Characterization and immunological effect of outer membrane vesicles from Pasteurella multocida on macrophages.

Pasteurella multocida is an important bacterial pathogen that can cause diseases in both animals and humans. Its elevated morbidity and mortality rates in animals result in substantial economic repercussions within the livestock industry. The prevention of diseases caused by P. multocida through immunization is impeded by the absence of a safe and effective vaccine. Outer membrane vesicles (OMVs) secreted from the outer membrane of Gram-negative bacteria are spherical vesicular structures that encompass an array of periplasmic components in conjunction with a diverse assortment of lipids and proteins. These vesicles can induce antibacterial immune responses within the host. P. multocida has been shown to produce OMVs. Nonetheless, the precise characteristics and immunomodulatory functions of P. multocida OMVs have not been fully elucidated. In this study, OMVs were isolated from P. multocida using an ultrafiltration concentration technique, and their morphology, protein constitution, and immunomodulatory properties in RAW264.7 cells were studied. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) revealed that the OMVs exhibited typical spherical and bilayered lipid vesicular architecture, exhibiting an average diameter of approximately 147.5 nm. The yield of OMVs was 2.6 × 1011 particles/mL. Proteomic analysis revealed a high abundance of membrane-associated proteins within P. multocida OMVs, with the capability to instigate the host's immune response. Furthermore, OMVs stimulated the proliferation and cellular uptake of macrophages and triggered the secretion of cytokines, such as TNF-ɑ, IL-1ß, IL-6, IL-10, and TGF-ß1. Consequently, our results indicated that OMVs from P. multocida could directly interact with macrophages and regulate their immune function in vitro. These results supported the prospective applicability of P. multocida OMVs as a platform in the context of vaccine development. KEY POINTS: • Preparation and characterization of P. multocida OMVs. • P. multocida OMVs possess a range of antigens and lipoproteins associated with the activation of the immune system. • P. multocida OMVs can activate the proliferation, internalization, and cytokine secretion of macrophages in vitro.

Isolation, Identification and Drug Resistance Rates of Bacteria from Pigs in Zhejiang and Surrounding Areas during 2019-2021.

This study aimed to determine the prevalence of bacterial diseases in pig farms in various regions of Zhejiang Province and surrounding areas. A total of 526 samples were collected from 85 pig farms in Zhejiang Province and surrounding areas. In this study, samples were analyzed using bacterial isolation and purification, Gram staining, PCR amplification, and antimicrobial susceptibility testing. A total of 36 Pasteurella multocida (Pm) isolates were detected, with an isolation rate of 6.84%; 37 Bordetella bronchiseptica (Bb) isolates were detected, with an isolation rate of 7.03%; 60 Glasserella parasuis (G. parasuis) isolates were detected, with an isolation rate of 11.41%; 170 Escherichia coli (E. coli) isolates were detected, with an isolation rate of 32.32%; 67 Streptococcus suis (SS) isolates were detected, with an isolation rate of 12.74%; 44 Actinobacillus pleuropneumoniae (APP) isolates were detected, with an isolation rate of 8.37%; and 7 Salmonella enteritis (SE) isolates were detected, with an isolation rate of 1.33%. Antimicrobial drug susceptibility testing against 21 types of antibiotics was carried out on the isolated strains, and the results showed that 228 strains had varying degrees of resistance to 21 antibiotics, including Pm, Bb, E. coli, and APP, with the highest resistance to lincomycin, at 100%. Pm and APP were the most sensitive to cephalothin, with resistance rates of 0. In terms of strains, Pm had the highest overall sensitivity to 21 antibiotics, and E. coli had the highest resistance. In short, bacterial diseases in Zhejiang and the surrounding areas were harmful, and the drug resistance situation was severe. This study provides scientific guidance for the clinical treatment of bacterial diseases.

Enhancing effect of chitosan nanoparticles on the immune efficacy of Bordetella bronchiseptica outer membrane vesicles.

The outer membrane vesicle (OMV) of bacteria is a bilayer membrane vesicle with a diameter of about 10-300 nm that is secreted during the growth of Gram-negative bacteria. OMV is considered as a high-quality vaccine candidate antigen because of its natural immunogenicity and non-replicability. Although the excellent antigenicity of OMV has been widely confirmed, its instability and heterogeneity greatly affect its immune effect. Many studies have demonstrated that in combination with nanoparticles can enhance the stability of OMV. In this study, OMVs were used to coat chitosan nanoparticles (CNPs) and obtain a stable OMV vaccine. The characteristics, including morphology, hydrodynamic size, and zeta potential were evaluated. The immune protection of CNP-OMV and anti-infection efficacy were examined and compared in vivo and in vitro. The results showed that the CNP-OMV were homogenous with a size of 139 nm and a stable core-shell structure. And CNP-OMV could significantly increase the cell proliferation, phagocytosis and TNF-α, IL-6 and IL-10 secretion of RAW264.7 in vitro. In vivo, CNP-OMV could significantly increase the levels of anti-Bb and OMV IgG antibodies. Levels of blood lymphocyte, and Th1 (IFN-γ, IL-12), Th2 (IL-4, IL-5), and Th17 (IL-17, TNF-α) type cytokines in the serum were all significantly increased. At the same time, CNP-OMV could significantly reduce the bacterial invading the lungs of challenged rabbits. And CNP-OMV could largely protect the lungs from injury. The above results showed that CNP-OMV had a good immune efficacy and could resist the infection of Bordetella bronchiseptica. This study provided a scientific basis for the development of novel effective and safe vaccine against Bordetella bronchiseptica, and also provided a new idea for the development of new bacterial vaccine.

Adjuvant Efficacy of the ECMS-Oil on Immune Responses against Bordetella bronchiseptica in Mice through the TLR2/MyD88/NF-κB Pathway.

Bordetella infection can be efficiently prevented through vaccination. The current study investigated the effects of an extract of Cochinchina momordica seed (ECMS) combined with oil on the immune responses to the inactivated Bordetella vaccine in mice. Serum IgG and IgG1 level was significantly increased in ECMS-oil group compared to any other group (P < 0.05) 2 weeks after immunization, while groups ECMS200 µg/400 µg-oil had a markedly higher level of serum IgG2b and IgG3 than any other groups (P < 0.05). Moreover, lipopolysaccharide/ConA-stimulated proliferation of splenocytes was significantly enhanced in ECMS 400 µg-oil immunized mice in comparison with mice in any other group (P < 0.05). RT-PCR assay revealed that while ECMS800 µg-oil group had significantly higher levels of serum IL-4, IL-10, Toll-like receptor (TLR)2, and IL-1 beta than any other group (P < 0.05), the levels of serum IL-2, IL-4, and IL-10 were markedly increased in ECMS 400 µg-oil group as compared to any other groups (P < 0.05). Blood analysis showed that ECMS800 µg-oil and oil groups had a significantly higher number of immunocytes than any other groups (P < 0.05). There were significant differences in the number of IgG+, IgG2b+, and IgA+ cells in the lung between ECMS800 µg-oil group and any other groups (P < 0.05). Western blot analysis demonstrated that stimulation with ECMS 25 µg/mL or 50 ng/mL led to a significant increase in the expression of TLR2, MyD88, and NF-κB in Raw264.7 cells (P < 0.05). Compared with any other group, the expression of MyD88 was markedly increased in the cells stimulated with ECMS 50 ng/mL, as indicated by the RT-PCR analysis (P < 0.05). Overall, we observed that ECMS-oil efficiently enhanced the humoral or cellular immune responses against Bordetella and suggested that the mechanism of adjuvant activity of ECMS-oil might involve TLR2/MyD88/NF-κB signaling pathway.

Effects of Dietary Coated Lysozyme on the Growth Performance, Antioxidant Activity, Immunity and Gut Health of Weaned Piglets.

The purpose of this study was to evaluate the effects of dietary coated lysozyme on growth performance, serum biochemical indexes, antioxidant activity, digestive enzyme activity, intestinal permeability, and the cecal microbiota in weaned piglets. In total, 144 weaned Large White × Landrace piglets were divided into six treatment groups, with 3 replicates and 8 piglets per replicate: CN, a basal diet; CL-L, CL-M, and CL-H, basal diet supplemented with 100, 150, 500 mg/kg coated lysozyme; UL, basal diet supplemented with 150 mg/kg lysozyme; and Abs, basal diet supplemented with 150 mg/kg guitaromycin for 6 weeks. Compared with the CN and UL diets, dietary CL-H inclusion increased the average daily gain (ADG) and decreased the feed/gain (F/G) ratio of piglets (p < 0.05). The addition of 500 mg/kg coated lysozyme to the diet significantly increased the total protein (TP) and globulin (Glob) plasma levels of weaned piglets (p < 0.05). Supplementation with 500 mg/kg coated lysozyme significantly increased the serum IgM concentration and increased lipase activity in the duodenum (p < 0.05). The addition of coated lysozyme and lysozyme significantly decreased the malondialdehyde (MDA) content, while the superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and total antioxidant capacity (T-AOC) levels all increased (p < 0.05). High-throughput sequencing results showed that CL-H treatment effectively improved the intestinal microbiome. The relative abundance of Terrisporobacter in the CL-H and CL-M groups was significantly lower than that in the other groups (p < 0.05). LEfSe analysis results showed that the relative abundance of Coprococcus_3 was higher in the CL-M treatment group. The marker species added to the CL-H treatment group was Anaerofilum. In summary, as a potential substitute for feed antibiotics, lysozyme is directly used as a dietary additive, which is inefficient. Therefore, we used palm oil as the main coating material to coat lysozyme. Lysozyme after coating can more effectively improve the growth performance of piglets by improving the intestinal flora, improving the activity of digestive enzymes, reducing the damage to intestinal permeability and oxidative stress in piglets caused by weaning stress, and improving the immunity of piglets.

Inhibitory Effect and Mechanism of Trichoderma taxi and Its Metabolite on Trichophyton mentagrophyte.

Trichophyton mentagrophytes is an important zoonotic dermatophyte, which seriously harms the skin of humans and animals. Chemical drugs are generally used for the prevention and treatment of the disease caused by T. mentagrophytes. Discovering new compounds from natural products is an important approach for new drug development. Trichoderma includes a variety of fungal species used for biological control of phytopathogenic fungi. However, the antifungal effects of Trichoderma and their metabolites on zoonotic fungal pathogens are largely unknown. Here, the effect of trichodermin, a metabolite derived from the plant endophytic fungus Trichoderma taxi, on T. mentagrophytes was examined, and the underlying mechanism was explored. T. mentagrophytes growth was suppressed significantly by trichodermin and completely inhibited under 1000 µg/mL trichodermin. The production and germination of T. mentagrophytes spores were remarkably reduced upon exposure to trichodermin, in comparison with control samples. Treatment of lesions caused by T. mentagrophytes on the rabbit skin with 1 mg/mL trichodermin prompted the healing process significantly; however, 20 mg/mL trichodermin was likely toxic to the skin. Under trichodermin treatment, the number of mitochondria in T. mentagrophytes increased significantly, while a few mitochondria-related genes decreased, indicating possible mitochondrial damage. In transcriptome analysis, the GO terms enriched by DEGs in the trichodermin-treated group included carbohydrate metabolic process, integral component of membrane, intrinsic component of membrane, and carbohydrate binding, while the enriched KEGG pathways comprised biosynthesis of secondary metabolites, glycolysis/gluconeogenesis, and carbon metabolism. By comparing the wild type and a gene deletion strain of T. mentagrophytes, we found that CDR1, an ABC transporter encoding gene, was involved in T. mentagrophytes sensitivity to trichodermin.

Safety and Efficacy of the Bordetella bronchiseptica Vaccine Combined with a Vegetable Oil Adjuvant and Multi-Omics Analysis of Its Potential Role in the Protective Response of Rabbits.

Infectious respiratory diseases caused by Bordetella bronchiseptica (Bb) are seriously endangering the development of the rabbit industry in China. Unfortunately, no licensed vaccines are available for this pathogen. The present study was designed to determine whether the inactivated Bb antigen formulated with vegetable oil adjuvant (named E515) which contains soybean oil, vitamin E, and ginseng saponins, functions as a safe and effective vaccine (E515-Bb) against Bb infection in rabbits. Based on local and systemic reactions, both the E515 adjuvant alone and the E515-Bb vaccine exhibited good safety in rabbits. Immune response analysis implies that rabbits immunized with the E515-Bb vaccine produced significantly higher, earlier, and longer-lasting specific antibody responses and activated Th1/Th2/Th17 cell responses than those immunized with the aluminum hydroxide (Alum)-adjuvanted Bb vaccine (Alum-Bb) or Bb antigen alone. Moreover, the E515-Bb vaccine effectively protected rabbits from Bb infection. Additionally, integrated multi-omics analysis revealed that the immunoprotective effect of the E515-Bb vaccine was achieved through upregulation of the complement and coagulation cascades and cell adhesion molecule (CAM) pathways, and the downregulation of the P53 pathway. Overall, these results indicate that the E515-Bb vaccine is safe, elicits an efficient immune response and provides good protection against Bb infection in rabbits. Thus, the E515-adjuvanted Bb vaccine can be considered a promising candidate vaccine for preventing Bb infection.

Outer Membrane Vesicles Coating Nano-Glycyrrhizic Acid Confers Protection Against Borderella bronchiseptica Through Th1/Th2/Th17 Responses.

PURPOSE: Outer membrane vesicles (OMVs) are spherical nano-sized proteolipids secreted by numerous pathogenic Gram-negative bacteria. Due to the immunostimulatory properties and protective efficacy, OMVs have received increasing attention as a candidate for the vaccine to prevent and treat bacterial infections. However, the immune response remains elusive due to the low structural stability and poor size homogeneity of the vesicles. In this study, OMVs were used to coat self-assembled glycyrrhizic acid nanoparticles (GANs) and obtain a stable OMV vaccine. The immunoprotective effects and anti-infection efficacy were evaluated in vivo and in vitro. METHODS: The OMVs were prepared by ultrafiltration method and fused with GAN through mechanical extrusion. The characteristics, including morphology, hydrodynamic size, zeta potential, and stability were evaluated. The in vitro immunological function of GAN-OMV on the macrophages and in vivo immune efficacy and anti-infection effect were examined and compared. RESULTS: The results showed that the GAN-OMV were homogenous with a size of 130 nm and a stable core-shell structure. Micropinocytosis-dependent and clathrin-mediated endocytotic pathways effectively internalized the GAN-OMV into the macrophages and promoted cell proliferation, cytokine secretion, and M1 polarization. Furthermore, subcutaneous GAN-OMV vaccination contributed to significantly higher Borderella bronchiseptica (Bb)-specific antibody production and lymphocyte proliferation. The splenic lymphocytes of mice immunized with GAN-OMVs displayed a higher ratio of CD4+/CD8+ T cells and CD19+ B cells and produced significantly higher levels of Th1/Th2/Th17 cytokines. GAN-OMV also effectively prevented Bb reinfection. CONCLUSION: In this study, GAN-OMV was developed successfully to stimulate Th1/Th2/Th17 immune responses against Bb and provide a promising strategy for novel vaccine development against the microbial pathogen.

Effects of Clostridium butyricum and a Bacteriophage Cocktail on Growth Performance, Serum Biochemistry, Digestive Enzyme Activities, Intestinal Morphology, Immune Responses, and the Intestinal Microbiota in Rabbits.

The objective of this study was to assess the effects of dietary supplementation with Clostridium butyricum (CB) and a bacteriophage cocktail (BP) on growth performance, serum biochemical parameters, intestinal digestive and oxidase enzymes, intestinal morphology, immune responses, and the cecum microbiota in rabbits. In total, 108 New Zealand rabbits (5 weeks old) were randomly and equally allotted into three dietary treatment groups (four replicates per treatment, n = 36/treatment): (1) the control (CN) group-rabbits fed the basal diet; (2) CB group-rabbits fed the basal diet supplemented with 100 mg/kg diet Clostridium butyricum; and (3) BP group-rabbits fed the basal diet supplemented with 200 mg/kg diet BP cocktail, respectively, for 6 weeks. Compared with the CN diet, dietary CB and BP inclusion increased the average daily gain (ADG) and average daily feed intake (ADFI) and decreased the feed/gain (F/G) ratio of rabbits. Furthermore, CB increased the digestive enzyme activity (α-amylase and trypsin in the ileum); the chymotrypsin activity was also significantly increased in the duodenum and jejunum. Supplementation with CB significantly enhanced antioxidant capacity (SOD and GSH-Px) in the jejunum and ileum and reduced MDA levels. Additionally, rabbits fed CB had significantly elevated villus height (V) and (V/C) ratios but reduced crypt depth (C). Moreover, dietary CB supplementation markedly increased the ileal expression of tight junction proteins (occludin, ZO-1, and claudin-1) and increased secretory immunoglobulin A (sIgA) production. High-throughput sequencing indicated that the microbiota in the rabbit intestine was altered by CB and BP. Venn diagrams and heatmap plots revealed that the gut microbial community composition varied obviously among rabbits fed different diets. Specifically, CB increased the relative abundance of beneficial bacteria to maintain intestinal barrier homeostasis, whereas BP decreased the relative abundance of Gammaproteobacteria, which included a plenty of pathogenic bacteria.

An Investigation of the Relationship between Cyniclomyces guttulatus and Rabbit Diarrhoea.

Cyniclomyces guttulatus is usually recognised as an inhabitant of the gastrointestinal (GI) tract in rabbits. However, large numbers of C. guttulatus are often detected in the faeces of diarrhoeic rabbits. The relationship of C. guttulatus with rabbit diarrhoea needs to be clearly identified. In this study, a C. guttulatus Zhejiang strain was isolated from a New Zealand White rabbit with severe diarrhoea and then inoculated into SPF New Zealand white rabbits alone or co-inoculated with Eimeriaintestinalis, another kind of pathogen in rabbits. Our results showed that the optimal culture medium pH and temperature for this yeast were pH 4.5 and 40-42 °C, respectively. The sequence lengths of the 18S and 26S ribosomal DNA fragments were 1559 bp and 632 bp, respectively, and showed 99.8% homology with the 18S ribosomal sequence of the NRRL Y-17561 isolate from dogs and 100% homology with the 26S ribosomal sequence of DPA-CGR1 and CGDPA-GP1 isolates from rabbits and guinea pigs, respectively. In animal experiments, the C. guttulatus Zhejiang strain was not pathogenic to healthy rabbits, even when 1 × 108 vegetative cells were used per rabbit. Surprisingly, rabbits inoculated with yeast showed a slightly better body weight gain and higher food intake. However, SPF rabbits co-inoculated with C. guttulatus and E. intestinalis developed more severe coccidiosis than rabbits inoculated with C. guttulatus or E. intestinalis alone. In addition, we surveyed the prevalence of C. guttulatus in rabbits and found that the positive rate was 83% in Zhejiang Province. In summary, the results indicated that C. guttulatus alone is not pathogenic to healthy rabbits, although might be an opportunistic pathogen when the digestive tract is damaged by other pathogens, such as coccidia.

Deubiquitination of MITF-M Regulates Melanocytes Proliferation and Apoptosis.

Microphthalmia-associated transcription factor-M (MITF-M) is the key gene in the proliferation and differentiation of melanocytes, which undergoes an array of post-translation modifications. As shown in our previous study, deubiquitinase USP13 is directly involved in melanogenesis. However, it is still ambiguous that the effect of USP13-mediated MITF-M expression on melanocytes proliferation and apoptosis. Herein, we found that MITF-M overexpressing melanocytes showed high cell proliferation, reduced apoptosis, and increased melanin levels. Besides, melanin-related genes, TYR, DCT, GPNMB, and PMEL, were significantly up-regulated in MITF-M overexpressing melanocytes. Furthermore, Exogenous USP13 significantly upregulated the endogenous MITF-M protein level, downregulated USP13 significantly inhibited MITF-M protein levels, without altering MITF-M mRNA expression. In addition, USP13 upregulation mitigated the MITF-M degradation and significantly increased the half-life of MITF-M. Also, USP13 stabilized the exogenous MITF protein levels. In conclusion, the MITF-M level was regulated by USP13 deubiquitinase in melanocytes, affecting melanocytes proliferation and apoptosis. This study provides the theoretical basis for coat color transformation that could be useful in the development of the new breed in fur animals.

Assessment of the mechanism of drug resistance in Trichophyton mentagrophytes in response to various substances.

BACKGROUND: Trichophyton mentagrophyte (TM), a zoonotic pathogen, has been endangering public health due to emerging drug resistance. Although increased attention is paid to this issue, there is very limited research available on drug resistance in TM. In this study, we studied the gene and proteomic changes, morphological changes, cellular fat localization, fat content changes, and biofilm of TM treated with different substances. RESULTS: The TM growth curve showed a positive correlation with the concentration of Fenarimol (FE), genistein (GE), clotrimazole (KM), and Miconazole nitrate salt (MK). The morphology of TM cells changed in different degrees after treatment with different substances as observed by TEM and SEM. The results showed that under KM and berberine hydrochloride (BB) treatment, a total of 3305 differentially expressed genes were detected, with the highest number in the KM-treated group (578 up-regulated and 615 down-regulated). A total of 847 proteins and 1850 peptides were identified in TM proteomics. Nile red staining showed that the fat content of TM was significantly higher in the BB-, ethidium bromide- (EB), FE-, KM-, Adriamycin hydrochloride- (YA), and MK-treated group compared to the control group. Results of the biofilm thickness showed that it gradually increased under treatment with specific concentrations of KM or BB, which may be related to the up-regulation of ERG25 and CYP related gene proteins. CONCLUSIONS: It is suggested that in order to effectively deal with dermatomycosis caused by TM, it is necessary to inhibit the expression of ERG25 and CYP related genes and fat metabolism, which can result in the inhibition of the production of biofilm by the fungus and solve the problem of fungal drug resistance in clinical settings.

PEGylated nano-Rehmannia glutinosa polysaccharide induces potent adaptive immunity against Bordetella bronchiseptica.

Vaccines, in many cases, stimulate only too weak immunogenicity to prevent infection. Therefore, adjuvants are required during their preparation to boost the immune response. We herein developed a PEGylated nano-adjuvant based on Rehmannia glutinosa polysaccharide (RGP). The addition of PEG layer exhibits enhanced immune performance of the nano-RGP. Stimulation of dendritic cells (DCs) with PEGylated nano-RGP (pRL) led to increased proliferation and cytokine production (IL-6, IL-12, IL-1ß and TNF-α). The pRL was internalized into DCs via a rapid and efficient method. The mice immunized with pRL exhibited enhanced antigen-specific serum IgG and Th1-(IFN-γ), Th2-(IL-4), and Th17-(IL-17, IL-6) cytokine production, contributing to a good anti-infection performance. Furthermore, the pRL could effectively deliver the antigen to the lymph nodes (LNs), activate DC in the LN and produce enhanced CD4+and CD8+ T-cells-derived memory (CD44high CD62Lhigh), and effector (CD44high CD62Llow) as well as functional phenotypes. Our results revealed that pRL can act as a promising adjuvant with targeted delivery of antigen due to its effective activation and robust adaptive immunity induction of DCs.

Comparative Proteomic Analysis Reveals Complex Responses to Bordetella bronchiseptica Infections in the Spleen of Rabbits.

Bordetella bronchiseptica (B. bronchiseptica) causes a respiratory disease in rabbits. To determine the proteins of B. bronchiseptica in rabbits related to the disease, differentially accumulated proteins in B. bronchiseptica-infected cells are identified by comparative proteomic analysis. Comparative proteomic analysis detects 5814 proteins and quantifies 4854 of these. Fifty eight upregulated and 38 downregulated proteins are identified in spleen tissue after B. bronchiseptica infection of rabbits (both p < 0.05). The significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways are ribosome, biosynthesis of amino acids, biosynthesis of amino acids, protein export, and carbon metabolism etc. (all p < 0.01). Significantly enriched KEGG pathways include 'ocu03010 ribosome' (a); 'ocu00260 glycine, serine threonine metabolism'. Analyses of control and infected spleen cells detect responses to B. bronchiseptica infection. Many differentially affected proteins are evident, and reflect different biological changes and diverse subcellular localizations between control and infected spleen cells. Infection markedly alters the expressions of proteins linked to the serine protease system, with the 'phagosome,' 'biosynthesis of amino acids,' 'glycine, serine threonine metabolism,' 'intestinal immune network for IgA production', and 'amino sugar and nucleotide sugar metabolism' associated with B. bronchiseptica infection. The result will inform studies of responses to B. bronchiseptica infections in rabbits.

KIT is involved in melanocyte proliferation, apoptosis and melanogenesis in the Rex Rabbit.

BACKGROUND: Melanocytes play an extremely important role in the process of skin and coat colors in mammals which is regulated by melanin-related genes. Previous studies have demonstrated that KIT is implicated in the process of determining the color of the coat in Rex rabbits. However, the effect of KIT on the proliferation and apoptosis of melanocytes and melanogenesis has not been clarified. METHODS: The mRNA and protein expression levels of KIT were quantified in different coat colored rabbits by qRT-PCR and a Wes assay. To identify whether KIT functions by regulating of melanogenesis, KIT overexpression and knockdown was conducted in melanocytes, and KIT mRNA expression and melanin-related genes TYR, MITF, PMEL and DCT were quantified by qRT-PCR. To further confirm whether KIT influences melanogenesis in melanocytes, melanin content was quantified using NaOH lysis after overexpression and knockdown of KIT. Melanocyte proliferation was estimated using a CCK-8 assay at 0, 24, 48 and 72 h after transfection, and the rate of apoptosis of melanocytes was measured by fluorescence-activated cell sorting. RESULTS: KITmRNA and protein expression levels were significantly different in the skin of Rex rabbits with different color coats (P < 0.05), the greatest levels observed in those with black skin. The mRNA expression levels of KIT significantly affected the mRNA expression of the pigmentation-related genes TYR, MITF, PMEL and DCT (P < 0.01). Melanin content was evidently regulated by the change in expression patterns of KIT (P < 0.01). In addition, KIT clearly promoted melanocyte proliferation, but inhibited apoptosis. CONCLUSIONS: Our results reveal that KIT is a critical gene in the regulation of melanogenesis, controlling proliferation and apoptosis in melanocytes, providing additional evidence for the mechanism of pigmentation of animal fur.

Analysis of Genome DNA Methylation at Inherited Coat Color Dilutions of Rex Rabbits.

Background: The dilution of color in rabbits is associated with many different genetic mechanisms that form different color groups. A number of previous studies have revealed potential regulatory mechanisms by which epigenetics regulate pigmentation. However, the genome-wide DNA methylation involved in animal coat color dilution remains unknown. Results: We compared genome-wide DNA methylation profiles in Rex rabbit hair follicles in a Chinchilla group (Ch) and a diluted Chinchilla group (DCh) through whole-genome bisulfite sequencing (WGBS). Approximately 3.5% of the cytosine sites were methylated in both groups, of which the CG methylation type was in greatest abundance. In total, we identified 126,405 differentially methylated regions (DMRs) between the two groups, corresponding to 11,459 DMR-associated genes (DMGs). Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that these DMGs were principally involved in developmental pigmentation and Wnt signaling pathways. In addition, two DMRs were randomly selected to verify that the WGBS data were reliable using bisulfite sequencing PCR, and seven DMGs were analyzed to establish the relationship between the level of DNA methylation and mRNA expression using qRT-PCR. Due to the limitation of small sample size, replication of the results with a larger sample size would be important in future studies. Conclusion: These findings provide evidence that there is an association between inherited color dilution and DNA methylation alterations in hair follicles, greatly contributing to our understanding of the epigenetic regulation of rabbit pigmentation.

Optimum Preparation Method for Self-Assembled PEGylation Nano-Adjuvant Based on Rehmannia glutinosa Polysaccharide and Its Immunological Effect on Macrophages.

BACKGROUND: Rehmannia glutinosa polysaccharide is the main reason that contributes to the immunological function of R. glutinosa. Due to its disadvantages in clinical use, here we designed the PEGylation nano-RGP (pRL) to improve the drug-targeting effect and the immunological function. Our present work aims to establish the optimum condition of preparing the pRL and to investigate its immunological function on macrophages. METHODS: pRL was prepared by thin film hydration method combined with ultra-sonication technique. And its preparation conditions were optimized with response surface methodology. Also, the lyophilization method was optimized. The characteristics of the pRL were evaluated, including particle size, drug loading, encapsulation efficiency and morphology. The immunological function of pRL on macrophage was investigated through CCK-8 test, ELISA and flow cytometry. RESULTS: The lipid-to-cholesterol molar ratio of 8:1, the addition of DSPE-PEG2000 of 9% and the lipid-to-drug ratio of 5.4:1 were the optimum preparation technology for pRL. The encapsulation efficiency (EE) of pRL under this preparation technology was 95.81±1.58%, with a particle size of 31.98 ± 2.6 nm. The lactose-to-lipid ratio (2:1) was the optimal lyophilization method. pRL promoted macrophage proliferation, which is significantly better than that of nano-RGP without PEGylation (RL). pRL-stimulated RAW264.7 cells showed a high secretion of pro-inflammatory cytokines, which is the characteristic indicator of M1 polarization. Enhanced cellular uptake through macropinocytosis-dependent and caveolae-mediated endocytosis was observed in pRL-treated RAW264.7 cells. CONCLUSION: Our study concluded that PEGylation effectively overcame the poor targeting effect of Rehmannia glutinosa polysaccharide (RGP) and significantly improved the immunological profile of its nano-formulation, which suggested that pRL could serve as an immune adjuvant in clinical application.

Effects of Trichophyton mentagrophytes infection on the immune response of rabbits.

BACKGROUND: Rabbit breeding has developed into a large-scale industry, and as such, the incidence of dermatophytosis in rabbits has become increasingly common. A rabbit model with Trichophyton mentagrophytes infection was established to study the changes within the immune responses after fungal infection. METHODS: After the T. mentagrophytes challenge on skin, pathogens on the skin were isolated from the rabbits in the fungal infection (FI) groups 20 days. Fungal observation under microscope were carried out. Identification of strains was achieved by polymerase chain reaction (PCR) using the CDR1 gene. The collected anticoagulant blood samples were analyzed for various blood cell parameters. The levels of antibodies, including IgM and IgA, cytokines, including IL-2, IL-6, and macrophage colony-stimulating factor (M-CSF), and soluble CD4 and CD8 in the serum of the FI group vs. the control group were determined independently. RNA isolation from blood samples and fluorescence-based quantitative PCR were carried out for the mRNA level of M-csf 20 days after fungal challenge. RESULTS: Our model resulted in typical symptoms of dermatophytosis on rabbit skin after challenged with fungus. Pathogens isolated from the infected rabbit skin were confirmed to be T. mentagrophytes by microscopic examination and PCR. The number of lymphocytes in the blood of the FI group was significantly decreased in comparison to the control group 2 days after the fungal challenge, but was significantly increased in comparison the control group 10 days after the fungal challenge (P < 0.01). Platelet counts of the FI group were significantly higher than in the control group at 2 (P < 0.05), 10 (P < 0.05), and 20 (P < 0.01) days after fungal challenge. The red blood cell distribution width of the FI group was significantly increased in comparison to that of the control group at 2, 10, and 20 days after fungal challenge (P < 0.01 for all days). The levels of antibodies (immunoglobulin (Ig) M and IgA (P < 0.01)), cytokines (interleukin (IL)-6 (P < 0.01), macrophage colony-stimulating factor (M-CSF) (P < 0.05)), and soluble CD4 (P < 0.01) and CD8 (P < 0.01) in the serum were significantly different between the FI and control groups. Serum M-csf mRNA level of the FI group was significantly higher than the control group 20 days after fungal challenge (P < 0.01). CONCLUSIONS: This study demonstrates how the immune system responds to infection with T. mentagrophytes and provides potential targets for the prevention and treatment of dermatophytosis.

Characterization and Establishment of an Immortalized Rabbit Melanocyte Cell Line Using the SV40 Large T Antigen.

Melanocytes (MCs) are specialized cells that synthesize melanin within the melanosome. Cultured MCs are useful in order to study their role in relation to pigmentation. However, MC isolation is laborious and the obtained cells have a limited culture time. In this study, we transformed lentivirus-mediated simian virus 40 Large T (SV40-LT) into primary rabbit melanocytes (Pri RMCs) to establish an immortalized cell line. Morphologically, the immortalized RMCs (Im RMC) were indistinguishable from the Pri RMCs, and dendrites were visible following Dopa staining. No significant differences in cell proliferation or growth between immortalized and primary RMCs were observed. Based on melanocyte-specific markers, the expression of MITF, TYR, and TYRP1 were detected by PCR, immunofluorescence staining, and western blot analysis. Through karyotype, soft agar, and tumorigenesis assays, the immortalized RMCs did not undergo malignant transformation. Our results show that Im RMCs can be used as a tool cell for future MC studies on the pigmentation mechanisms of fur animals.