鸡传染性喉气管炎活疫苗生产工艺的改进——鸡胚绒毛尿囊膜渗透法接种应用实验

采用鸡胚绒毛尿囊膜渗透法给鸡胚接种病毒,收获感染的鸡胚绒毛尿囊膜制备鸡传染性喉气管炎活疫苗,相比于传统的人工气室绒毛尿囊膜接种法和尿囊腔接种法可降低鸡胚的早死亡率,提高鸡胚利用率,降低生产成本,提高单产量。     

鸡传染性法氏囊病毒鸡胚适应株的培育

将鸡传染性法氏囊病毒（IBDV）野毒株分别经绒毛膜和尿囊腔途径接种于鸡胚进行传代培养，应用AGP法和Dot－ELISA法分别对每代收取的不同培养物（绒毛膜和尿囊液）进行检测。结果显示，经绒毛膜途径传至第3代，尿囊腔途径传至第4—5代，即成为鸡胚完全适应株，可作为鸡胚培养病毒用毒种，方法应选择操作简便、且收获物质量高。数量多的尿囊腔接种途径。     

鸡传染性喉气管炎弱毒活疫苗生产工艺的优化及改进试验

《中华人民共和国兽用生物制品规程》规定,在生产鸡传染性喉气管炎弱毒活疫苗时,采用鸡胚尿囊腔接种和人工气室绒毛尿囊膜接种工艺生产成品。由于人工气室绒毛尿囊膜接种早死胚多,而且工艺比较繁琐,所以我国大多数生产企业在生产中多采用尿囊腔接种方法,但尿囊腔接种后病毒繁殖时间要比人工气室绒毛尿囊膜接种后延长1~2 d,增加了孵化成本,而且绒毛尿囊膜病变(增厚、水肿等)也不如人工气室绒毛尿囊膜接种后产生的病变严重(膜重)。为此,借鉴了鸡痘鹌鹑化弱毒活疫苗的新生产工艺———鸡胚绒毛尿囊膜渗透法[1],并将此方法用于鸡传染性喉气管炎…     

鸡传染性喉气管炎疫苗生产工艺改进初探

在鸡传染性喉气管炎活疫苗生产中，用尿囊腔接种法代替绒毛尿囊膜接种法，收获的胚液和绒毛尿囊膜的含毒量(EID5D)均可达到《中华人民共和国兽用生物制品规程》对制苗的要求，提高了种蛋的利用率。     

鸭瘟活疫苗生产工艺改进试验

利用尿囊腔接种法替代绒毛尿囊膜接种法生产鸭瘟活疫苗,收获的胚液和绒毛尿囊膜及胎儿的病毒含量(ELD50)均高于<中华人民共和国兽用生物制品规程>2000年版中的病毒含量标准,而且降低了早死率,提高了鸡胚利用率.     

鸡胚和雏鸡接种ALV-J相关急性纤维肉瘤浸出液的致病性比较

为了比较鸡胚接种ALV-J相关急性纤维肉瘤浸出液对胚体及雏鸡的致病性,将含ALV-J相关病毒的肉瘤浸出液分别经5日龄胚卵黄囊、11日龄胚绒毛尿囊膜、1日龄雏鸡腹腔接种,比较不同接种方式对SPF鸡胚及雏鸡的致病性.结果表明,5日龄卵黄囊接种的鸡胚在18～22日龄死胚率为14/30,肿瘤发生率为8/14; 11日龄绒毛尿囊膜接种的鸡胚在18～22日龄引起鸡胚死亡率为17/30,肿瘤发生率为6/17.对雏鸡的致病性比较表明,绒毛尿囊膜接种的13只出壳雏鸡全部死亡,有11只出现肿瘤.结果提示,绒毛尿囊膜接种的致病性不仅高于卵黄囊接种,也高于1日龄雏鸡接种.绒毛尿囊膜接种不仅肿瘤发生率高,且发生得更早、更快,可作为这种急性纤维肉瘤进一步作人工造病的实验模型.     

一种新的鸡痘鹌鹑化弱毒疫苗生产工艺改进试验--绒毛尿囊膜渗透法

采用一种新的鸡胚绒毛尿囊膜接种方法,即绒毛尿囊膜渗透法来制备鸡痘鹌鹑化弱毒活疫苗.此法不必做人工气室,减少了鸡胚早死率及人为污染率,减化了生产工艺并使鸡胚利用率大大提高.     

鸡传染性喉气管炎病毒培养特性的研究

本试验从发病鸡群分离一株鸡传染性喉气管炎病毒(命名为NM98a)，并用分离株和ILT王岗株对鸡胚绒毛尿囊膜、鸡胚尿囊腔及鸡胚肾细胞上培养进行比较研究。结果表明：ILTV经不同途径接种鸡胚和鸡胚肾细胞上培养，在鸡胚绒毛尿囊膜和鸡胚肾细胞上引起的病变不同，但最终增殖达到的感染性病毒量没有明显区别。鸡胚尿囊腔接种是ILTV增殖最佳途径。     

鸡胚绒毛尿囊膜渗透法在鸡新城疫活疫苗(Ⅰ系株)生产中的应用试验

与传统尿囊腔接种方法相比，用绒毛尿囊膜渗透接种的方法感染鸡胚，收获胚液制备疫苗，解决了接种后部分鸡胚在规定时间内不死的问题，可明显提高鸡胚利用率和降低生产成本。     

鸡传染性法氏囊病的实验室诊断

采集病死鸡法氏囊经处理后绒毛尿囊膜途径接种5枚SPF鸡胚,接种胚均于接种后48~96 h死亡,死亡胚病变特征为体表和皮下出血,胚水肿,肾脏出血,肝脏有坏死灶,心肌苍白。琼扩试验结果显示法氏囊样品孔、绒毛尿囊膜样品孔与标准血清孔间均可出现明显的沉淀线,说明样品中含有传染性法氏囊病病毒。根据实验室检查情况,诊断为鸡传染性法氏囊病。     

鸡痘鹌鹑化弱毒活疫苗生产工艺改进试验

采用人工气室绒毛尿囊膜和尿囊腔两种接种方法制备鸡痘活疫苗，结果表明经尿囊腔接种的结果和经人工气室绒毛尿囊膜接种的结果相同。因此，以尿囊腔接种代替人工气室绒毛尿囊膜接种，可以降低成本，简化工艺。本试验为以后采用机械化接种生产鸡痘苗提供了可靠的数据。     

鸡胚尿囊液量与鸡种的相关性研究

选择９日龄非免疫的罗曼蛋鸡胚和淮南麻黄鸡胚各１８０个,按不同胚重各分成３组,同时接种鸡新城疫病毒,观察病毒对不同品种鸡胚尿囊液量的影响。结果表明：罗曼蛋鸡胚的尿囊液量极显著高于淮南麻黄鸡胚;罗曼蛋鸡胚的尿囊液量／胚蛋重大于淮南麻黄鸡胚。罗曼蛋鸡胚的失水率小于淮南麻黄鸡胚。罗曼蛋鸡胚和淮南麻黄鸡胚尿囊液量与胚重均呈极显著正相关。     

Production of quail-chick chimaeras by blastoderm cell transfer

1. Quail-chick chimaeras were produced by injecting dissociated quail blastoderm cells into chick embryos. 2. Quail blastoderms were removed from the yolk and the cells were dispersed by trypsin treatment or pipetting. The cell suspension (1 to 5 microliters) was injected into the subgerminal cavity of unincubated chick embryos. The chick embryos were then cultured in recipient eggshells. 3. Quail blastoderm cells injected into the chick embryos adhered to the chick embryonic cells. The rates of hatching were 8.6% (38 chicks from 441 eggs) and 40.3% (48 chicks from 119 eggs) when the volumes of the cell suspension injected were 3 to 5 microliters and 1 microliter, respectively. 4. Seven out of 86 hatched birds were clearly identified as being chimaeric because part of the feather colouring was of quail specificity. In addition to these chimaeric birds, there were 8 chimaeric embryos which died before hatching. The distribution patterns of the quail feathers were varied among the chimaeric birds and embryos. 5. This technique provides a basis for the investigation of chick embryo cryopreservation, genetic transformation and analysis of cell lineage of chickens.     

复方中药禽康散对接种新城疫病毒鸡胚的保护效应

为了研究复方中药禽康散提取液对鸡新城疫病毒活性的影响,试验采用鸡胚法以不同浓度中药提取液与新城疫F48E9毒株悬液混合后各接种8枚鸡胚,观察各组鸡胚的存活情况。结果表明：中药浓度为1．0g/mL、0．5g／mL、0．25g／mL时活胚率分别是87．5％、75．0％、50．0％,而不加中药组鸡胚96h后全部死亡。说明复方中药禽康散具有抗新城疫F48E9毒株的作用,且随着中药浓度的增加抗病毒作用增强,呈现一定的量效关系。     

Development in culture of the chick embryo from cleavage to hatch

1. Early uterine embryos were obtained from hens by induced oviposition 7.5-8.0 h after the preceding egg was laid. They were cultured in vitro and then in recipient shells to hatch. As controls, embryos from freshly laid eggs were cultured in recipient shells to hatch. 2. For embryos cultured from uterine eggs, the hatch rate was 22.5%, and for embryos cultured from laid eggs, the hatch rate was 62.5%. 3. The weight of the chicks hatched from culture was about 60% of the weight of the preceding egg, or donor egg. Male and female chicks reached maturity and have produced viable offsprings. 4. The results show that it is possible to grow chick embryos in culture from the early cleavage stage (stage II) to hatch. They extend earlier findings on the culture of embryos from the blastoderm stage (Stage X) to hatch. The technique provides a basis for investigations on chick embryo cryopreservation.     

Pathogenicity of aerobically sporulating microorganisms: Bacillus cereus]

The pathogenicity of the metabolites of B. cereus was determined by bioassays with white mice, chick embryos, rabbits, and kittens. Cell-free filtrates of the cultures of several strains of B. cereus were used for application; in the mice the application was done i. v., i. p. and p. o., in the chick embryos via the allantoic sac, in the kittens p. o. and i. p., in the rabbits via tied-up sections of the small intestine by the loop-test method. It was revealed that some strains of B. cereus produced substances of exotoxin nature under suitable conditions. White mice and chick embryos were the best materials for their detection.     

Developmental fall in whole body protein turnover of chick embryos during incubation

Whole body protein turnover was measured in chick embryos during incubation to investigate whether or not there is a fall in fractional rates of protein synthesis and degradation during development. Stable isotopically labelled [15N]phenylalanine was injected intraperitoneally into embryos on days 12 and 19. From 60 to 90 min after injection the isotope enrichment in free and protein-bound phenylalanine was measured with a selected-ion gas-chromatograph mass-spectrometer. The results showed that from days 12 to 19 of incubation, there was a remarkable reduction in fractional rates of protein synthesis and degradation in the whole body of chick embryos. During embryonic growth, protein synthesis per unit of RNA that is, the minimum amino acid translation rate of RNA, did not change significantly, whereas the RNA:protein ratio was reduced to one-third from days 12 to 19 of incubation. It was concluded, therefore, that the dramatic fall in fractional synthesis rate in chick embryos would be entirely attributable to the rapid increase in protein content, thereby changing the RNA:protein ratio in parallel with the fractional synthesis rate.     

Sexing chick embryos: a rapid and simple protocol

1. Analysis of gene expression in the developing chick gonads requires the collection of male and female tissues from embryos between 3.5 d and 8.5 d of development. However, male and female chick embryos are indistinguishable by morphological examination before d 7.5 of development. 2. Sex identification of earlier embryos is only possible by molecular methods, which at present are laborious and time consuming. 3. We have devised a PCR-based sexing protocol which combines both sex specific and control reactions in a single tube assay. The assay is rapid and effective over a wide range of DNA concentrations and is tolerant of poor quality DNA. 4. Procedures are described for identifying the sex of individual embryos using either tissue samples or a small number of cells recovered from amniotic fluid.     

中草药抑制H_5亚型禽流感病毒对鸡胚毒性的研究

根据中医利用解表、清瘟、凉血止血的中草药防治人类流感的原理,选取金银花、北板蓝根、贯叶连翘、双黄连、贯众等五种中草药所制成的制剂进行H5亚型禽流感病毒感染鸡胚后毒力作用的抑制实验。首先对鸡胚接种禽流感H5亚型病毒,然后对感染鸡胚分别注射上述几种中药制剂,再抽取胚液进行HA和HI实验,将其中对鸡源H5-AIV有抑制作用的中草药挑选出来,测定中草药对禽流感病毒的治疗指数(Therapeutic index,TI)。然后采用寇氏法计算药物半数有效量ED50。结果表明,金银花、北板蓝根、贯叶连翘、双黄连、贯众五种中草药制剂在对H5亚型禽流感病毒的鸡胚毒性方面均有较好的抑制作用。     

Protein synthesis measured in the whole body of chick embryos cultured in vitro

1. Whole body protein synthesis was measured in chick embryos cultured in vitro. On day 7 of incubation chick embryos were cultured for 60 min in synthetic serum‐free medium containing 4‐[³H]phenylalanine. Specific radioactivities in free and protein‐bound phenylaline in the whole embryo were measured, starting 2 min after commencement of the culture process.

2. The values for fractional synthesis rate (FSR) estimated in vitro at 20, 30, 45 and 60 min during the embryo culture agreed well, ranging from 35 to 40%/d, suggesting that the method would serve as a useful model for studying the effect of growth promoters in chick embryos.

3. Bovine insulin in the synthetic medium did not affect FSR of protein in chick embryos cultured in vitro.