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.     

用免疫荧光、酶标技术检测赭曲霉毒素A的研究

该项研究采用本课题组提纯的赭曲霉毒素A(Ochratoxin A·简称OA)和研制的兔抗OA抗体等,进行间接荧光法和ELISA间接法检测OA的试验。通过对不同浓度的OA纯品和含毒饲料浸提液及中毒死亡鸡内脏的间接荧光染色,均能检出分布均匀、大小有别的黄绿色荧光亮点;用OA为被检抗原和兔抗OA抗体与羊抗兔IgG-HRP进行ELISA间接法试验,对含不同浓度OA的试验孔和对照孔的检测,结果均正确稳定。研究结果证明,以上两种检测方法检测OA均具有灵敏、特异、快速等优点。     

狗枣猕猴桃果实软化过程中阶段性专一酶的研究

狗枣猕猴桃果实采后软化过程分两个阶段，第一阶段软化较快，起主要作用的阶段性专一酶是淀粉酶；第二阶段软化较慢，起主要作用的阶段性专一酶是多聚半乳糖醛酸酶。乙烯释放对果实软化有促进作用；保护性酶（过氧化物酶。过氧化氢酶）出现在果实软化后期，因而不是果实软化的阶段性专一酶。     

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.     

Investigations on the Occurrence of Hereditary Diseases in the Danish Cattle Population 1989-1991

Agerholm, J.S., A. Basse and K. Christensen: Investigations on the occurrence of hereditary diseases in the Danish cattle population 1989-1991. Acta vet. scand. 1993, 34, 245-253.– The methods of the Danish Bovine Genetic Disease Programme are outlined, and the results obtained during the first 3 years in function are described. The most common disease reported was spinal muscular atrophy in calves of the Red Danish Dairy breed with 312 reports. Necropsy was performed on 162 cases, and spinal muscular atrophy was diagnosed in 82 of these. Bovine progressive degenerative mye-loencephalopathy, rectovaginal constriction, syndrome of arthrogryposis and palatoschisis, hereditary chondrodysplasia (2 different types), syndactylism, epitheliogenesis imperfecta, and osteogenesis imperfecta was diagnosed with 1 case each. Lethal trait A46 was diagnosed in 4 calves. Some of these diseases have not previously been described in Denmark, and epitheliogenesis imperfecta was for the first time diagnosed in the Hereford breed. Chromosome translocation 1/29 was detected in the Blonde d’Aquitaine (BAQ), Limousine, and Red Danish Dairy breed. The aberration occurred frequently in BAQ. Furthermore, a complex chromosome translocation t(l;8;9)(q45;ql3;q26) was detected in the Red Danish Dairy breed.     

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。