Effect of in ovo administration of Newcastle disease vaccine conjugated with Astragalus polysaccharide on growth performance, intestinal development, and mucosal immunity in broiler chickens.

This study was conducted to investigate the effect of in ovo administration of a mixture of Astragalus polysaccharide (APS) and Newcastle disease vaccine (NDV) on growth performance, intestinal development, and mucosal immunity in newly hatched chicks. Six hundred specific-pathogen-free (SPF) Leghorn fertilised eggs were incubated in a commercial hatchery and divided into four groups: (a) control group injected with 1 ml of 0.9% physiological saline, (b) APS group injected with 1 ml of 1 mg/ml APS solution, and (c) NDV group injected with 1 ml of 104.0 EID50 /dose of NDV solution, and (d) APS + NDV group injected with a mixture of 0.5 ml of 2 mg/ml APS plus 0.5 ml 104.0 EID50 /dose ND vaccine (NDV) on Day 18.5 of incubation. The results showed that in ovo injection of APS or the mixture of APS and NDV increased the body weight at 1 day (IW) and final weight (FW) at 28 days and increased the feed conversion ratio (FCR) at 1-7, 8-14, 15-21, and 1-28 days of age. The villus height (VH) was increased (p < 0.05), and the crypt depth (CD) was decreased (p < 0.05) in the duodenum compared with the control group. The VH/CD ratios were increased (p < 0.05) in the APS + NDV group compared with controls, NDV group, and APS group on d3. The levels of slgA in washings were increased (p < 0.05) on Days 3, 7, 14, 21, and 28, and the number of IgA+ cells in the duodenum was increased on Days 7, 14, 21, and 28. In addition, the IgA+ cells were promoted from the villus root to the apex in the APS + NDV group. It can be concluded that in ovo administration of NDV conjugated with APS compared with NDV alone may be more effective in promoting growth performance and intestinal mucosal immunity.

The construction of recombinant Lactobacillus casei expressing hemagglutinin-neuraminidase protein and its immune response in chickens.

Newcastle disease virus (NDV) is one of the most important diseases in poultry. The present study generated recombinant surface-displayed Lactobacillus casei (L. casei) expressing the hemagglutinin-neuraminidase (HN) of NDV (Lc-pPG-HN) and a live pPG vector (Lc-pPG) and evaluated their immunogenicity. A 1670 bp HN gene fragment was successfully amplified and cloned into a prokaryotic protein expression system. Protein expression in the resulting recombinant Lc-pPG-HN (surface displayed) strain was verified using Western blotting and indirect immunofluorescence. A single band was observed on the Western blots, and the molecular weight of the corresponding protein was 63 kDa. A fluorescent signal for Lc-pPG-HN was observed using fluorescence microscopy. A total of 270 healthy chicks were divided into three treatment groups. Five replicates were used for each treatment, while six chicks were used per replicate. The following three treatment groups were used: physiological saline group (Control), Lc-pPG group and recombinant vaccine group (Lc-pPG-HN). The primary immunization and booster immunization of the chicks were performed via oral administration on 1 and 10 days old. Tissue and blood samples were collected from chickens that received oral recombinant L. casei strains on 1, 7, 14 and 21 days post-immunization for immune-related index analyses. Chickens orally immunized with Lc-pPG-HN showed significantly increased body weights and immune organ indices. Oral immunization with Lc-pPG-HN also enhanced the concentrations of serum interleukin-2 (IL-2), interferon-γ (IFN-γ), intestinal lavage fluid secretory immunoglobulin A (SIgA) and histomorphological development of the small intestine. Our results also indicated that recombinant L. casei significantly increased Lactobacillus and Bifidobacterium colonization and decreased the relative abundance of Escherichia coli (E. coli) in the chicken caecum. Similar enhancement effects from hemagglutination inhibition were also observed in the antibody titers. Oral administration of Lc-pPG-HN effectively protected against NDV and alleviated the symptoms of the NDV challenge. In summary, recombinant L. casei had positive impacts on the performance, immunological function, gut development, and microbiota of growing chicks and may be a potential therapeutic candidate against NDV.

Construction and immune efficacy of recombinant Lactobacillus casei strains expressing Malt from Aeromonas veronii.

Aeromonas veronii is an important zoonotic pathogen that causes significant economic losses in the aquaculture industry. The use of probiotics in aquaculture is a practical alternative to antibiotics to promote animal health and aid in disease prevention. In the present study, we aimed to construct a recombinant Lactobacillus casei(surface-displayed or secretory) strain containing Malt from A. veronii TH0426 and assess its potential as an oral vaccine. A 1314-bp Malt gene fragment was successfully amplified and cloned into a prokaryotic protein expression system. Protein expression in resulting recombinant strains Lc-MCS-Malt (surface-displayed) and Lc-pPG-Malt (secretory) was then verified by Western blotting and indirect immunofluorescence. A single band was observed on the Western blots, with the molecular weight of the corresponding protein shown to be 48 kDa. Furthermore, a fluorescent signal for Lc-MCS-Malt was observed by fluorescence microscopy. At 0, 7, 16, 25, and 34 days post-immunization, tissue and blood samples were collected from common carp orally administered with the recombinant L. casei strains for immune-related index analyses. Treatment of common carp with the recombinant vaccine candidate stimulated high serum or skin mucus specific antibody titers and induced a higher lysozyme, ACP, SOD activity, while fish fed with Lc-pPG or PBS had no detectable immobilizing immune responses. Expression of IL-10, IL-1ß, TNF-α, and IFN-γ genes in the group immunized with recombinant L. casei were significantly (P < 0.05) up regulated as compared with control groups, indicating that inflammatory response and cell immune response were triggered. Results also showed that recombinant L. casei could stimulate the mucosa through colonization of the intestine, resulting in increased transcription of IL-10, IL-1ß, TNF-α, and IFN-γ. Immunity and colonization assays also showed that after 34 days of fasting, recombinant L. casei were still present in the intestines of the immunized fish. Common carp that received Lc-MCS-Malt(53.3%) and Lc-pPG-Malt (46.7%) exhibited higher survival rates than the controls after challenge with the pathogen A. veronii. Our findings suggested that recombinant L. casei can adequately protect fish and improve immunity, providing a theoretical basis for the future development of an oral Lactobacillus vaccine for use in aquaculture.

The immune adjuvant effect of Astragalus polysaccharide on in ovo injection of Newcastle disease vaccine.

In this study, immunogenic efficacies of in ovo administration of Astragalus polysaccharide (APS) along with live Newcastle disease vaccine (live ND vaccine) (live VG/GA strain) were evaluated. Four hundred fertilized eggs were randomly divided into four groups (n = 100/group), and vaccinated in ovo, respectively, with solutions of APS, live ND vaccine, live ND vaccine combined with APS, and 0.9% physiological saline into their amniotic fluid on d 18.5 of incubation. Significant improvement of chicks' development was displayed in those vaccinated with live ND vaccine adjuvanted with APS in ovo, manifested as enhanced hatchability and gaining weight. Moreover, in ovo administration of live NDV vaccine plus APS could significantly enhance the serum anti-NDV antibody titres and interferon gamma (IFN-γ), interleukin (IL)-2, IL-4 and IL-6 concentrations, promote lymphocyte proliferative capability as well as improve the frequencies of CD4+ and CD8+ T cells in peripheral blood. Overall results indicated in ovo administration of live ND vaccine adjuvanted with APS could stimulate stronger humoral and cellular responses in newly hatched chicks.

Effect of the oral administration of astragalus polysaccharides on jejunum mucosal immunity in chickens vaccinated against Newcastle disease.

Astragalus polysaccharides (APS) are a traditional Chinese medicine with a therapeutic effect by enhancing immune function; however, the underlying functional mechanism is still unclear. The aim of the present study was to determine the effect of oral administration of APS on jejunum mucosal immunity in chickens vaccinated against Newcastle disease (ND). One-day-old Hy-Line male chickens were divided into five groups of 20 chicks each: three APS groups, one vaccinated control (VC) group and one non-vaccinated negative control (NC) group. On d 10, the APS groups were orally administered 0.5 mL of APS at doses of 1 mg/mL (APSL), 2 mg/mL (APSM) and 4 mg/mL (APSH) daily for 4 consecutive days. The chicks in the control groups were administered 0.5 mL saline for those 4 days. All groups except NC were administered a ND virus (NDV) vaccine on day 14. The jejunum was removed from 4 randomly selected chickens of each group at 1, 7, 14 and 28 days after vaccination. The jejunal villus height (VH) and crypt depth (CD) were measured and the VH:CD ratio calculated. Immunohistochemistry was used to analyze the differences of IgA+ cells in the jejunum. NDV specific secretory IgA (sIgA) levels in jejunal contents were detected using an indirect ELISA. At most time points, VH:CD ratios, number of IgA+ cells, and sIgA levels were significantly higher in the APS groups than those in VC and NC groups, but there were little differences among the three doses of APS groups. These results indicate that oral administration of APS could enhance the intestinal mucosal immune function of chickens, and APS could be used as a vaccine enhancer.

Generation and functional evaluation of a DNA vaccine co-expressing Cyprinid herpesvirus-3 envelope protein and carp interleukin-1 beta.

Cyprinid herpesvirus 3 (CyHV-3) infection in carp causes a fatal and highly contagious disease that results in huge economic losses in common and koi carp aquaculture worldwide. Thus the development of an effective vaccine to protect carp stocks against the CyHV3 virus is imperative. In this study, we immunized common carps with a DNA vaccine consisting of a plasmid that co-expresses the CyHV-3 envelope protein ORF25 and the carp IL-1ß gene in order to evaluate the adjuvant potential of IL-1ß. Our result shows that antibodies specific to ORF25 can be detected as early as one week after intramuscular injection of the DNA vaccine at low dosage. Moreover, the co-expression of IL-1ß can enhance the potency of the vaccine, as demonstrated by a higher antibody level after the third immunizations. Importantly, the DNA vaccine reduced mortality in carps when they were immunized prior to a CyHV-3 challenge, as compared to negative control groups. However, despite being able to induce higher neutralizing antibody titres, the co-expression of IL-1ß in the DNA vaccine did not significantly improve the overall survival of immunized fish following virus challenge. Furthermore, the DNA vaccine can protect carps from tissue damage and histopathological alteration caused by viral infection. These strongly suggests that the vaccine can efficiently elicit protective immunity against CyHV-3 infection. In conclusion, the DNA vaccine formulated with the pIRES-ORF25-IL-1ß DNA construct can protect carp against CyHV-3 infection and has potential applicability in the aquaculture industry.

TRIM28 down-regulation on methylation imprints in bovine preimplantation embryos.

SummaryTRIM28/KAP1/TIF1ß was identified as a universal transcriptional co-repressor and is critical for regulating post-fertilization methylation reprogramming in preimplantation embryos. In this study, three siRNAs (si647, si742, and si1153) were designed to target the TRIM28 mRNA sequence. After transfection of the mixture of the three siRNA (siMix) into bovine fibroblast cells, the most effective one for TRIM28 knockdown was selected. By injecting RNAi directed against TRIM28 mRNA, we found that TRIM28 knockdown in oocytes had the most effect on the H19 gene, in which differentially methylated region (DMR) methylation was almost completely absent at the 2-cell stage (1.4%), while control embryos showed 74% methylation. In addition, global H3K9me3 levels at the 2-cell stage were significantly higher in the in vitro fertilization (IVF) group than in the TRIM28 knockdown group (P<0.05). We further show that TRIM28 is highly expressed during oocyte maturation and reaches peak levels at the 2-cell stage. In contrast, at this stage, TRIM28 expression in somatic cell nuclear transfer (SCNT) embryos decreased significantly (P<0.05), suggesting that Trim28 transcripts are lost during SCNT. TRIM28 is required for the maintenance of methylation imprints in bovine preimplantation embryos, and the loss of TRIM28 during SCNT may contribute to the unfaithful maintenance of imprints in cloned embryos.

[The roles of maternal-effect proteins in the maintenance of genomic imprints].

Genomic imprinting is a mechanism of differentially epigenetic modification that restricts monoallelic expression to either the maternally or paternally inherited copy of the gene during gametogenesis. Imprinted methylation undergoes a process of erasure, acquisition, and maintenance during gametogenesis and early embryogenesis. Disruptions in any of these steps may lead to imprinting disorders, resulting in the aberrant development of embryogenesis, placentation and postnatal growth. Recent studies have shown that maternal-effect proteins are important for the regulation of imprinted gene during the development of preimplantation embryos. In order to obtain a better understanding for the mechanism of maternal-effect proteins in the maintenance of genomic imprints, the recent study progress of maternal-effect proteins, such as DPPA3, ZFP57, TRIM28 and DNMT1, are summarized, and the regulation mechanism of these maternal-effect proteins for genomic imprints are discussed.

A novel phenotype of B cells associated with enhanced phagocytic capability and chemotactic function after ischemic stroke.

Accumulating evidence has demonstrated the involvement of B cells in neuroinflammation and neuroregeneration. However, the role of B cells in ischemic stroke remains unclear. In this study, we identified a novel phenotype of macrophage-like B cells in brain-infiltrating immune cells expressing a high level of CD45. Macrophage-like B cells characterized by co-expression of B-cell and macrophage markers, showed stronger phagocytic and chemotactic functions compared with other B cells and showed upregulated expression of phagocytosis-related genes. Gene Ontology analysis found that the expression of genes associated with phagocytosis, including phagosome- and lysosome-related genes, was upregulated in macrophage-like B cells. The phagocytic activity of macrophage-like B cells was verified by immunostaining and three-dimensional reconstruction, in which TREM2-labeled macrophage-like B cells enwrapped and internalized myelin debris after cerebral ischemia. Cell-cell interaction analysis revealed that macrophage-like B cells released multiple chemokines to recruit peripheral immune cells mainly via CCL pathways. Single-cell RNA sequencing showed that the transdifferentiation to macrophage-like B cells may be induced by specific upregulation of the transcription factor CEBP family to the myeloid lineage and/or by downregulation of the transcription factor Pax5 to the lymphoid lineage. Furthermore, this distinct B cell phenotype was detected in brain tissues from mice or patients with traumatic brain injury, Alzheimer's disease, and glioblastoma. Overall, these results provide a new perspective on the phagocytic capability and chemotactic function of B cells in the ischemic brain. These cells may serve as an immunotherapeutic target for regulating the immune response of ischemic stroke.

"Red Spitz Tumor" on the Ear: Case Report and Review of the Literature.

Spitz nevus (SN) is a benign melanocytic lesion with cytologic and architectural atypia. It is sometimes difficult to distinguish SNs from atypical Spitz tumor (AST), Spitz melanoma, or conventional melanoma. SNs frequently develop in Caucasians and appear on the skin of the head and lower extremities. Lesions on the ear in Asian populations are rare. Here, we report a "red Spitz tumor" on the ear of a Chinese 18-year-old boy. Dermoscopic examination revealed possibly malignant features presented as polymorphous vessels along with central white area, pseudo-network depigmentation and atypical peripheral globular pattern. The results of histopathological examination strongly suggested that the neoplasm was a compound SN and no recurrences or metastases occurred during 1-year follow-up post-surgery. Further, we review the literature on 4 previously reported cases of SN on the ear and summarize the main points of SN diagnosis and differential diagnosis with atypical Spitz tumors and melanoma.

Microglia Impede Oligodendrocyte Generation in Aged Brain.

PURPOSE: Age-related increase in myelin loss may be responsible for brain atrophy, and the mechanism is not completely understood. We aim to comprehensively delineate oligodendrocyte heterogeneity in young and aged mice and to reveal the underlying mechanism for myelin loss during aging. METHODS: Diffusion tensor imaging and immunofluorescent staining were performed to verify the demyelination in the aged brains of both rodents and human. Further, the single-cell RNA sequencing data of all brain cells from young and aged mice were deeply analyzed to identify the subsets of oligodendrocyte lineage cells. Cell-to-cell interaction analysis was performed to detect the mechanism of observed changes in oligodendrocyte generation. RESULTS: Oligodendrocytes were observed to up-regulate several senescence associated genes in aged brain. Four clusters of oligodendrocyte precursor cells (OPCs) were identified in both young and aged brains. The number of those OPCs in basal state was significantly increased, while the OPCs in the procedure of differentiation were immensely decreased in aged brain. Furthermore, it was identified that activated microglia in the aged brain released inflammatory factors to suppress OPC differentiation. Stat1 might be a potential target to transform senescent microglia into tissue repair type to promote oligodendrocyte generation. CONCLUSION: These results provided a perspective on how age activated microglia could impede remyelination and might give a new therapeutic target for age-related remyelinating diseases.

Immunomodulatory effect of Acanthopanax senticosus polysaccharide on immunosuppressed chickens.

The immunomodulatory effect of Acanthopanax senticosus polysaccharide (ASPS) on immunosuppressed chickens induced by cyclophosphamide (Cy) was observed in this study. Four hundred 7-day-old chickens were randomly divided into 4 groups: vaccinated control group (VC group), Cy-challenged control group (Cy group), Cy-challenged + low-dose ASPS group (ASPSL + Cy group), and Cy-challenged + high-dose ASPS group (ASPSH + Cy group). All groups except the VC group were injected with Cy at a dose of 80 mg/kg/day of BW for 3 successive days to induce immunosuppression. At the age of 10 d, the ASPSL + Cy group and ASPSH + Cy group were intramuscularly injected with 0.2 mL of ASPS at the dose of 100 and 200 mg/mL/day, respectively, once a day for 3 successive days. The Cy group was injected with saline solution in the same way as the 2 ASPS groups. At the age of 14 d, the chickens were vaccinated with Newcastle disease (ND) vaccine in all groups. On day 7, 14, 21, and 28 after the vaccination, BW, lymphocyte proliferation, the serum antibody titers of the ND vaccine, the proportion of CD4+ and CD8+ T lymphocytes, and the concentrations of interferon gamma and IL-2 were determined. The results showed that chickens were injected with Cy at a dose of 80 mg/kg of BW for 3 d displayed lower immune responses than the control group, indicating that the immunosuppressive model was successfully established. At most time points, both high and low doses of ASPS could significantly promote lymphocyte proliferation; enhance BW, antibody titers, and the proportion of CD4+ and CD8+ T lymphocytes; and raised the concentrations of interferon gamma and IL-2 in Cy-treated chickens compared with those in the Cy control group (P < 0.05). These results indicated that ASPS could resist immunosuppression induced by Cy and may be a new-type immune adjuvant to improve vaccination in normal and immunosuppressed chickens.

The Effects of In Ovo Injection of Synbiotics on the Early Growth Performance and Intestinal Health of Chicks.

In this study, the effects of synbiotic inclusion at the intra-amniotic stage in layer chicks were evaluated with different parameters, such as performance, immunological function, intestinal development, and cecal microflora content. A total of 1,200 eggs with fertile embryos were allocated into four treatment groups. For every treatment, five replicates were used, and 60 eggs were included in each replicate. The following four treatment groups were established: the non-injected group, 0.9% physiological saline injection (saline) group, 1 × 106 CFU/egg Lactobacillus plantarum injection (probiotic) group, and 1 × 106 CFU/egg L. plantarum + 2 mg/egg Astragalus polysaccharide injection (synbiotic) group. In ovo injection was carried out at 18.5 days of incubation. The results showed that in ovo injection of probiotics or synbiotics did not affect the hatching or growth performance of the chicks but significantly increased their feed intake (FI), body weight (BW), and the feed conversion ratio (FCR). Additionally, in ovo injection of synbiotics enhanced the levels of serum interleukin-2 (IL-2), interferon-γ (IFN-γ), and secretory immunoglobulin A (SIgA) in intestinal lavage fluid and the histomorphological development of the small intestine. Our results also indicated that intra-amniotic synbiotic injection significantly increased Lactobacillus and Bifidobacterium colonization while decreasing the relative abundance of Escherichia coli in the chicken cecum (P < 0.05). In summary, in ovo injection of synbiotics had positive impacts on the performance, immunological function, gut development, and microbiota of growing chicks.

Effects of in ovo Injection of Astragalus Polysaccharide on the Intestinal Development and Mucosal Immunity in Broiler Chickens.

The purpose of this study was to examine the effects of in ovo injection of Astragalus polysaccharide (APS) on hatchability, body weight (BW), intestinal histomorphology, the number of IgA+ cells and sIgA content in intestine, and the expression of intestinal immune-related genes in broiler chickens. On day 18 of the incubation, a total of 960 live embryo eggs were weighed and randomly divided into 4 treatment groups: a control group and three APS groups. The eggs in the control group were injected with 0.5 mL physiological saline. The eggs in the APS groups were injected with 3 different amounts of APS in 0.5 mL physiological saline: 1 mg (APSL), 2 mg (APSM) and 4 mg (APSH). The solution was injected into the amnion of each egg. The results showed that in ovo injection of APS did not affect the hatchability but increased the body weight of the 14 d and 21 d chickens, with a significant increase observed in the APSM group (P < 0.05). At most time points, the villus height (VH) was increased (P < 0.05) and the crypt depth (CD) was decreased (P < 0.05) in the small intestine of the broilers, with higher VH/CD ratios in the APSL and APSM groups compared with the control group. The number of IgA+ cells in the mucosa and the secretory immunoglobulin A (sIgA) levels in the intestinal washings were higher in the APSM and APSH groups than in the APSL and control groups. The gene expression levels of interleukin (IL)-2, interleukin (IL)-4, interferon gamma (IFN-γ), and Toll-like receptor (TLR)-4 were significantly enhanced by APS stimulation at most time points (P < 0.05). These results indicated that in ovo injection of APS has the potential of promoting intestinal development and enhancing intestinal mucosal immunity of broiler chickens in the early stage after hatching.

A TaqMan-based real-time PCR assay for the detection of Ungulate bocaparvovirus 2.

Ungulate bocaparvoviruses (UBoV) 2-5 are recently discovered porcine bocaparvoviruses belonging to the family Parvoviridae, and are considered to be a potentially major cause of swine diseases. In order to detect local UBoV2 epidemics in China, we developed a TaqMan-based real-time PCR (qPCR) assay targeting the UBoV2 VP1 gene and compared the results of qPCR with conventional PCR (cPCR). The qPCR reproducibly detected a recombinant DNA plasmid containing the VP1 gene over a range of eight orders of magnitude, from 9.97 × 10-1-106 copies/µL, with a lower limit of detection of 9.97 copies/µL, compared with approximately 9.97 × 102 copies/µL for cPCR. The qPCR assay showed no cross-reactivity with other UBoVs or other porcine viruses. This qPCR assay detected UBoV2 in 18.1% (84/463) of pig samples collected from Chinese swine herds, with the highest infection rate of 35.3% (53/150) in loose stools. UBoV2 was not detected in liver samples. The TaqMan-based qPCR assay established in this study was highly sensitive and specific for the diagnosis and quantification of UBoV2. The results of this study will further our understanding of the etiology, epidemiology, and pathogenesis of UBoV2 infection.

Fibroblast Growth Factor 21 Deficiency Attenuates Experimental Colitis-Induced Adipose Tissue Lipolysis.

AIMS: Nutrient deficiencies are common in patients with inflammatory bowel disease (IBD). Adipose tissue plays a critical role in regulating energy balance. Fibroblast growth factor 21 (FGF21) is an important endocrine metabolic regulator with emerging beneficial roles in lipid homeostasis. We investigated the impact of FGF21 in experimental colitis-induced epididymal white adipose tissue (eWAT) lipolysis. METHODS: Mice were given 2.5% dextran sulfate sodium (DSS) ad libitum for 7 days to induce colitis. The role of FGF21 was investigated using antibody neutralization or knockout (KO) mice. Lipolysis index and adipose lipolytic enzymes were determined. In addition, 3T3-L1 cells were pretreated with IL-6, followed by recombinant human FGF21 (rhFGF21) treatment; lipolysis was assessed. RESULTS: DSS markedly decreased eWAT/body weight ratio and increased serum concentrations of free fatty acid (FFA) and glycerol, indicating increased adipose tissue lipolysis. eWAT intracellular lipolytic enzyme expression/activation was significantly increased. These alterations were significantly attenuated in FGF21 KO mice and by circulating FGF21 neutralization. Moreover, DSS treatment markedly increased serum IL-6 and FGF21 levels. IL-6 pretreatment was necessary for the stimulatory effect of FGF21 on adipose lipolysis in 3T3-L1 cells. CONCLUSIONS: Our results demonstrate that experimental colitis induces eWAT lipolysis via an IL-6/FGF21-mediated signaling pathway.