Characterization of bovine ileal epithelial cell line for lectin binding,susceptibility to enteric pathogens,and TLR mediated immune responses

In this study, primary and immortalized bovine intestinal epithelial cells (BIECs) were characterized for the expression of surface carbohydrate moieties. Primary BIEC-c4 cells showed staining greater than 90 % for 16 lectins but less than 50 % staining for four lectins. Immortalized BIECs showed significantly different lectin binding profile for few lectins compared to BIEC-c4 cells. BIEC-c4 cells were studied for infectivity to E. coli, Salmonella enterica, bovine rotavirus, bovine coronavirus, and bovine viral diarrhea virus. Bovine strain E. coli B41 adhered to BIEC-c4 cells and Salmonella strains S. Dublin and S. Mbandaka showed strong cell invasion. BIEC-c4 cells were susceptible to bovine rotavirus. LPS stimulation upregulated IL-10, IL-8, and IL-6 expression and Poly I:C upregulated TLR 8 and TLR 9 expression. This study provides important knowledge on the glycoconjugate expression profile of primary and immortalized BIECs and infectivity and immune responses of primary BIECs to bacterial and viral pathogens or ligands.     

Replication of neurovirulent equine herpesvirus type 1 (EHV-1) in CD172a+ monocytic cells

Equine herpesvirus type 1 (EHV-1) is responsible for respiratory disorders, abortion and myeloencephalopathy (EHM) in horses. Two pathotypes of EHV-1 strains are circulating in the field: neurovirulent (N) and non-neurovirulent (NN). For both strains, CD172a⁺ monocytic cells are one of the main carrier cells of EHV-1 during primary infection, allowing the virus to invade the horse’s body. Recently, we showed that EHV-1 NN strains showed a restricted and delayed replication in CD172a⁺ cells. Here we characterize the in vitro replication kinetics of two EHV-1 N strains in CD172a⁺ cells and investigate if the replication of these strains is similarly silenced as shown for EHV-1 NN strains. We found that EHV-1 N replication was restricted to 7–8% in CD172a⁺ cells compared to 100% in control RK-13 cells. EHV-1 N replication was not delayed in CD172a⁺ cells but virus production was significant lower (10^3.0 TCID₅₀/10⁵ inoculated cells) than in RK-13 cells (10^8.5 TCID₅₀/10⁵ inoculated cells). Approximately 0.04% of CD172a⁺ cells produced and transmitted infectious EHV-1 to neighbour cells compared to 65% of RK-13 cells. Unlike what we observed for the NN strain, pretreatment of CD172a⁺ cells with histone deacetylases inhibitors (HDACi) did not influence the replication of EHV-1 N strains in these cells. Overall, these results show that the EHV-1 replication of N strains in CD172a⁺ cells differs from that observed for NN strains, which may contribute to their different pathogeneses in vivo.     

Comparative effect of advanced soy products or corn protein concentrate with porcine meal on growth,body composition,and distal intestine histology of Florida pompano,Trachinotus carolinus

The present study was designed to investigate the effects of diets containing advanced soy products (enzyme‐treated soy and fermented soy) or corn protein concentrate (CPC) in combination with porcine meal (PM) to completely replace poultry byproduct meal (PBM) on growth performance, body composition, and distal intestine histology of Florida pompano, Trachinotus carolinus. Four experimental diets were formulated to be isonitrogenous and isolipidic, to contain 400 g/kg crude protein and 80 g/kg lipid. A reference diet (PBM diet [PBMD]) contained 150 g/kg PBM and 495 g/kg soybean meal (SBM), and three test diets were formulated replacing PBM with 15 g/kg of CPC (CPC diet [CPCD]) or replacing all SBM and PBM with 535 g/kg fermented soy (fermented soybean meal diet [FSBMD]) or 451.3 g/kg enzyme‐treated soy (enzyme‐treated soybean meal diet [ESBMD]). All three test diets were supplemented with 38 g/kg of PM. Diets were fed based on a percentage of bodyweight adjusted after sampling the fish every 2 weeks to triplicate groups of Florida pompano juveniles (mean weight 8.06 ± 0.22 g). After 8 weeks of feeding, fish fed CPCD and ESBMD performed equally well in terms of final body weight, thermal growth coefficient, and percentage weight gain in comparison to fish fed PBMD. In all cases, feeding FSBMD resulted in poor feed conversion and lower feed intake compared to other treatments. Protein retention efficiency, whole‐body proximate composition, phosphorus, sulfur, potassium, magnesium, calcium, sodium, and zinc contents were not significantly influenced by the dietary treatments. The results obtained in the present histological study showed no significant differences in the thickness of serous layer, muscular layer, and submucosal layer of the intestine among treatments. Fish fed CPCD showed a significant widening of the lamina propria with an increase of cellular infiltration and higher presence of goblet cells compared to other dietary treatment. Based on these results, 451 g/kg ESBM or combination of 150 g/kg of CPC and 495 g/kg SBM supplemented with 38 g/kg PM can be utilized to develop a practical diet for juvenile Florida pompano without impacting growth, nutritive parameters, and several distal intestine health parameters.     

铜对生长猪肝中IGF—Ⅰ基因mRNA表达的影响

采用生长试验、免疫放射分析和定量PCR方法，观察了生长猪血液中类胰岛素生长因子（IGF—Ⅰ）及其结合蛋白（IGFBP3）数量的变化和IGF—Ⅰ基因在肝中表达量的变化。结果显示，每1kg饲料中添加125-250mg铜，能够上调IGF-Ⅰ基因的表达量，显著提高猪的平均日增重，促进生长猪循环血液中IGF—Ⅰ浓度的增加。试验结果证实，铜是通过促进生长激素一胰岛素样生长因子轴相关因子的合成和分泌来发挥促生长作用的。     

中国巴马小型猪内源性反转录病毒的检测

目的 :对我国特有巴马小型猪内源性反转录病毒 ( porcine endogenous retrovirus,PERV)的存在与 m RNA的表达情况进行检测 ,了解巴马小型猪内源性反转录病毒的携带情况。方法 :根据以往建立的 PCR、RT-PCR检测方法 ,对来自于巴马小型猪外周血淋巴细胞的 DNA和RNA样品进行 PERV核心蛋白基因 ( gag)、多聚酶基因 ( pol)及囊膜基因 ( env)的存在与表达进行检测 ;同时 ,根据目前通用的 env基因分型方法检测 PERV env-A、env-B、env-C的存在与表达。结果 :在 1 2个被检的 DNA样品中均检出了PERV特异性 DNA的存在 ;同样 ,在 1 2个被检的 RNA样品中均有 PERV特异性 RNA的表达 ,且所表达的 PERV均为 A型和 B型 ;其中有9个 DNA样品检测出 PERV-C型的存在 ,所有样品中均未检出 C型 PERV的表达。结论 :检测结果表明 1 2个被检巴马小型猪基因组中存在着内源性反转录病毒序列 ,且能以 m RNA的形式表达 ,这一结果为我国特有小型猪的开发、利用及其病毒安全性评价奠定了基础。     

牛皮肤成纤维细胞的体外培养与冻存

利用牛皮肤组织块直接培养法，得到牛皮肤细胞的原代培养物，再用酶消化法和反复贴壁法处理，能够纯化成纤维细胞。成纤维细胞的冻存是通过选用6种分别含有二甲基亚砜（DMSO）、甘油（GL）及乙二醇（EG）的保护液，以相同的冻前处理方法，对牛皮肤成纤维细胞进行缓慢冷冻，冰箱预冷平衡1－2h，逐步投入液氮（－196℃）中保存，再经37℃水浴解冻，Hanks液脱保护剂，以贴壁率评价冻存效果。结果表明，20％DMSO保护液对牛皮肤成纤维细胞表现出较好且稳定的冷冻保护效果，其平均贴壁率达87．9％。     

Feline Ubiquitin Fusion Protein Genes

Using cDNA from a CRFK cell line as a template, PCR amplification was performed with the Ub1S and poly(dT) primers to isolate feline ubiquitin genes. Sequencing of the 495 bp PCR fragment revealed that the putative amino acids induced by this fragment gave a fusion protein consisting of a ubiquitin polypeptide (76 amino acids) and an extension protein of ribosomal proteins L40 (52 amino acids). The putative amino acid sequence of ubiquitin was identical to those of humans, rats and pigs.The recombinant glutathione S-transferase (GST)–feline ubiquitin fusion proteins were produced in Escherichia coli and purified. The fusion proteins had a molecular weight of about 42 kDa and were detected by immunoblot assay with rabbit anti-ubiquitin antiserum.The mRNAs from heat-shocked and non-heat-shocked cells were subjected to RT-PCR (Ub1S and poly(dT) primers) analysis. The molecular weights of the ubiquitinated proteins in heat-shocked CFRK cells were between 18 kDa and 24 kDa by immunoblot assay.These results suggested that there were more ubiquinated proteins in the heat-shocked CRFK cells than in the pre-heat-shocked cells.     

桑悬浮细胞原生质体培养的研究

采用继代培养三个月后的桑子叶悬浮细胞为材料,进行了原生质体的分离和培养。桑悬浮细胞在纤维素酶、果胶酶、半纤维素酶的混合溶液中,酶解获得产量高、活力强的原生质体。原生质体在K8p(附加6—BA、NAA、2,4—D、LH)液体培养基中,再生细胞经多次分裂,得到肉眼可见的小愈伤组织。再通过增殖继代培养,获得浅黄色、具有明显颗粒结构的愈伤组织,转至各种激素含量的MSB固体培养基中,尚未获得绿苗分化。     

猪血免疫球蛋白的热稳定性及动力学研究

为了使猪血免疫球蛋白在各类食品中得到较好的应用 ,对猪血免疫球蛋白 (PIgG)的热稳定性及热变性动力学进行了分析研究。结果表明 ,PIgG在 6 0℃以下很稳定 ,6 0～ 6 5℃比较稳定 ,大于 70℃变性明显加快 ,超高温杀菌 (12 0℃ ,4s)条件下 ,PIgG几乎失活 ;其热动力学反应为 1 2级 ;在 6 0 ,6 5 ,70 ,75 ,80 ,85和 90℃ ,PIgG热变性的D值分别为 33 333× 10 3 ,14 2 86× 10 3 ,3 333× 10 3 ,0 4 76× 10 3 ,0 2 2 2× 10 3 ,0 16 9× 10 3 和 0 0 76× 10 3 s ,在此温度范围内的Z值为 10 81℃ ,表观活化能为 2 2 7 31kJ·mol-1。