We used liquid chromatography mass spectrometry to study the metabolomes of germ-free, gnotobiotic, and specific-pathogen-free mice, while also evaluating the influence of age and sex on metabolite profiles. Microbiota modified the metabolome of all body sites and accounted for the highest proportion of variation within the gastrointestinal tract. Microbiota and age explained similar amounts of variation the metabolome of urine, serum, and peritoneal fluid, while age was the primary driver of variation in the liver and spleen. Although sex explained the least amount of variation at all sites, it had a significant impact on all sites except the ileum. Collectively, these data illustrate the interplay between microbiota, age, and sex in the metabolic phenotypes of diverse body sites. This provides a framework for interpreting complex metabolic phenotypes and will help guide future studies into the role that the microbiome plays in disease.

Evaluation of exhaust air dust polymerase chain reaction as a supplement method for soiled bedding sentinel monitoring in specific pathogen free mouse facility using two different individually ventilated cage racks.

Health monitoring is essential for ensuring animal health and reliable research results. Each animal facility should establish adequate health monitoring methods, and microbiological quality control should be implemented through regular health surveillance. Recently, specific pathogen free (SPF) mice have been housed in individually ventilated cage (IVC) racks in the majority of mouse facilities globally, and health monitoring is implemented using a soiled bedding sentinel (SBS). Even though SBS monitoring is a standard method, it has a limitation in that some pathogens are not sufficiently transmitted to the sentinel housed in the IVC. The exhaust air dust polymerase chain reaction (EAD PCR) method has been reported to be a reliable complementary method to SBS monitoring based on research findings. In Korea, health monitoring programs using EAD PCR have not yet been applied to laboratory animal facilities. The microbiological status of mouse colonies housed in the two IVC racks was compared using SBS and EAD PCR monitoring in our SPF mouse facility. Except for Helicobacter spp. and Staphylococcus aureus, the detection of 16 pathogens did not differ between the two methods. In the detection of Helicobacter spp., EAD PCR was found to be more sensitive than SBS. Helicobacter spp. were not found by SBS, whereas four S. aureus positive samples were detected by either SBS or EAD PCR test. According to our findings, EAD PCR can be used as a supplement to SBS monitoring. Moreover, EAD PCR can reduce the number of animals used, making it a 3R (Replacement, Reduction, Refinement)-consistent method.

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.

The Interaction between B87 Vaccine Strain and BC6/85 of Infectious Bursa Disease Virus in SPF Chickens.

This study was initiated to determine the interaction between two infectious bursal disease virus (IBDV) strains in the early stages of infection by detection and quantification of IBDV RNA in lymphoid and non-lymphoid tissues. SPF chickens were inoculated with single infection or dual infection by the mild strain B87 followed by the pathogenic strain BC6/85 at 0, 1, 2, and 3 days post-inoculation (dpi) with B87. Real-time RT-PCR assays were developed to examine the viral loads of the tissues collected at various time intervals. The results reveal that B87 could delay the time point of positive detection of the BC6/85 strain in the bursa of Fabricius from 1 dpi to 3 dpi, indicating that B87 interfered with the replication of BC6/85. The interference occurred when BC6/85 was inoculated at 2 dpi and 3 dpi with the B87 strain. Moreover, BC6/85 could affect the proliferation and duration of B87 in SPF chickens. The rates of positive detection for B87 decreased significantly during dual infection. The investigation of the interaction between the two strains is important for the implementation of appropriate control measures.

Multi-omics analysis reveals the effects of microbiota on oral homeostasis.

The oral epithelium's normal morphological structure and function play an important role in maintaining oral homeostasis, among which microbiota and chronic stress are key contributing factors. However, the effects of microbiota and chronic stress on the morphological structures and molecular function of oral homeostasis remain unclear. In this study, morphological staining was used to compare the tongue structure of specific pathogen-free and germ-free mice, and an integrated multi-omics analysis based on transcriptomics, proteomics, and metabolomics was performed to investigate the regulatory mechanisms of microbiota and chronic stress on oral homeostasis. We found that the morphological structure of the tongue in germ-free mice was disordered compared with in specific pathogen-free mice, especially in the epithelium. Multi-omics analysis indicated that differentially expressed molecules of the tongue between germ-free and specific pathogen-free mice were significantly enriched in the mitochondrial metabolic process and immune response. Interestingly, microbiota also significantly influenced the permeability of the oral epithelial barrier, represented by the differential expression of keratinization, and cell adhesion molecules. It was worth noting that the above changes in the tongue between specific pathogen-free and germ-free mice were more significant after chronic stress. Collectively, this is the first study to reveal that the microbiota might maintain oral homeostasis by reshaping the structure of the oral epithelial barrier and changing the function of molecular biology, a process that may be driven by the immune response and mitochondrial metabolic process of oral tissue. Furthermore, chronic stress can enhance the regulatory effects of microbiota on oral homeostasis.

Experimental infection of Toxoplasma gondii in specific pathogen-free and commercial broiler chicks.

Toxoplasma gondii is an intracellular protozoan parasite that infects most warm-blooded animals, including humans, and causes toxoplasmosis. In this study, the pathogenicity of RH strain of T. gondii in broiler chicks was investigated and the susceptible tissues of chicks were clarified. Fourteen-day-old broiler chicks were injected intraperitoneally with 1 × 107, 1 × 106, and 1 × 105 tachyzoites of T. gondii, respectively. The results showed that the spleen and lungs were frequently positive for T. gondii DNA. Moreover, in chicks, infection with only 1 × 107 tachyzoites resulted in clinical symptoms, with lower weight gain, higher body temperature, anorexia and apathy, indicating that chicks are insusceptible to T. gondii infection. Then, this study investigated the relationship between T. gondii survival and chick body temperature. In the experiment, the invasion and proliferation of T. gondii were evaluated at 37 °C and 41 °C, respectively, and the survival of the parasites was significantly inhibited at 41 °C. In conclusion, broiler chicks are insusceptible to T. gondii infection, and the higher body temperature compared to other susceptible animals is a key factor in the reduction of T. gondii pathogenicity.

Characterization of Chronic Hepatitis E Virus Infection in Immunocompetent Rabbits.

Chronic hepatitis E virus (HEV) infection is frequently reported in immunocompromised patients, but has also been increasingly reported in non-immunocompromised individuals. We characterized the course of chronic HEV infection in immunocompetent rabbits. In two independent experiments, 40 specific-pathogen-free rabbits were infected with a rabbit HEV genotype 3 strain in serial diluted titers (108 to 104 copies/mL). Serum and fecal samples were collected weekly and were tested for HEV RNA, antigen, anti-HEV and liver enzymes. Rabbits that spontaneously cleared the infection before 10 weeks post-inoculation (wpi) were kept to the end of the study as recovery control. Liver tissues were collected from HEV-infected rabbits at 5, 10 and 26 wpi for histopathological analysis. Nineteen rabbits (47.5%) developed chronic HEV infection with persistent viraemia and fecal HEV shedding for >6 months. Seroconversion to anti-HEV was observed in 84.2% (16/19) of the chronically infected rabbits. Serum levels of aminotransferase were persistently elevated in most of the rabbits. Characterizations of chronic HEV infection in immunocompetent settings could be recapitulated in rabbits, which can serve as a valuable tool for future studies on pathogenesis.