Inhibition of chicken leukocyte migration in vitro: A direct agarose plate assay

Leukocyte migration inhibition (LMI) is a widely used in vitro correlate of delayed type hypersensitivity (DTH) in mammals. This report describes the development of a direct agarose LMI assay for studying DTH in avian species. Optimum demonstration of LMI was found with leukocytes isolated on a Ficoll-diatrizoate gradient solution. The agarose culture plates were maintained at pH 7.2–7.4 in a water-vapor saturated, 39°C incubator with 2% CO₂ tension.

Antigen specific LMI was demonstrated in chickens with DTH to purified protein derivative of Mycobacterium (PPD) and ferritin. A good comparison between LMI and DTH, as measured by the delayed wattle reaction (DWR), was demonstrated. The effect of bacterial lipopolysaccharide (LPS) on LMI was examined and LPS in microgram quantities was found to inhibit in vitro migration of chicken leukocytes. Contamination of antigen preparations with LPS is a probable explanation for occasional nonspecific inhibition of leukocyte migration since endotoxin is an almost ubiquitous contaminant of antigen preparations.     

Experimental study of antibiotic-induced immunosuppression in mice—I. Humoral and cell-mediated immune responsiveness related to in vivo antibiotic treatment

By using CBA/J mice as responders, the immunodepressive effect of seven antibacterial chemotherapeutic agents was tested, i.e. penicillin (pen), streptomycin (str), erythromycin (erm), kanamycin (kan), tetracycline (tet), colistin (col) and chloramphenicol (chl). Lymphocytotoxicity power, as well as the ability of each drug to influence secondary humoral (against sheep red blood cells or diphtheria anatoxin) or cell-mediated (against PPD and Coxsackie A₉ virus) immunity were searched.

Erm, col and chl markedly depressed humoral and cell-mediated immune responsiveness in vitro after in vivo treatment with non-cytotoxical amounts. The Th lymphocyte supplementation of B reactive spleen cell population recovered the immune capacity in col and chl in vivo-treated groups, but not in erm-treated group.     

A new freeze-dried living virus vaccine against sheep-pox

A sheep-pox virus strain has been adapted and multiplied in primary lamb kidney cell cultures. The main characteristics of the strain have been verified in vitro after clones were isolated, and the results confirmed its identity. The safety and the potency of the strain have been investigated in sheep.

The inoculation of the strain to sheep was followed by a post-vaccinal reaction materialised by a nodule at the site of inoculation and an increase of temperature by about 1°C. No reactions adversely affecting pregnancy have been noted. Immunisation was demonstrated by an increase in the level of neutralising serous antibodies and protection against the pathogenic virus. The immunity tended to decrease during the second year after primovaccination and a yearly booster vaccination appeared to be necessary. Primovaccination of lambs over 2 months of age produced a better immunity, especially when the lambs were born from vaccinated ewes.

This strain forms the active principle of a freeze-dried vaccine containing no adjuvant of the immunity.     

Pathogenesis of Bovine herpesvirus-1 (BHV-1) infections: Interactions of the virus with peripheral bovine blood cellular components

Bovine herpesvirus-1 (BHV-1) is believed to reach the placenta via the hematogenous route as the virus has been recovered from the blood buffy coat cells of experimentally infected cattle. However, the manner in which the virus relates to these and other blood cells while in transit in the blood is not known. The nature of this relationship was investigated in the present study.

An in vitro correlate of the in vivo cell-virus interactions was simulated by isolating peripheral blood cells on a Ficoll-Hypaque gradient and testing their capacity to adsorb and act as host cells for BHV-1 replication. It was shown that all leukocytes, but not red cells, can adsorb virus readily. Of the leukocytic fraction, monocytes and lymphocytes adsorb the virus most effectively. Monocytes supported viral replication while lymphocytes could do so only after stimulation with phytohemagglutinin.

It is concluded that in vivo replicating BHV-1 is transported in monocytes, and is adsorbed to all leukocytes from which it finally reaches the placenta, leading to abortion.     

Studies on the mechanism of vaccinal immunity to Marek''s disease

Current knowledge of the nature of the antigens and of the host immune responses in vaccinal immunity to Marek's disease is reviewed. It is suggested that a two-step mechanism of resistance operates. The first step involves humoral and cell-mediated responses directed against viral antigens; the second step occurs after challenge with Marek's disease virus and consists of cellmediated responses directed against tumour cells.     

The pathogenesis and immunology of bluetongue virus infection of ruminants

Bluetongue (BLU) virus is transmitted from infected to susceptible ruminants by hematophagous vector midges (Culicoides species). Cattle are important reservoir hosts of the virus because infection typically is asymptomatic and characterized by prolonged cell associated viremia, and because at least some species of insect vector preferentially feed on cattle. Interaction of BLU virus with the cell membrane of erythrocytes in infected cattle likely facilitates both prolonged viremia as well as infection of the insect vector. BLU disease is most common in sheep and some wildlife species. A variety of host, agent and environmental factors clearly can influence expression of disease in these species. The pathogenesis of BLU virus infection of cattle and sheep is remarkably similar, thus the basis for expression of disease in sheep but not cattle remains to be firmly established. Some difference in susceptibility of endothelial cells to infection in the two species is one potential explanation.

Ruminants develop a variety of antiviral responses after BLU virus infection. Antibodies to outer capsid protein VP2 are responsible for virus neutralization, and confer resistance to reinfection with the homologous serotype of BLU virus. Antibodies to epitopes on proteins which are common to all viruses of the BLU serogroup form the basis of current diagnostic serologic tests. Cell mediated responses have been incompletely characterized, in part because BLU virus replicates within dividing lymphocytes and virus-mediated cytolysis inhibits in vitro blastogenesis. Immunological competence of ruminants to BLU virus arises prior to midgestation, and suggestions that persistent immune tolerant BLU virus infection occurs after in utero exposure of cattle have not been substantiated and are not consistent with recent findings.     

Thymocyte growth factor: A progression growth factor for cycling immature cortical thymocytes

A growth factor for cultured guinea pig thymocytes was isolated from an extract of calf thymus. This thymocyte growth factor (TGF) was purified to apparent homogeneity employing reverse phase high performance liquid chromatography as the final step. TGF was characterized as an N-terminal blocked hydrophilic peptide with an apparent M_r of 1600. The amino acid composition revealed a high content of acidic amino acids but no apparent relationship with previously defined growth factors and thymic differentiation hormones. TGF was active in nanomolar concentration and stimulated the DNA synthesis and mitotic activity specifically in a subpopulation of thymocytes with immature cortical phenotype. The responsive thymocytes were recruited from G₁ into S phase within 2 h after addition of TGF. In the absence of the growth factor, the target cells were blocked at a putative restriction point in G₁, prior to the onset of the S phase. It is proposed that TGF is a progression growth factor participating in the regulation of the intense proliferation of immature thymocytes in the thymus cortex in vivo.     

CRISPR/Cas9体外酶切检测猪生长抑素基因定点修饰靶点活性的研究

本试验旨在通过CRISPR/Cas9体外酶切法检测猪生长抑素(SST)基因定点修饰靶点的活性。试验设计5个长20 bp的单链导向RNA(sgRNA),即SST-sgRNA-g1、SST-sgRNA-g2、SST-sgRNA-g3、SST-sgRNA-g4和SST-sgRNA-g5。化学合成sgRNA寡核苷酸序列,将寡核苷酸序列连接到可同时表达Cas9和sgRNA的质粒中,挑选正确克隆的质粒作为模板进行体外转录形成SST-sgRNA。利用CRISPR/Cas9体外酶切含靶点的DNA片段,根据酶切条带的灰度换算成sgRNA活性。结果显示目的sgRNA寡核苷酸双链成功插入到质粒中且序列正确,以质粒为模板体外转录SST-sgRNA成功。靶位点经Cas9蛋白酶切后与标准sgRNA1和sgRNA2酶切活性作比较,确定靶点SST-sgRNA-g1、SST-sgRNA-g4和SST-sgRNA-g5活性较高,可为在细胞水平、胚胎水平做基因定点修饰提供依据。     

高通量SNP芯片在牛体外早期胚胎染色体质量鉴定中的初步应用

旨在基于高通量SNP芯片建立一种可评估牛早期胚胎染色体质量和生产性能的方法,为胚胎牛育种提供技术支撑。本研究通过胚胎切割技术分别对荷斯坦奶牛体内囊胚(n=27)、体外囊胚(n=21)、体外2细胞胚胎(n=6)和8细胞胚胎(n=5)进行切割取样并统计发育率,其中体内囊胚组分为1/2体内胚组(切取半个胚胎)和体内滋养层(trophoblast cells, TE)组(切取体内胚胎少量TE),体外囊胚组、体外2和8细胞胚胎分别为体外TE组(切取体外胚胎少量TE)、体外2细胞(切取体外2细胞胚胎的1个卵裂球)和8细胞组(切取体外8细胞胚胎的1个卵裂球)。切割取样后进行全基因组扩增(whole-genome amplification, WGA),对扩增成功且DNA量大于1 000 ng的样品进行SNP芯片检测,芯片检出率大于90%的进行生产性能评估,低于90%的进行染色体片段缺失分析和数据填充。结果显示：1)切割部分TE后,体内TE组剩余部分和体外TE组剩余部分继续培养24 h后的发育率分别为(94.4±5.6)%和(90.5±6.6)%,而1/2体内胚组剩余部分的发育率显著降低,仅为(22....     

Lymphoblastic transformation assay of sheep peripheral blood lymphocytes: A new rapid and easy-to-read technique

A new micro-method was used to evaluate in vitro sensitivity of ovine peripheral blood lymphocytes (PBL) to different non specific mitogens (pHA, Con A, PWM) and to investigate the interest of a colorimetric assay for measurement of transformed lymphocytes.

The results showed that sheep PBL in flat-bottomed microplates responded optimally at a cell density of 8 × 10⁶ cells/ml to PHA (2.5 μg/ml), Con A (5 μg/ml) and PWM (5 μg/ml).

The colorimetric assay using a tetrazolium salt (MTT), for measuring the transformed lymphocytes, is very well correlated with the classical method of [³H]thymidine incorporation.

This new revelation technique of the mitogenic response improve the technical value of the assay, which is more rapid and easy-to-read, without diminishing the biological value.     

文心兰组织培养及转基因研究进展

综述了文心兰组织培养及转基因研究的主要进展。外植体、培养基及其添加物、植物生长调节剂和培养方式对文心兰类原球茎或丛芽诱导均产生重要影响,大田植株的花梗、花芽、茎尖、根尖等均可用作初代培养的外植体,其中花梗及花芽为最常用的外植体,试管苗的所有组织器官均是有效的外植体;文心兰离体培养以固体培养方式为主,1/2MS、MS是用于文心兰离体培养最常用的基本培养基,培养基中植物生长调节剂种类、浓度及其配比是文心兰离体培养成功与否的关键因素。文心兰类原球茎形成过程是典型的体细胞胚胎发生发育过程,体细胞胚胎发生受植物生长调节剂、基因型、培养条件等影响较大。文心兰转基因研究尚处于初步阶段。     

牛体外胚胎冷冻保存的研究进展

体外胚胎冷冻保存技术是胚胎移植技术的重要组成部分,在辅助生殖技术中发挥重要作用,同时对种质资源保存、加强遗传改良和促进优质种源国际交流等方面具有重要意义。然而,体外胚胎冷冻过程中存在脂质含量过高、活性氧水平升高及机械损伤等问题,导致体外胚胎冷冻效率低,这极大地限制了体外胚胎冷冻保存技术的广泛应用。大量研究表明,通过去脂质、优化体外胚胎培养液、人工塌陷囊胚腔和优化冷冻程序等手段,可以有效提高冷冻后胚胎的存活率和发育能力。因此,本文概述了体外胚胎冷冻保存技术的研究进展和胚胎冷冻过程中存在的问题,总结了提高体外胚胎冷冻效率的方法措施,旨在为提高体外胚胎冷冻保存效率提供一定参考。     

Research Progress on Primary Culture,Purification and Identification Technique of Bovine Mammary Epithelial Cells

The bovine mammary epithelial cells not only have the function of synthesis and secretion of milk, but also play an important role in innate immunity system of the mammary gland, and there is great significance of them on studying the mechanism of lactation, mastitis pathogenesis and drug screening.Primary cultured bovine mammary gland epithelial cells are suitable for setting up cell model which can be used as the dielectric for physiological, pathological and pharmacological researching, avoiding the difficulties of in vivo test, such as the long cycle, the high cost, the individual difference, etc.The author summarized the latest researches of cell primary culture in vitro, cultivation technology, purification and identification method in order to provide reference for the studies of bovine mammary epithelial cells culture.     

Corynebacterium equi: An interhost review with emphasis on the foal

A review of the recent and/or significant literature concerning Corynebacterium equi, including its morphologic, biochemical, immunological, and pathological characteristics in the foal, humans and other animals is presented. The similarity in the tissue responses of mammalian hosts to C. equi and Mycobacterium spp. is discussed.

The antigenic structure and virulence factors associated with C. equi. other corynebacteria and mycobacteria are compared.

The immunological aspects of resistance to C. equi are considered. The evidence suggests that the major immune response elicited in the foal by C. equi is cell-mediated. However, the immunopathogenic mechanism needs clarification. Areas of future research are suggested.     

Efficacy of vaccines against bacterial diseases in swine: what can we expect?

This paper discusses what can be expected with regard to efficacy of antibacterial vaccines used in swine, based on the present knowledge of pathogen–host interactions. First, vaccination against bacteria that mainly cause disease by production of exotoxins is considered. Vaccines containing the inactivated toxin or a non-toxic but antigenic recombinant protein derived from the exotoxin can be expected to provide protection against disease. The degree of protection induced by such vaccines varies, however, depending amongst other things on the pathogenesis of the disease. Vaccination against clostridial infections, Actinobacillus pleuropneumoniae infections, progressive atrophic rhinitis and enterotoxigenic Escherichia coli, is considered. The second part of the article deals with vaccination against extracellular bacteria. Protection against these bacteria is generally mediated by antibodies against their surface antigens and certain secreted antigens, but cellular immunity may also play a role. Efficacy of vaccines against swine erysipelas, Streptococcus suis infections, Mycoplasma hyopneumoniae infections and swine dysentery is discussed. Finally, vaccination against facultatively intracellular bacteria is considered. For protection against these bacteria cell-mediated immunity plays an important role, but antibodies may also be involved. It is generally accepted that live-attenuated vaccines are more suitable for induction of cell-mediated immunity than inactivated vaccines, although this also depends on the adjuvant used in the vaccine. As an example, vaccination against Salmonella enterica serotype Typhimurium is discussed.     

抗马链球菌马亚种组方加味优选及其体内抑菌作用研究

【目的】筛选可抑制马腺疫主要致病菌——马链球菌马亚种的最优中药组方,为临床马腺疫的防治提供参考。【方法】选用赤芍、大黄、山豆根等27种中药,首先通过体外抑菌试验(牛津杯法、最小抑菌浓度(MIC)、最小杀菌浓度(MBC)、联合药敏试验)结合L₈(2⁶)正交试验初步筛选抗马链球菌马亚种中药组方的最佳优选因素,再通过L₁₈(3⁶)正交试验进一步筛选基础组方最佳配比;最后在基础组方的基础上,根据中兽医方剂配伍理论,添加黄芪和当归2味补虚药,通过马腺疫小鼠模型体内抑菌试验筛选加味组方配比,结合组方保护率结果及组织脏器病理变化验证加味组方的体内抑菌作用。【结果】试验筛选出了马链球菌马亚种超敏和高敏的7味中药：浙贝母、赤芍、甘草、大黄、雪白睡莲、山豆根和苦参。大黄、雪白睡莲、山豆根、甘草、苦参和浙贝母的MIC分别为0.078、0.078、0.125、0.156、0.156和0.250 mg/mL。雪白睡莲、赤芍、山豆根、甘草、大黄、苦参和浙贝母的MBC分别为0.078、0.125、0.125、0.156、0.15...     

奶牛乳腺上皮细胞原代培养、纯化及鉴定技术研究进展

奶牛乳腺上皮细胞不仅具有合成和分泌乳汁的功能,而且在乳腺的先天免疫中扮演着重要角色,对泌乳机制、乳房炎发病机制的研究,以及药物筛选具有重要意义。原代培养的奶牛乳腺上皮细胞适宜建立细胞模型,可作为生理、病理、药理等方面研究的良好介质,解决体内试验周期长、成本高、个体差异大的难题。作者主要从奶牛乳腺上皮细胞原代培养的发展历程、培养技术、纯化技术及鉴定方法等方面的最新研究情况进行综述,以期为奶牛乳腺上皮细胞培养相关研究提供参考。     

Influence of sodium diethyldithiocarbamate (Imuthiol) on lymphocyte function and growth in weanling pigs

Mitogen-stimulated lymphocyte proliferation, delayed-type hypersensitivity (DTH) reactions, interleukin-2 (IL-2) production, and growth performance were evaluated in 3-week-old pigs treated with imuthiol. Lymphocyte proliferative responses to Con A and PWM were reduced (P < 0.05) in pigs treated with imuthiol at 25 mg/kg; PHA proliferative responses were not influenced by imuthiol treatment. Imuthiol at 2.5 mg/kg and 25 mg/kg lowered IL-2 production when compared to saline-treated controls. Delayed-type hypersensitivity responses to PHA were higher in 25 mg/kg imuthiol-treated pigs; however, 2.5 mg/kg imuthiol-treated pigs had lower DHT reactions. Imuthiol at 2.5 mg/kg and 25 mg/kg reduced (P < 0.05) average daily feed intake. These data suggest that in vivo imuthiol treatment in pigs lowers lymphocyte proliferative responses, IL-2 production, and growth performance.     

干扰素-ε研究进展

干扰素-ε（IFN-ε）是新发现的一种Ⅰ型干扰素（IFNs），是由多种细胞分泌的一类细胞因子，其本质是一种糖蛋白，可间接发挥抗病毒、免疫调节和抗肿瘤的生物学活性。但与其他Ⅰ型干扰素不同，IFN-ε的产生不需要病毒诱导，其由黏膜上皮细胞组成型表达，并受激素调节，在黏膜免疫中具有重要作用。目前相关研究报道较少，本文就人和不同动物IFN-ε的起源、体内分布规律，以及对生殖系统的保护、神经系统的调节及抗病毒等研究进展进行综述，以便进一步了解和开发新型干扰素。