黄芩苷对BALB/c小鼠无乳链球菌性乳腺炎治疗作用的研究

在建立小鼠乳腺炎标准动物模型的基础上,通过细菌计数、HE染色、酶联免疫吸附试验等方法研究了乳腺炎模型中无乳链球菌感染诱发的乳腺免疫反应,并在此基础上研究黄芩苷对乳腺炎的治疗效果。结果表明,模型组母鼠乳腺内的无乳链球菌在攻菌后24h达到峰值,随后逐渐下降。模型组乳腺组织在攻菌后12h出现明显脂肪变性,并随感染时间延长有加重趋势。模型组IFN-γ水平在攻菌后72h达到峰值,和生理盐水组相比差异极显著（P〈0.01）;模型组IL-4水平在攻菌后48h达到峰值后逐渐下降。IL-4水平急剧升高可能是促使乳腺急性炎症的主要原因之一。治疗组乳腺内细菌数在攻菌后24h、48h极显著低于模型组（P〈0.01）,此后治疗组细菌数逐渐下降,乳腺炎症明显减轻,全身症状逐渐缓解;治疗组IFN-γ水平在感染后48h、72h极显著高于模型组（P〈0.01）;治疗组IL-4水平在感染后24h、48h极显著低于模型组（P〈0.01）;由此可推断,黄芩苷能够抑制无乳链球菌在体内增殖来降低乳腺感染程度,并通过调节Th1/Th2平衡来减轻乳腺局部的炎症表现,说明黄芩苷对试验性小鼠乳腺炎有良好的治疗作用。     

粗糙型布鲁菌M111菌壳的安全性和免疫原性

以粗糙型布鲁菌M111株和重组裂解质粒制备出布鲁菌菌壳,利用小鼠模型对布鲁菌菌壳、布鲁菌M111活菌和福尔马林灭活菌的安全性和免疫原性进行比较研究。结果显示,与布鲁菌弱毒菌株M111比较而言,布鲁菌菌壳具有更好的安全性,免疫小鼠后能产生与弱毒菌株相似的血清抗体水平、脾CD3+和CD4+T淋巴细胞反应,甚至产生更高水平的IFN-γ。这些结果表明,布鲁菌菌壳具有与弱毒菌株相似的体液免疫和细胞免疫能力,将来可能作为预防布鲁菌感染的新型候选疫苗,但布鲁菌菌壳疫苗的有效性和特异性免疫机制还有待深入研究。     

布鲁菌外膜蛋白的糖基化及免疫效果检测

为研制安全、有效、新型靶向化布鲁菌外膜蛋白疫苗,采用去污剂连续抽提与电洗脱法分离纯化出布鲁菌外膜蛋白,并进行α-D-甘露吡喃异硫氰酸苯酯糖基修饰、佐剂乳化.依据统计学中完全随机实验设计试验,免疫BALB/c小鼠,收集不同时间点血液样品,ELISA法检测血清中IgG和IFN-γ含量.结果显示分离纯化出37.5 kD与47.2 kD的目的蛋白并成功糖基化修饰;纳米颗粒佐剂乳化后的试验用疫苗免疫小鼠,血清IFN-γ水平呈显著升高趋势(P＜0.05);抗体水平略显优势,但差异不显著(P＞0.05).表明试验用疫苗能激发机体的细胞免疫应答,且产生了免疫记忆.本研究为研制布鲁菌新型靶向化疫苗的研发和推广提供了研究依据.     

布鲁菌转录调节因子HFQ诱导机体免疫反应的分析

旨在分析布鲁菌(Brucella)转录调节因子HFQ诱导机体产生的免疫反应。以热灭活牛种布鲁菌S2308为模板,根据GenBank登录的S2308 hfq基因序列(BAB1_1134)设计引物,PCR扩增hfq基因片段后,将其克隆至原核表达载体pET-32a,转化大肠杆菌BL21(DE3)感受态细胞,诱导HFQ蛋白表达;利用SDS-PAGE电泳以及Western blot对重组HFQ蛋白(rHFQ)进行分析;pET-32a空载体、rHFQ和疫苗株M5-90刺激小鼠巨噬细胞RAW 264.7,利用ELISA试剂盒检测细胞因子IFN-γ和IL-4的表达水平;将pET-32a、rHFQ和M5-90免疫小鼠后,检测小鼠脾细胞中IFN-γ和IL-4的水平,以及小鼠血清中IgG抗体水平。结果显示,hfq基因大小为237 bp,编码79个氨基酸,rHFQ大约在25.8 ku处出现蛋白条带,纯化后为单一条带。Western blot结果显示,rHFQ具有较好的反应原性。rHFQ刺激RAW 264.7后,诱导IFN-γ和IL-4的水平与M5-90组相似,显著高于PBS组和pET-32a空载体组,且随着刺激时间的延长而升高。rHFQ免疫小鼠后,诱导脾细胞产生IFN-γ和IL-4的水平,小鼠血清中IgG的水平与M5-90组相似,显著高于PBS组和pET-32a空载体组。布鲁菌HFQ蛋白具有较好的反应原性,并能诱导机体产生较高的细胞免疫和体液免疫水平,是布鲁菌亚单位疫苗研制较理想的候选抗原。     

地锦草总黄酮对小鼠免疫功能及细胞因子mRNA表达影响

探讨地锦草总黄酮对小鼠免疫功能及细胞因子IL-2、IL-12、IFN-γ、TNF-αmRNA表达影响。将小鼠随机分成4组：地锦草总黄酮高剂量组（H组）、中剂量组（M组）、低剂量组（L组）和纯水对照组（C组）,连续灌胃9 d后,检测其单核巨噬细胞吞噬功能、2%绵羊红细胞诱导的小鼠迟发型变态反应、肝脏指数和脾脏指数,并用逆转录酶-聚合酶链式反应的方法检测脾脏IL-2、IL-12、IFN-γ和TNF-αmRNA表达水平。结果显示,地锦草总黄酮3个剂量组脾脏指数、廓清指数（K）、吞噬指数（α）、24 h足跖增厚值均显著高于C组;M组和H组肝指数显著高于C组（P〈0.01）;3个剂量组均能提高IL-2、IL-12、IFN-γ和TNF-αmRNA的表达水平,与C组相比,H组最显著。试验结果表明,地锦草总黄酮能有效提高机体的免疫功能及IL-2、IL-12、IFN-γ和TNF-αmRNA表达。     

铜绿假单胞菌oprL和oprF基因二价组合DNA疫苗初免-灭活疫苗加强免疫效果的研究

为探索铜绿假单胞菌oprL和oprF基因的二价组合DNA疫苗pOPRL+pOPRF初免-灭活疫苗加强免疫的效果,本实验以铜绿假单胞菌二价组合DNA疫苗pOPRL+pOPRF免疫BALB/c小鼠后再以灭活疫苗加强免疫,根据两种疫苗的免疫次数分为初免-加强免疫1组和初免-加强免疫2组,同时设置二价组合DNA疫苗单独免疫组、灭活疫苗单独免疫组以及生理盐水对照组。免疫后间接ELISA法测定小鼠血清抗体水平、MTT法检测小鼠脾淋巴细胞增殖水平,双抗体夹心ELISA测定小鼠脾淋巴细胞分泌的IFN-γ、IL-2和IL-4的水平。三免两周后将铜绿假单胞菌以1.35×10~(10)cfu/只的剂量对各组小鼠进行攻毒试验,测定攻毒后各组小鼠的保护率。结果显示DNA疫苗初免-灭活疫苗加强免疫诱导的小鼠血清抗体水平、刺激指数(SI)值及小鼠脾淋巴细胞分泌的IFN-γ和IL-2含量显著高于DNA疫苗单独免疫组(p0.05)。且初免-加强免疫1组小鼠的SI值及分泌的IFN-γ和IL-2含量显著高于灭活疫苗组(p0.05),但各组小鼠分泌的IL-4含量无显著差异(p0.05)。初免-加强免疫1组、初免-加强免疫2组、灭活疫苗组和DNA疫苗单独组的保护率分别为92%、80%、84%和72%。表明二价组合DNA疫苗初免-灭活疫苗加强免疫策略可以诱导小鼠产生较高水平的抗体和Th1型细胞免疫应答,并可为小鼠提供较好的保护率。     

负载重组口蹄疫病毒VP4蛋白的树突状细胞启动的T细胞应答

为研究负载口蹄疫病毒VP4蛋白的树突状细胞对淋巴结T细胞的活化效应,构建了pET32a-VP4原核表达载体,经过诱导表达和纯化获得重组VP4蛋白.同时制备骨髓源树突状细胞(BMDCs)和淋巴结T细胞,以VP4蛋白负载BMDCs后与淋巴结T细胞共培养.收集不同时间点的共培养上清液,用ELISA法检测其IFN-γ的含量.结果显示,负载VP4蛋白的BMDCs与T细胞共培养后3、9和48 h,试验组上清液中IFN-γ含量与对照组相比,均有极显著差异.这表明负载口蹄疫病毒VP4蛋白的BMDCs可有效激活淋巴结T细胞,使其分泌大量IFN-γ.     

含粘膜佐剂的鸡新城疫疫苗诱导雏鸡非特异性免疫应答的研究

为研究分析重组大肠杆菌不耐热肠毒素（labile enterotoxin,LT）作为粘膜佐剂,配伍鸡新城疫疫苗的冻干样品对免疫鸡非特异性免疫应答的影响,在前期已经构建的LTR72/G192基础上,选择2、4μg LTRG蛋白作为每羽份疫苗添加量,及降低新城疫病毒（Newcastle disease virus,NDV）50%抗原量等配伍条件。按照生产条件制备冻干疫苗,进行雏鸡免疫试验,免疫后24、48、72 h采集脾脏、胸腺淋巴结,检测几种非特异性细胞因子表达水平。结果表明：IL-6、INF-γ水平在各疫苗免疫后24h即升高,48 h达最高值;添加2、4μg LTRG蛋白的NDV疫苗组IFN-β、TNF-α水平在48 h左右显著升高,其中含4μg LTRG疫苗组与其他组差异具有极显著统计学意义（P〈0.01）;IL-6、IFN-γ转录水平有所增强,但各组间差异不具有显著统计学意义（P〉0.05）,72 h左右几种细胞因子水平均迅速下降,与NDV疫苗接种组相近。由此可见,LTRG能辅助抗原诱导免疫雏鸡的非特异性免疫应答,有利于对感染病毒的清除。     

阴道免疫对羊布鲁氏菌病疫苗抗体变化的影响

正布鲁氏菌病(简称布病)是严重危害养殖业和人体健康的一种人畜共患传染病,接种布鲁氏菌疫苗是防控该病的主要措施之一。在布鲁氏菌疫苗使用说明书中标明可以通过口服或肌肉注射等方式进行免疫。董炳梅等[1]经阴道途径给小鼠接种布鲁氏菌S2疫苗,然后检测树突状细胞等免疫因子的变化,认为阴道接种疫苗后树突状细胞明显增多,并能刺激免疫细胞产生γ干扰素(IFN-γ)。本研究尝试通过阴道途径对羊只进行布鲁氏菌病S2疫苗的免疫,然后检     

深度测序技术分析布鲁菌感染巨噬细胞RAW264.7早期转录组学变化

本研究试图寻找参与布鲁菌胞内感染相关的宿主相关基因,为从感染宿主角度阐述布鲁菌的致病机制奠定基础.布鲁菌感染小鼠巨噬细胞后,利用数字基因表达谱技术筛选小鼠巨噬细胞感染布鲁菌16M株的差异表达基因,并利用荧光定量PCR对差异表达基因进行验证.差异表达基因经GO Term、KEGG分析,识别感染后显著富集的信号通路.在感染后4h,筛选出差异表达基因3 576个,其中58％的基因表现上调.并且NOD凋亡信号通路、溶酶体信号通路、NOD受体信号通路、FcγR-介导的吞噬通路、p53信号通路、内质网蛋白处理相关通路被显著富集.利用数字基因表达谱技术成功分析巨噬细胞感染布鲁菌后转录组学变化,为布鲁菌致病机制的逐步阐述奠定基础.     

布鲁氏菌M5侵染小鼠巨噬细胞一氧化氮和非对称性二甲基精氨酸含量的测定

本试验旨在研究布鲁氏菌侵染小鼠巨噬细胞过程中一氧化氮(NO)对布鲁氏菌的抑制作用,以及NO和非对称性二甲基精氨酸(ADMA)的相互作用关系。以布鲁氏菌标准疫苗株M5侵染小鼠巨噬细胞,采用Griess试剂法和酶联免疫吸附法(ELISA)测定细胞内外NO含量和ADMA水平,并对各个侵染时间段进行CFU计数。结果显示,被布鲁氏菌侵染后巨噬细胞的NO含量呈上升趋势,且胞外含量显著高于胞内(P<0.05),与对照组相比均差异显著(P<0.05);而巨噬细胞的ADMA含量随着侵染时间的增加与NO含量呈负相关,与对照组相比均差异极显著(P<0.01)。结合CFU计数结果,表明NO对布鲁氏菌的抑制作用只发生在侵染前期(12 h前),在侵染后期(12 h后)NO对布鲁氏菌的生长并未起到抑制作用,而ADMA在布鲁氏菌侵染小鼠巨噬细胞过程中对NO的生成有一定的抑制作用。     

Alternative strategy for visceral leishmaniosis control: HisAK70-Salmonella Choleraesuis-pulsed dendritic cells

Here, we describe a novel approach that exploits an attenuated mutant of Salmonella enterica serovar Choleraesuis as carrier to deliver a plasmid encoding protein HisAK70. Subsequently, dendritic cells (DCs) were pulsed with this vaccine vector. The aim of this study was to evaluate the effectiveness of the prepared HisAK70-S. Choleraesuis-pulsed DCs (HisAK70-SAL DCs) against visceral leishmaniosis (VL). In our ex vivo model of infection, the prepared formulations could decrease parasite growth by up to 80% by augmenting the production of IL-12p40 and by reducing arginase activity (ARG). Also, BALB/c mice when immunised with this formulation showed significant reduction in parasite burden in both spleen (20% of reduction) and liver (75% of reduction). The balance of the immune ratios IFN-γ/IL-10, TNF-α/IL-10, and IgG2a/IgG1 reflected the acquisition of an improved resistant phenotype in HisAK70-SAL DCs vaccinated mice compared to control mice. Our results suggest that HisAK70-SAL DCs could be a promising alternative approach for vaccine delivery that has the potential to fight Leishmania infantum (L. infantum) infection.     

非洲猪瘟病毒P72蛋白合成肽疫苗的构建及其免疫效力评估

【目的】构建非洲猪瘟病毒CAS19-01/2019株(GenBank登录号:MN172368.1)结构蛋白P72的合成肽疫苗,通过免疫小鼠评估合成肽疫苗的免疫效力。【方法】利用ProtParam、SOPMA等软件分析P72蛋白的理化性质与结构信息,通过ABCpred、SVMtrip、IEDB预测P72蛋白的T细胞与B细胞抗原表位,筛选出显著的表位多肽区域,合成多肽辅以弗氏佐剂腹腔注射免疫小鼠,检测免疫组小鼠产生的特异性抗体、T淋巴细胞亚群、脾脏淋巴细胞增殖、细胞因子白介素4(IL-4)、IL-2、γ干扰素(IFN-γ)、免疫球蛋白(IgG),从体液免疫与细胞免疫角度评估合成肽的免疫效力。【结果】综合分析得出P72蛋白是稳定性亲水蛋白,二级结构中α-螺旋、β-转角、延伸链、无规则卷曲分别占19.35%、5.42%、25.08%和50.15%。筛选出了P72蛋白的8个优势抗原表位,626－634、520－528、298－306、203－211位氨基酸处为T细胞抗原表位,587－606、232－251、110－129、39－58位氨基酸处为B细胞抗原表位。整合优势表位合成2个多肽P72-1与P72-2,首次免疫小鼠14 d时可检测到P72-1与P72-2的特异性抗体,首免后28 d达到最高值,其最高抗体效价分别为1∶25 600与1∶12 800;免疫后小鼠T淋巴细胞亚群CD4^＋/CD8^＋显著上升(P＜0.05);脾脏淋巴细胞增殖试验结果显示,免疫组淋巴细胞数量均极显著升高(P＜0.01);细胞因子IL-4、IL-2、IFN-γ含量均极显著增加(P＜0.01)。【结论】本研究成功研制2种合成肽疫苗,在免疫效力上P72-2高于P72-1,二者都能产生高水平的特异性抗体,刺激脾脏淋巴细胞增殖,诱导产生细胞因子IL-4、IL-2、IFN-γ,本研究为非洲猪瘟合成肽疫苗研制奠定技术基础。     

布鲁氏菌Ⅳ型分泌系统对树突状细胞自噬、胞内生存和炎症反应的影响

为探究Ⅳ型分泌系统在布鲁氏菌(Brucella)感染过程中的作用,深入了解布鲁氏菌Ⅳ型分泌系统在疫苗开发中的潜力,本研究以牛种布鲁氏菌A19疫苗株为研究对象,使用A19 VirB启动子缺失株感染小鼠树突状细胞(DCs),通过菌落计数(CFU)评估Ⅳ型分泌系统对布鲁氏菌黏附侵袭及胞内生存的影响,同时对感染的细胞进行RNA和总蛋白的提取,分别通过实时荧光定量PCR和Western blotting检测自噬基因Beclin-1的转录和表达情况;收集感染后的细胞上清液,利用ELISA检测炎症因子白细胞介素-6(IL-6)和IL-10的分泌水平。黏附侵袭结果显示,布鲁氏菌VirB启动子缺失株与亲本株A19的黏附侵袭水平无显著差异(P>0.05);胞内生存试验发现,感染的4 h,布鲁氏菌VirB启动子缺失株的胞内存活能力显著低于亲本株A19(P<0.05),感染后0、24和48 h极显著低于亲本株A19(P<0.01);实时荧光定量PCR和Western blotting结果显示,感染后4、8和12 h,布鲁氏菌VirB启动子缺失株刺激细胞产生Beclin-1的水平极显著高于亲本株A19(P<0.01);ELISA结果显示,感染后8和12 h,布鲁氏菌VirB启动子缺失株刺激细胞产生IL-6的水平显著高于亲本株A19(P<0.05),而在感染后8和12 h,布鲁氏菌VirB启动子缺失株刺激细胞分泌IL-10的水平显著低于亲本株A19(P<0.05),感染24 h时极显著低于亲本株A19(P<0.01)。综上所述,当VirB启动子缺失后,布鲁氏菌对DCs的黏附侵袭能力并未明显改变,但显著降低了布鲁氏菌在DCs内的存活能力,提升了DCs的自噬水平,促进了DCs IL-6的分泌,抑制了IL-10的分泌。本研究初步探究了布鲁氏菌Ⅳ型分泌系统在感染DCs过程中的生物学作用,为后续布鲁氏菌疫苗改造研究奠定了理论基础。     

Bacterial survival, lymph node changes, and immunologic responses of cattle vaccinated with standard and mutant strains of Brucella abortus.

Forty-eight cattle were used in 4 experiments; 6-week-old calves in experiments 1-3 (n = 24) and 10-month-old heifers in experiment 4 (n = 24). In experiments 1-3, 7 groups of 3 calves each were inoculated SC with 5 strains of Brucella abortus: virulent strain 2308 (2 groups), vaccine strain 19 (2 groups), and mutant strains RB51. 19 delta 31K, and 19 delta SOD. Sera and lymph node tissues were examined at 2-week intervals for evidence of infection. At postinoculation (PI) week 12, 2 calves in each group were given dexamethasone for 5 days. Calves were then euthanatized and lymphoid tissue, spleen, liver, and bone marrow were examined for evidence of B abortus. Calves given strain 2308 had large numbers of bacteria in their lymph nodes, marked granulomatous lymphadenitis in the deep cortex, and loss of lymphoid cells in superficial cortical areas. In addition, they had high serum antibody titers at PI week 16. Calves given strain 19, or genetic mutants derived from strain 19, cleared bacteria from lymph nodes more rapidly, had less lymphoid destruction, and developed antibody titers that did not persist for 16 weeks. The RB51 strain (rough) was cleared most rapidly from lymphoid tissues and induced serum antibody responses only to the core of the lipopolysaccharide molecule. Treatment of calves with dexamethasone did not cause B abortus to reappear in tissues of any calves, nor did serum antibody titers increase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diagnostic characterization of a feral swine herd enzootically infected with Brucella.

Eighty feral swine were trapped from a herd that had been documented to be seropositive for Brucella and which had been used for Brucella abortus RB51 vaccine trials on a 7,100-hectare tract of land in South Carolina. The animals were euthanized and complete necropsies were performed. Samples were taken for histopathology, Brucella culture, and Brucella serology. Brucella was cultured from 62 (77.5%) animals. Brucella suis was isolated from 55 animals (68.8%), and all isolates were biovar 1. Brucella abortus was isolated from 28 animals (35.0%), and isolates included field strain biovar 1 (21 animals; 26.3%), vaccine strain Brucella abortus S19 (8 animals, 10.0%), and vaccine strain Brucella abortus RB51 (6 animals, 7.5%). Males were significantly more likely to be culture positive than females (92.9% vs. 60.6%). Thirty-nine animals (48.8%) were seropositive. Males also had a significantly higher seropositivity rate than females (61.9% vs. 34.2%). The relative sensitivity rates were significantly higher for the standard tube test (44.6%) and fluorescence polarization assay (42.6%) than the card agglutination test (13.1%). Lesions consistent with Brucella infection were commonly found in the animals surveyed and included inflammatory lesions of the lymph nodes, liver, kidney, and male reproductive organs, which ranged from lymphoplasmacytic to pyogranulomatous with necrosis. This is the first report of an apparent enzootic Brucella abortus infection in a feral swine herd suggesting that feral swine may serve as a reservoir of infection for Brucella abortus as well as Brucella suis for domestic livestock.     

Treatment of cattle with DNA-encoded Flt3L and GM-CSF prior to immunization with Theileria parva candidate vaccine antigens induces CD4 and CD8 T cell IFN-γ responses but not CTL responses

Theileria parva antigens recognized by cytotoxic T lymphocytes (CTLs) are prime vaccine candidates against East Coast fever in cattle. A strategy for enhancing induction of parasite-specific T cell responses by increasing recruitment and activation of dendritic cells (DCs) at the immunization site by administration of bovine Flt3L and GM-CSF prior to inoculation with DNA vaccine constructs and MVA boost was evaluated. Analysis of immune responses showed induction of significant T. parva-specific proliferation, and IFN-γ-secreting CD4(+) and CD8(+) T cell responses in immunized cattle. However, antigen-specific CTLs were not detected. Following lethal challenge, 5/12 immunized cattle survived by day 21, whereas all the negative controls had to be euthanized due to severe disease, indicating a protective effect of the vaccine (p<0.05). The study demonstrated the potential of this technology to elicit significant MHC class II and class I restricted IFN-γ-secreting CD4(+) and CD8(+) T cells to defined vaccine candidate antigens in a natural host, but also underscores the need to improve strategies for eliciting protective CTL responses.     

猪圆环病毒核酸疫苗pcDNA-PCV-ORF2-ISS的构建及分子佐剂联合免疫研究

用PCR方法扩增猪圆环病毒PCVwhn株编码CAP蛋白的基因ORF2,同时人工合成一段CpG序列,并设计上下游引物,进行扩增,定向插入到真核表达载体pcDNA3.1(+)中,构建新型核酸疫苗(pcDNA-PCV-ORF2-ISS),并进行了酶切和测序鉴定.用构建的pcDNA-PCV-ORF2-ISS质粒以及pcDNA-PCV-ORF2与5种细胞因子分子佐剂质粒(IL-6/4、IL-2、IL-4、IL-6、IFN-γ真核表达质粒)联合对仔猪进行免疫试验.免疫后用间接ELISA法测定猪圆环病毒Ⅱ型特异性抗体IgG,用流式细胞仪测定猪血液CD3+、CD4+和CD8+T细胞亚类的数量.结果显示,成功构建含CpG免疫刺激序列的猪圆环病毒Ⅱ型核酸疫苗;pcDNA-PCV-ORF2-ISS在仔猪免疫实验能诱导细胞免疫并产生特异性抗体(P<0.05);用5种细胞因子分子佐荆质粒与pcDNA-PCV-ORF2疫苗同时免疫能显著提高DNA疫苗的免疫效果(P<0.05).分子佐剂免疫增强效果次序为:IL-6、CpG、IL-6/4、IL-2、IL-4和IFN-γ,表明IL-6和CpG最佳.     

Cell-mediated immune response in swine infected with Mycobacterium avium subsp. avium

The zoonotic characteristic of Mycobacterium avium subsp. avium (MAA) represents a veterinary and economic problem in infected pigs. In this study, we analysed cell-mediated immunity six months after experimental infection by measuring interferon-γ (IFN-γ) production and by performing lymphocyte transformation tests after in vitro re-stimulation with the MAA-derived antigen. At the same time, IFN-γ-producing cells were characterised by flow cytometry. In MAA-infected animals, the production of IFN-γ increased in response to the MAA antigen in the blood, spleen and mesenteric lymph nodes. Similarly, a positive antigen-driven response was detected by the proliferation assay. In contrast, IFN-γ production and proliferation was undetectable after stimulation with the MAA antigen in uninfected control animals. These results indicate that both methods can be used for the identification of individual MAA-infected pigs. Using flow cytometry, we found that double-positive CD4(+)CD8(+) lymphocytes were the major T lymphocyte subset producing IFN-γ after in vitro re-stimulation.