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.     

Construction of recombinant capripoxviruses as vaccine vectors for delivering foreign antigens: Methodology and application

Goatpox (GTP), sheeppox (SPP) and lumpy skin disease (LSD) are three severe diseases of goat, sheep and cattle. Their typical clinical symptoms are characterized by vesicles, papules, nodules, pustules and scabs on animal skins. The GTP, SPP and LSD are caused by goatpox virus (GTPV), sheeppox virus (SPPV) and lumpy skin disease virus (LSDV), respectively, all of which belong to the genus Capripoxvirus in the family Poxviridae. Several capripoxvirus (CaPV) isolates have been virulently attenuated through serial passaging in vitro for production of live vaccines. CaPV-based vector systems have been broadly used to construct recombinant vaccines for delivering foreign antigens, many of which have been demonstrated to induce effective immune protections. Homologous recombination is the most commonly used method for constructing recombinant CaPVs. Here, we described a methodology for generation of recombinant CaPVs by the homologous recombination, and further reviewed CaPV-vectored vaccines for delivering foreign antigens.     

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。     

鸡沙门氏菌弱毒冻干苗的研制

由减毒鸡沙门氏苗97A疫苗株作为制苗用菌种，经普通琼脂培养，冷冻真空干燥等工艺生产鸡沙门氏菌弱毒疫苗4批，每批疫苗分别以免疫剂量接种6日龄AA肉鸡，免疫第14天时，用强毒株Sg9和Sp4攻击，免疫组死亡保护率均达90％以上，试验结果表明，该苗具有良好的安全性和免疫效果。     

怀孕后期感染PRRS病毒母猪所产新生仔猪的免疫反应

将PRRS病毒BJ－4株感染怀孕后期（约90天）的抗体阴性和阳性母猪，待自然分娩后，观察新生仔猪的免疫应答。结果显示，接种PRRS病毒BJ－4的母猪没有表现出明显的临床症状，没有出现流产死产。新生仔猪浦被子前血清中PRRS病毒核酸TR－PCR检测和ELISA抗体阴性，哺乳后特异性抗体出现，5－6周母源抗体逐渐下降；20日龄猪瘟疫苗免疫后疫苗抗体维持时间短，仔猪在40日龄后进入野毒感染的危险期。RT－nested PCR检测血清中PRRS病毒核酸和易感仔猪病毒特异性抗体监测的结果提示仔猪群内可能存在水平传播。流式细胞术检测外周血淋巴细胞亚群发现CD3^ 细胞减少，CD4^ 细胞显著下降，CD8^ ，CD4^ CD8^ 和SLA－DR^ 表达细胞升高。以上结果表明在感染后病毒能够长期持续性存在，猪场内新生仔猪母源抗体逐渐下降后，通过水平传播受到感染，感染后免疫应答受到不利影响。     

鸡传染性法氏囊病继发大肠杆菌感染的诊断研究

笔者饲养的200多只蛋鸡用种公鸡于50日龄左右突然发病，主要症状为食欲剧减甚至废绝，羽毛逆立，缩颈嗜眠。倒提时口内流出大量黏液状液体，排恶臭白色或微黄带泡沫稀粪，呈水样。发病率达80％，死亡率达32％。剖检可见部分鸡胸肌和腿肌出血，法氏囊水肿出血，个别鸡法氏囊内有黄色较硬的球状分泌物，有的鸡有心包炎和肝周炎，肠道、脾脏和肾脏有出血点。综合以上症状和实验室病毒学和细菌学检测，确诊为传染性法氏囊病继发大肠杆菌感染。