感染IBDV雏鸡血液激素水平和ANAE阳性淋巴细胞动态变化

为探讨内分泌活动及细胞免疫反应在鸡抗传染性法氏囊病毒（ＩＢＤＶ）感染中的调节作用机制而进行了本研究。结果表明,攻毒后１～５ｄ内,未免疫攻毒鸡（Ａ组）、免疫攻毒鸡（Ｂ组）血浆皮质酮均明显上升,而未免疫未攻毒鸡（Ｃ组）则否。Ａ、Ｂ、Ｃ３组血浆Ｔ４水平攻毒前后无明显变化,Ｔ３除在攻毒后第３天Ａ组明显高于Ｂ、Ｃ组外,其他时间无明显差异性变化。血液ＡＮＡＥ阳性淋巴细胞（％）在攻毒后的１～５ｄ内,Ａ组和Ｂ组明显下降,以后回升,至２８ｄ回复到攻毒前水平,并接近于Ｃ组。Ａ组的ＡＮＡＥ阳性淋巴细胞（％）与皮质酮呈显著负相关,与Ｔ３和Ｔ４无明显相关性。     

Effects of Compound Chinese Herbal Medicinal Polysaccharides on Immune Signal Molecules Expression in Different MHC B-LβⅡ Genotypes Chickens Lymphocyte

This study was aimed to explore the effect of compound Chinese herbal medicinal polysaccharides (cCHMPS) on immunomodulatory in different MHC B-LβⅡ genotype chickens.MHC B-LβⅡ genotype was analyzed by PCR-SSCP method in 500 chickens.Collected the blood from different MHC B-LβⅡ genotype chickens,isolated peripheral blood lymphocytes,and added cCHMPS with the final concentration of 200,100,75,50,25 and 0 μg/mL cocultured for 24 h.Then the lymphocyte supernatant cAMP,cGMP,Ca²⁺,NO and iNOS content were detected by ELISA.The results showed that cCHMPS could increase cAMP,cGMP,Ca²⁺,NO and iNOS levels in chicken lymphocyte supernatant compared with control group in each MHC B-LβⅡ genotype chickens.And when the concentration of cCHMPS was 50 μg/mL,cAMP,Ca²⁺,NO and iNOS levels of AA and BC genotypes chicken lymphocyte supernatant were higher than that of the same genotype chicken,cGMP level of BC genotype chicken lymphocyte supernatant was higher than that of the same genotype chicken;When cCHMPS concentration was 75 μg/mL,cGMP level of AA genotype chicken lymphocyte supernatant was higher than that of the same genotype chicken;When cCHMPS concentration was 100 μg/mL,cAMP,cGMP,Ca²⁺,NO and iNOS levels of BB genotype chicken lymphocyte of the supernatant were higher than that of the same genotype chicken.cCHMPS could increase cAMP,cGMP,Ca²⁺,NO and iNOS levels in different MHC B-LβⅡ genotypes chickens,and the cCHMPS optimum immunomodulatory doses were different in each MHC B-LβⅡ genotypes chickens.     

Effects of Compound Chinese Herbal Medicinal Polysaccharide Extraction on NF-κB,TNF-α and IL-6 mRNA Expression in Different MHC B-Lβ Ⅱ Genotype of Chickens Lymphocyte

In order to explore the effect of compound Chinese herbal medicines polysaccharides(cCHMPS) on immunomodulatory in different MHC B-Lβ Ⅱ genotype chickens,200 White feather broiler were chosen and PCR-SSCP technique was applied to analyze the polymorphism of MHC B-Lβ Ⅱ gene. The peripheral blood were collected according to the different MHC B-Lβ Ⅱ genotype,and the lymphocytes were isolated from peripheral blood and the cCHMPS were added with a final concentration of 100,75,50 and 0 μg/mL for co-culturing 24 h.Then the expression of NF-κB,TNF-α,IL-6 mRNA in lymphocyte using Real-time PCR method were detected. The results showed that:Compared with the control group,different does of cCHMPS could significantly improve NF-κB,TNF-α,IL-6 mRNA expression levels in chicken with different MHC B-Lβ Ⅱ genotypes (P<0.05), and when the cCHMPS concentration was 50 μg/mL,the NF-κB,TNF-α,IL-6 mRNA expression levels in lymphocyte of AB and AA genotype chicken were significantly higher than that of other groups (P<0.05).The NF-κB,IL-6 mRNA expression levels of AC genotype chicken were significantly higher than the other groups (P<0.05). The TNF-α mRNA expression levels of AC genotype chicken were significantly higher than the other groups when cCHMPS was 100 μg/mL (P<0.05).There results indicated that the cCHMPS could stimulate NF-κB,TNF-α,IL-6 mRNA expression in different MHC B-Lβ Ⅱ genotype chickens,and the optimum immunomodulatory does were different in each MHC B-Lβ Ⅱ genotype chicken.     

中药对柔嫩艾美尔球虫感染鸡外周血T细胞亚群的影响

为研究中药对柔嫩艾美尔球虫感染鸡免疫水平的影响,本实验采用流式细胞技术检测柔嫩艾美尔球虫感染鸡外周血CD4+T细胞亚群,以及CD8+T细胞亚群数目的动态变化。结果表明,接种球虫后,感染给中药组鸡CD4+、CD8+T细胞亚群数目及百分比(CD4+T/CD8+T)均高于感染不给药组,9d时,感染给中药组最高,分别为9.77和7.53,与感染不给药组差异显著(p0.05)。感染球虫组CD4+T/CD8+T比值均低于不感染组,感染给中药组在接种后第6d和第9d均高于感染不给药组。感染给中药组、不感染给中药组和不感染不给药组CD4+T/CD8+T平均值均在2-1之间,分别为1.11、1.33和1.25,而感染不给药组仅为0.93。     

The Influence for Chickens' T Lymphocyte Subsets in Peripheral Blood after Inoculated with the Recombinant Chicken Interferon-α/Chicken Interleukin-2 Complex Protein

To explore the influence of recombinant chicken interferon-α/chicken interleukin-2 fusion protein (rChIFN-α-Linker-ChIL-2 protein, recombinant fusion protein) on the percentage content of peripheral T lymphocyte subsets (PBLC) of SPF chicken,the flow cytometry was used to detected the percentage contents of CD3⁺CD4⁺, CD3⁺CD8⁺ and the CD4⁺/CD8⁺ ratio in PBLC at different days post injection in the 14-day-old SPF chicken injected with rChIFN-α-Linker-ChIL-2 protein (group 2) and recombinant chicken interferon α (rChIFN-α) protein (group 3). The results indicated that the recombinant fusion protein and rChIFN-α protein could increase the percentage content of CD3⁺CD4⁺ T lymphocyte, decrease the percentage content of CD3⁺CD8⁺ T lymphocyte, improve CD4⁺ /CD8⁺ T lymphocyte ratio during 3 to 14 d post injection. The percentage contents of CD3⁺CD4⁺, CD3⁺CD8⁺ and CD4⁺/CD8⁺ ratio in chickens of group 2 were significantly different from the PBS control group (group 1) (P<0.01), and were notably different from group 3 during 3 to 7 d post injection (P<0.05). These results showed that both the rChIFN-α-Linker-ChIL-2 protein and rChIFN-α protein could affect the percentage contents of lymphocyte subsets, improve CD4⁺/CD8⁺ ratio, enhance the cellular immune function in chicken. The influences of rChIFN-α-Linker-ChIL-2 protein on the percentage contents of CD3⁺CD4⁺ and CD3⁺CD8⁺ T lymphocyte, and CD4⁺/CD8⁺ value were notable higher than rChIFN-α, which suggested that recombinant fusion protein play a synergistic role of interferon alpha and interleukin-2 on the cellular immune function by this pathway in chicken.     

藏鸡、茶花鸡染色体核型分析

运用改进的鸡外周血淋巴细胞培养-空气干燥法,分析了藏鸡、茶花鸡染色体核型.研究结果表明:藏鸡和茶花鸡的染色体形态十分相近,染色体2n众数均为78,但No.4、No.6染色体的形态存在一定的差别,即藏鸡分别为sm和m型,而茶花鸡分别为st和sm型.同时收集丝羽乌骨鸡、鹿苑鸡、狼山鸡、萧山鸡、北京油鸡的核型资料进行分析,发现鸟类核型进化可能是由不对称向对称发展的,着丝粒以及着丝粒周边区域的染色体重排可能是地方鸡种分化和形成的重要内在因素.     

以MTT比色法检测鸡脾淋巴细胞转化效果

本研究运用ＭＴＴ比色法以正交试验对鸡脾脏淋巴细胞转化的最佳条件（ｃｏｎＡ量、血清种类和浓度）进行了研究。结果表明，无论血清种类和浓度如何，低剂量ＣｏｎＡ（２．５～１０μｇ／ｍｌ）可以获得较好的转化效果。当ＣｏｎＡ的量确定时，低浓度鸡血清（０．５％～１．２５％）的转化效果优于高浓度（２％～３％）：５％犊牛血清优于任何浓度的鸡血清；无血清培养基优于血清培养基。其最佳组合为无血清ＲＰＭＩ１６４０培养基加ＣｏｎＡ２．５μｇ／ｍｌ。     

Effects of DDA, CpG-ODN, and plasmid-encoded chicken IFN-�� on protective immunity by a DNA vaccine against IBDV in chickens

This study examined the adjuvant effects of dimethyl dioctadecyl ammonium bromide (DDA), CpG oligodeoxynucleotides (CpG-ODN), and chicken interferon-γ (ChIFN-γ) on a DNA vaccine (pcDNA-VP243) against the infectious bursal disease virus (IBDV). A plasmid encoding chicken IFN-ã was constructed. Twice at 2-week intervals, two-week-old chickens were injected intramuscularly and intraperitoneally with either a DNA vaccine alone or a DNA vaccine together with the respective adjuvants. On week 2 after the second immunization, the chickens were orally challenged with the highly virulent IBDV. The groups that received the DNA vaccines plus either DDA or CpG-ODN showed significantly lower survival rates than the group that received the DNA vaccine alone. However, the survival rates for the DNA vaccine alone and for the DNA vaccine plus ChIFN-γ were similar. The chickens had no detectable antibodies to the IBDV before the challenge but all the surviving chickens in all groups except for the normal control group showed the induction of antibodies to the IBDV at day 10 after the challenge. As judged by the lymphocyte proliferation assays using the a WST-8 solution performed on the peripheral blood and splenic lymphocytes, the stimulation indices (SI) of the peripheral blood lymphocytes in all groups except for the normal control group were similar immediately before the challenge. At 10 days post-challenge, the SI for DNA vaccine plus either CpG-ODN or ChIFN-γ was similar to that of the DNA vaccine control group. For splenic lymphocytes, the SI in the DNA vaccine plus CpG-ODN and DNA vaccine plus ChIFN-γ groups were higher than for the DNA vaccine control. These results suggest that DDA actually compromises the protection against the IBDV by DNA vaccine, and CpG-ODN and IFN-γ had no significant effect.     

复方中药成分对新城疫疫苗免疫雏鸡外周血T淋巴细胞转化和血清抗体效价的影响

选择4种中药成分（黄芪多糖、淫羊藿多糖、峰胶黄酮、人参皂甙）组成2个复方，各按3个剂量水平与新城疫疫苗混合后免疫雏鸡，以无佐剂苗和油乳苗为对照，分别于免疫后7、14、21、28、35、42d采血，用MTT法和微量血凝法测定T淋巴细胞转化和血清抗体效价的动态变化。结果表明。中药佐剂能显著促进淋巴细胞转化。提高血清抗体效价，多数时间点与油佐剂的效果相当，部分时间点显著强于油佐剂。综合判定方1高剂量和方2中剂量的效果最好。     

鸡防御素Gal-13对雏鸡淋巴细胞增殖功能的影响

为观察鸡防御素Gal-13对雏鸡淋巴细胞增殖功能的影响,以Gal-13为研究对象,在体外培养雏鸡淋巴细胞,运用MTT法首先研究不同质量浓度的Gal-13对抗原刺激的雏鸡外周血淋巴细胞增殖功能的影响,然后选取1mg/L的Gal-13饲喂雏鸡,观察其各免疫器官和外周血淋巴细胞增殖功能的变化.结果发现:体外Gal-13(0.625～1.250 mg/L)提高淋巴细胞的增殖功能,但过高剂量的Gal-13(5～10 mg/L)抑制淋巴细胞的增殖.1 mg/L的Gal-13能够提高雏鸡中枢免疫器官淋巴细胞增殖功能,抑制脾脏淋巴细胞的增殖功能.结果表明,Gal-13能够提高中枢免疫器官的免疫反应,对免疫器官的作用具有一定选择性.     

维生素E缺乏对雏鸡淋巴细胞膜抗氧化及膜流动性的影响

用低维生素E（VE）和添加不饱和脂肪酸的日粮饲喂雏鸡，建立VE缺乏雏鸡模型。分离全血淋巴细胞并制备细胞膜，检测淋巴细胞膜超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活力及丙二醛(MDA)含量；采用荧光偏振法检测淋巴细胞膜流动性。结果表明，VE缺乏雏鸡淋巴细胞膜SOD和GSH-Px活力及膜流动性低于对照组，MDA含量高于对照组，组间差异显著或极显著 (P<0.05、P<0.01)。说明VE对维持雏鸡淋巴细胞膜功能起重要作用。     

中药多糖对雏鸡淋巴细胞转化和新城疫抗体水平的影响

观察不同浓度的复方多糖、黄芪多糖（APS）、当归多糖（ASP）及淫羊藿多糖（EPS）对正常罗曼雏鸡免疫功能的影响。将260只1d龄健康罗曼雏鸡随机分为13组,每组20只。分别于1d龄皮下注射生理盐水以及复方多糖、APS、ASP、EPS,连续注射7d。于7、14、21、28、35、42、49、56d龄采血,检测淋巴细胞转化率和ND—HI抗体效价。试验结果表明：使用复方多糖、APS、ASP、EPS后,各试验组外周血淋巴细胞转化率和ND—HI抗体效价明显升高。复方多糖可使外周血淋巴细胞转化率和ND—HI抗体效价显著高于其他各多糖组。复方多糖、APS、ASP、EPS均可提高鸡的外周血淋巴细胞转化率和ND—HI抗体效价,其中复方多糖效果最好。     

参青球净散和桂枝治疗鸡球虫病的效果观察

考察自拟中药组方参青球净散和桂枝对鸡球虫病的治疗效果。采用人工感染小肠球虫混合卵囊制造鸡球虫病模型,将试验鸡分为6组,即参青球净散高剂量组、中剂量组、低剂量组,桂枝组,感染对照组,健康对照组。除感染对照组和健康对照组外,其余各组试验鸡于攻虫的当天开始给药,连用7 d。检测试验鸡相对增重率、血便记分、卵囊值、病变记分、抗球虫指数及血清相关酶活性等指标,综合评价药物疗效。结果表明,参青球净散组和桂枝组均可防止感染鸡体质量下降,但在精神状态、血便数量、采食量方面参青球净散组显著优于桂枝组(P<0.05);参青球净散高剂量组抗球虫指数大于160,达到良好水平,中剂量组、低剂量组和桂枝组的抗球虫指数均小于160,大于120,属于有效范围;各组血清乳酸脱氢酶(LDH)的活性显著升高(P<0.05);除对照组以外,其余各组的血清碱性磷酸酶(ALP)活性显著提高(P<0.05)。提示自拟中药组方参青球净散对鸡球虫病疗效确实。     

和田黑鸡与普通商品鸡淋巴细胞转化形态差异分析

根据普通商品鸡与未注射疫苗的和田黑鸡淋巴细胞转化率的对比,分析和田黑鸡机体与普通商品鸡免疫力的强弱。用密度梯度离心法分离出鸡外周血淋巴细胞,于37℃、5%的CO2培养箱中体外培养,如果其受到某种诱导剂,如有丝分裂原植物凝集素(PHA)、刀豆蛋白A(ConA)的诱导后,就会表现为细胞体积增大、代谢旺盛、蛋白质和核酸合成增加,即向淋巴母细胞转化和增殖,淋巴细胞转化率的高低则可以反映机体的免疫水平。试验发现,随着时间的递增,当用植物凝集素(PHA)、刀豆蛋白A(ConA)诱导和田黑鸡和普通商品鸡时转化率也呈现上升趋势,普通商品鸡淋巴细胞的转化率要大于和田黑鸡,且在72h后普通商品鸡与和田黑鸡的淋巴细胞LPS诱导后转化率都呈现递减趋势。该试验为动物疫苗免疫效果检测和细胞因子制剂的研究奠定了基础。     

Immune profile of infectious bursal disease. III. Effect of infectious bursal disease virus on the lymphocyte responses to phytomitogens and on mixed lymphocyte reaction of chickens

A whole-blood-culture technique was used to sequentially evaluated peripheral blood lymphocyte responses to phytohemagglutinin (PHA) and concanavalin A (Con A) of normal chickens and chickens infected at 1 day or 3 weeks of age with infectious bursal disease virus (IBDV). This method had numerous advantages over the more conventional techniques. A comparative study was made on the percentage of inhibition of responses of peripheral blood lymphocytes to PHA and Con A of 1-day- and 3-week-old IBDV-infected chickens. In both groups, there was a minimum inhibition between 3 and 4 weeks postinfection (PI) and a maximum inhibition at 6 weeks PI. A one-way mixed lymphocyte reaction (MLR) was performed using mitomycin-C-treated cells as stimulator cells obtained from chickens of genetically different strains. Lymphocytes from the experimental birds (control, 1-day-infected, and 3-week-infected groups) were used as the responder cells. The results showed that MLR response of the IBDV-infected chickens was significantly reduced compared with those of the uninfected controls. The degree of lowered response was much more severe in chickens infected at 1 day of age than in those infected at 3 weeks of age.     

阻断淋巴细胞糖皮质激素受体对鸡免疫功能的影响

为进一步证明糖皮质激素受体（ＧＲ）在介导淋巴细胞免疫功能中的作用,观察ＧＲ阻断鸡对马立克氏病病毒（ＭＤＶ）的感受性,用ＲＵ４８６阻断鸡淋巴细胞ＧＲ,当ＧＲ被阻断６０％以上时（ＲＵ４８６剂量为５０ｍｇ／ｋｇ）,鸡脾细胞诱生ＩＬ－２和ＩＦＮ活性及Ｔ、Ｂ细胞丝裂原刺激增殖活性均明显降低;外周血淋巴细胞、单核细胞、ＡＮＡＥ＋细胞减少;脾脏、法氏囊、胸腺重量与体重之比的相对重量减轻,淋巴细胞系统免疫功能呈低下状态。此时雏鸡对ＭＤＶ的易感性增高。当ＧＲ被阻断不足３５％时（ＲＵ４８６剂量为２５ｍｇ／ｋｇ）,对淋巴细胞系统免疫功能无影响。     

中药复方多糖对雏鸡B淋巴细胞免疫功能的影响

360只刚出壳的健康雏鸡,随机分为4组,每组90只,从出壳后第5天分别灌喂中药复方多糖、中药复方水煎剂、黄芪多糖、生理盐水,连喂7 d。于12、15、20、25、30、37、44、51日龄,每组随机取10只鸡。通过HA-HI实验、EA玫瑰花环实验,检测雏鸡B淋巴细胞的免疫功能;将鸡宰杀,完整摘取法氏囊,称重,测定法氏囊指数。实验结果表明,灌服中药复方多糖、复方水煎剂以及黄芪多糖的实验组,其抗体效价、EA玫瑰花环形成率、法氏囊指数值均提高,其中中药复方多糖效果较好,复方水煎剂效果优于黄芪多糖;中药复方多糖与复方水煎剂、黄芪多糖差异显著(P<0.05),与生理盐水对照组比较差异极显著(P<0.01)。     

Genetic Diversity Analysis of Three Newly Discovered Native Chicken Breeds in Yunnan Using mtDNA D-loop Region Sequences

This study was aimed to evaluate the information of their genetic background of Frizzle chicken (FM),Naked-neck chicken (CB) and YN chicken (YN) that were three newly discovered native chicken genetic resources with excellent characteristics,which had been found in Nujiang prefecture and mountainous area of Yunnan province.The variation in a total of 168 individuals sampled from the three chicken populations was assessed using the mitochondrial DNA (mtDNA) D-loop region sequences as genetic marker.The results showed that there were a total of 27 haplotypes were defined in the three native chicken as well as the haplotype diversity of these three chicken were 0.947,0.938 and 0.596,respectively.The nucleotide diversity of these three chicken were 0.01268,0.01434 and 0.00239,respectively.Phylogenetic tree displayed that all 168 individuals distributed in maternal lineage A,B,C,E,F and G.Frizzle chicken contained lineages E,F and G,and Naked-neck chicken contained all 6 lineages,of which the main lineages were E,F and G.YN chicken included lineages E,F and G,and lineage E was the highest percentage in this population.Also,it found that YN chicken was closely related to Gallus gallus murghi,White Plymouth Rock chicken,White Leghorn chicken and New Hampshires chicken,while Naked-neck chicken was closely related to Gallus gallus spadiceus,Gallus gallus jabouillei,Gallus gallus gallus Linnaeus,Indonesian cockfight and Laos chicken.The Frizzle chickens shared more haplotypes with Naked-neck chicken,and the evolutionary relationship between the two species was closer.This study provided a basis for the origin and genetic assessment of three newly discovered Yunnan local chicken breeds.     

免疫调节剂对球虫感染鸡的免疫学指标的影响

为探讨免疫调节剂对鸡球虫病的免疫反应调节作用 ,设计并进行了免疫调节剂对球虫感染鸡的免疫学指标影响的研究。测定指标包括 :淋巴细胞玫瑰花环细胞形成率 ,淋巴细胞转化率 ,红细胞免疫复合物花环形成率 ,红细胞 C3b受体花环形成率 ,脾脏重 /体重相对重 ;血清中 Ig G,Ig M,Ig A的 EL ISA P/ N值。结果各项免疫学指标的差异显著。表明胸腺素、黄芪多糖和卡介苗等免疫调节剂可提高球虫感染鸡的淋巴细胞玫瑰花形成率、淋巴细胞转化率。