Niubie is derived from a fresh filtrate of semi-digested rumen contents from herb-fed cattle and serves as the distinctive hotpot base in Southwest China. The interplay of ingested herbs and ruminal microbiota shapes its distinctive flavor and functional compound profile. Nonetheless, its quality deteriorates during cold storage, and the underlying molecular mechanisms remain unclear. This study presents an integrated metagenomic and metabolomic framework for dissecting covert spoilage in refrigerated Niubie. Results showed that key physicochemical parameters remained stable during storage. Metagenomic analysis revealed a staged microbial succession. Specifically, genera with lipolytic and putrefactive potential (e.g., Anaerovibrio, Clostridium) increased from day 3 onward. This shift was accompanied by a corresponding rise in the abundance of virulence factor and antibiotic resistance genes. Subsequently, classic spoilage bacteria Pseudomonas and opportunistic pathogens Streptococcus became markedly enriched by day 7. Metabolomic profiling further showed that most beneficial functional metabolites declined, whereas lipid degradation pathways were upregulated, resulting in the accumulation of lipid oxidation markers and biogenic amines. Spearman correlation analysis linked methanogenic archaea, lipolytic bacteria, and amino acid degraders to spoilage metabolites, revealing convergent deterioration networks. These findings demonstrate that, despite stable conventional quality indices, critical microbial and metabolic deterioration commenced as early as day 3 of cold storage, offering molecular-level insights for the early detection and targeted preservation of Niubie.

Mycoplasma synoviae (MS) stands as a pivotal pathogen, responsible for triggering arthritis and airsacculitis in both chickens and turkeys. Given the pressing need for safe and efficacious vaccine candidates, we engineered recombinant adenoviruses expressing a fusion antigen. This antigen consisted of the pyruvate dehydrogenase E1 subunit beta (pdhβ) and dihydrolipoyl dehydrogenase (pdhD) of MS. We then systematically evaluated the immune effect and protective efficacy of these recombinant adenoviruses against MS challenge in a chicken model. Our results demonstrated the successful construction of recombinant adenoviruses rAd-pdhβ, rAd-pdhD, and rAd-pdhβ-pdhD. The pdhβ, pdhD, and pdhβ-pdhD proteins were efficiently expressed in cells infected with the respective recombinant adenoviruses. Animal experiments further revealed that vaccination with recombinant adenoviruses rAd-pdhβ, rAd-pdhD, and rAd-pdhβ-pdhD elicited significant specific humoral and cellular immune responses (P < 0.05). Notably, rAd-pdhβ-pdhD exhibited superior immunogenicity compared to rAd-pdhβ and rAd-pdhD. Moreover, all three recombinant adenovirus vaccine candidates conferred partial protection to chickens against MS challenge. They effectively alleviated MS-induced footpad and joint swelling, as well as inflammation. Among them, rAd-pdhβ-pdhD demonstrated a better protective effect. In conclusion, vaccination with recombinant adenoviruses rAd-pdhβ, rAd-pdhD, and rAd-pdhβ-pdhD can evoke immune responses and provide partial protection against MS in chickens. In particular, rAd-pdhβ-pdhD holds greater potential as a vaccine candidate against MS.

为了快速、准确检测鸡滑液囊支原体(MS),对MS标准株vlhA基因进行分析,设计并合成特异性引物和探针,优化反应条件,构建了基于vlhA基因的TaqMan实时荧光定量PCR快速检测方法.结果显示,所构建方法标准曲线所对应的相关系数R2=1.000,斜率S=-3.452,截距I=43.950,表明标准质粒浓度与Ct值的线性关系良好;该方法除MS外,其他参考菌(毒)株均无目的基因扩增,其最小检测浓度为1×101copies/μL,组内重复及组间重复的变异系数均低于0.1;运用所建立方法与普通PCR方法对4份疑似MS临床样本进行检测,检测结果完全相符合;应用该方法对贵州省7个地区107份疑似MS样本进行检测,发现总体阳性率为26.17％.说明建立的方法具有较好的特异性、敏感性和重复性,可应用于临床检测.

The full length of PstS gene was firstly amplified by SOE-PCR and cloned into expression vector pColdⅠ. Then the vector pCold-PstS was transformed into BL21（DE3） to express the fusion protein PstS by IPTG induction, based on which polyclonal rabbit anti-serum was generated by immunizing New Zealand rabbit with the purified PstS protein. The total protein, membrane protein and cytoplasmic protein of M.gallisepticum were extracted to analyze the distribution of PstS by Western blot with the rabbit anti-serum. The PstS gene was 1 065 bp in length, and its encoded protein had a transmembrane region between amino acid 9 and 31. The recombinant protein PstS obtained by the induced expression of pCold-PstS was about 43 ku in size and had good antigenicity. The PstS existed on the membrane and in the cytoplasm of M.gallisepticum. To summarize, PstS was an immunogenic protein on the membrane of M.gallisepticum, which provided theoretical support for further research on its biological function and genetic engineering vaccine.

MG-132, an aldehyde-based peptide proteasome inhibitor (PI) that binds to the proteasome and reversibly inhibits proteasome activity, has been widely used in experimental research. However, it is not clear whether MG-132 has anti-inflammatory effects on liver injury. The molecular mechanism of the anti-inflammatory effect of the PI MG-132 on Con A-induced acute liver injury (ALI) mice was investigated by ELISA, HE, q RT-PCR, and IHC. The results showed that the serum activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and TNF-α and IL-6 contents of mice in the high and medium dose groups were reduced compared with those in the ALI group. The superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels in liver tissues were significantly increased, and the malondialdehyde (MDA) content was decreased. The pathological sections of mice in the ALI group showed typical ALI manifestations such as significant central venous stasis of liver tissues, cell swelling, and inflammatory cell infiltration. The pathological damage of liver tissues was relieved significantly in the three dose groups, especially in the high-dose group. The transcriptional level of TLR4/NF-κB pathway key factors mRNA was significantly reduced, and the expression of TLR4 and NF-κB P65 protein in liver tissues was significantly and positively correlated with the contents of TNF-α and IL-1β (p < 0.01). Our findings suggest that MG-132 can alleviate the inflammatory response to Con A-induced ALI and exert a hepatoprotective effect, and its anti-inflammatory effect is related to the inhibition of TLR4/NF-κB signaling pathway activation.

Dried distillers’ grains with solubles (DDGS) are rich in nutrients, and partially alternative feeding of DDGS effectively reduces cost of feed and improves animals’ growth. We used 16S rDNA gene sequencing and LC/MS-based metabolomics to explore the effect of feeding cattle with a basal diet (BD) and a Jiang-flavor DDGS diet (replaces 25% concentrate of the diet) on microbiome and metabolome of ruminal and cecal contents in Guanling yellow cattle. The results showed that the ruminal and cecal contents shared the same dominance of Bacteroidetes , Firmicutes and Proteobacteria in two groups. The ruminal dominant genera were Prevotella_1 , Rikenellaceae_RC9_gut_group , and Ruminococcaceae_UCG-010 ; and the cecal dominant genera were Ruminococcaceae_UCG-005 , Ruminococcaceae_UCG-010 , and Rikenellaceae_RC9_gut_group . Linear discriminant analysis effect size analysis (LDA > 2, P < 0.05) revealed the significantly differential bacteria enriched in the DDGS group, including Ruminococcaceae_UCG_012 , Prevotellaceae_UCG_004 and Anaerococcus in the ruminal contents, which was associated with degradation of plant polysaccharides. Besides, Anaerosporobacter , Anaerovibrio , and Caproiciproducens in the cecal contents were involved in fatty acid metabolism. Compared with the BD group, 20 significantly different metabolites obtained in the ruminal contents of DDGS group were down-regulated ( P < 0.05), and based on them, 4 significantly different metabolic pathways ( P < 0.05) were enriched including “Linoleic acid metabolism,” “Biosynthesis of unsaturated fatty acids,” “Taste transduction,” and “Carbohydrate digestion and absorption.” There were 65 significantly different metabolites (47 were upregulated, 18 were downregulated) in the cecal contents of DDGS group when compared with the BD group, and 4 significantly different metabolic pathways ( P < 0.05) were enriched including “Longevity regulating pathway,” “Bile secretion,” “Choline metabolism in cancer,” and “HIF-1 signaling pathway.” Spearman analysis revealed close negative relationships between the top 20 significantly differential metabolites and Anaerococcus in the ruminal contents. Bacteria with high relevance to cecal differential metabolites were Erysipelotrichaceae_UCG-003 , Dielma , and Solobacterium that affect specific metabolic pathways in cattle. Collectively, our results suggest that feeding cattle with a DDGS diet improves the microbial structure and the metabolic patterns of lipids and carbohydrates, thus contributing to the utilization efficiency of nutrients and physical health to some extent. Our findings will provide scientific reference for the utilization of DDGS as feed in cattle industry.

为了研究鸡滑液囊支原体(Mycoplasma synoviae,MS)膜蛋白EF-Tu和pdhβ的免疫应答效果,试验针对目的基因设计特异性引物,经PCR扩增并构建原核表达载体,应用相关软件对其核苷酸和氨基酸序列进行生物信息学分析,对目的蛋白进行诱导表达并进行动物免疫试验.结果表明:PCR扩增EF-Tu和pdhβ基因得到特异性条带,大小为1 185 bp和993 bp;成功构建了重组质粒pET32a-EF-Tu和pET32a-pdhβ;滑液囊支原体贵州分离株(MS-GZ-ZJ株)同其他流行株的EF-Tu基因同源性在99.7％～100％之间,pdhβ基因同源性在99.6％～100％之间,说明MS-GZ-ZJ株EF-Tu、pdhβ基因高度保守;EF-Tu、pdhβ蛋白的分子质量分别为43 ku和35 ku,是稳定的亲水蛋白;EF-Tu蛋白二级结构以α-螺旋、无规则卷曲和延伸链为主,pdhβ蛋白以α-螺旋和无规则卷曲为主.表达的蛋白大小与预期相符,EF-Tu蛋白主要为可溶性表达,pdhβ蛋白主要为包涵体表达;EF-Tu、pdhβ重组蛋白均能与MS阳性血清特异性结合.EF-Tu重组蛋白不能单独诱导细胞免疫应答,而pdhβ重组蛋白能刺激鸡群产生少量的白细胞介素4(IL-4),两个重组蛋白混合能诱导少量的IL-2和IL-4,但比疫苗组低很多;体液免疫应答效果不明确.说明单独使用经预测免疫原性好的蛋白质不一定能诱导动物机体产生良好的反应原性.

为查明贵州省惠水县某养殖场鸡发病死亡的原因,对送检鸡进行剖检和病理学观察,并应用qRT-PCR方法对病原核酸进行检测.结果:病(死)鸡肝脏肿大,有明显粟粒状细胞瘤增生,均匀分布于肝实质中,呈灰白色;组织病理切片观察发现大量圆形嗜酸性颗粒的髓细胞样瘤细胞和广泛的肝细胞空泡变性;病原核酸检测显示禽白血病病毒阳性,确诊为禽白血病病毒感染.

为研究p24蛋白在鸡毒支原体中的定位,对p24基因序列进行分析,PCR扩增p24基因并构建原核表达载体,表达p24蛋白,制备多克隆兔抗血清,ELISA测定其效价,提取鸡毒支原体总蛋白、膜蛋白、胞浆蛋白通过Western blot进行亚细胞定位.结果显示:p24基因高度保守,全长576 bp,编码192个氨基酸;成功构建表达载体pCold-p24,经转化、诱导表达后获得重组蛋白rp24,大小约为35kDa;ELISA检测兔抗rp24血清效价为1:12800,说明rp24蛋白具有很好的免疫原性;Western blot显示rp24蛋白在膜蛋白免疫印迹条带较亮,在胞浆蛋白中条带弱,证明p24主要分布在膜表面,是鸡毒支原体的膜蛋白.试验结果为后续基因工程疫苗研制、诊断试剂盒研发、致病机理研究奠定基础.

[目的]获得鸡滑液囊支原体(Mycoplasma synoviae,MS)贵州流行株,以进一步开展其病原学及免疫学相关研究.[方法]从贵州省织金县某肉鸡场共采集107份疑似MS感染病鸡的咽拭子、肿胀关节和脚垫、肿胀组织渗出物进行PCR检测,对PCR筛选的阳性组织样本进行MS分离培养,并进行瑞氏染色、生化试验及VlhA基因序列分析.[结果]PCR检测筛选到20份阳性样本,从关节和爪垫阳性样本中分离到1株疑似MS,分离株在平板上生长出"煎蛋状"菌落;瑞氏染色可见蓝色细小的球形菌;生化试验中,分离株分解葡萄糖和麦芽糖,但不分解精氨酸、尿素等,与MS标准株生化结果一致;分离培养物经PCR扩增得到821 bp的目的片段;分离株VlhA基因核苷酸相似性分析表明该分离株为MS,与GenBank登录的国内其他地区流行株相似性有地域差异.与安徽、重庆、福建、广东、广西、湖北、湖南、江苏和云南等地区流行株相似性在90.3％～99.7％之间;该分离株与湖南MS流行株相似性最高,达到99.7％;但与国外参考流行株的相似性低;VlhA基因遗传进化树分析显示,该分离株与国内参考株亲缘关系较近,与国外参考株亲缘关系较远.[结论]本研究成功从引起关节和足垫肿胀的鸡中分离鉴定了1株MS贵州流行株,该分离株存在一定变异,为贵州省MS的分子生物特征及本病的诊断和防控研究提供了基础依据.

为查明贵州省铜仁市某蛋鸡场11月龄蛋鸡发病死亡的原因,采集病死鸡病料进行细菌分离培养,以及禽流感病毒、新城疫病毒、鸡安卡拉病毒、鸡毒支原体的核酸检测.结果:病料中未分离出细菌;鸡安卡拉病毒和鸡毒支原体核酸检测均为阳性,禽流感病毒、新城疫病毒核酸检测为阴性.结论:鸡场病例存在鸡毒支原体与鸡安卡拉病毒混合感染.

为查明贵州省贵定县某养殖场猪群发病的原因,分别采集2头病猪的肺脏、肝脏、扁桃体、淋巴结、脾脏、肾脏组织进行细菌分离培养及猪瘟病毒、猪繁殖与呼吸综合征病毒、支原体的核酸检测.结果:无细菌感染情况;猪瘟病毒、猪繁殖与呼吸综合征病毒、支原体核酸检测均为阳性.结论:养殖场病例存在猪瘟病毒、猪繁殖与呼吸综合征病毒、支原体混合感染.

为查明贵州省龙里县某养殖场猪发病的原因,采集5头病死猪病料进行细菌分离培养鉴定,并进行猪繁殖与呼吸综合征病毒、猪伪狂犬病病毒、猪圆环病毒2型、弓形虫的核酸检测.结果:5头病死猪病料均未分离出细菌;3头病死猪组织样品的猪繁殖与呼吸综合征病毒核酸检测为阳性;5头病死猪组织样品的猪圆环病毒2型核酸检测为阳性;2头病死猪组织样品的弓形虫核酸检测为阳性;所有样品的猪伪狂犬病毒核酸检测均为阴性.结论:猪场病例存在猪繁殖与呼吸综合征病毒、圆环病毒2型、弓形虫混合感染,需要加强猪群的免疫防控和净化.