鸡食管扁桃体的组织学和免疫组织化学研究

本研究通过组织学和免疫组织化学方法,观察鸡新型肠相关性淋巴组织-食管扁桃体的组织结构特征。并研究其中T淋巴细胞及其亚群和抗体生成细胞的定位分布。研究发现,食管扁桃体的粘膜形成数目和结构与食管一致的皱襞,粘膜固有层中分布大量的淋巴组织,并存在于前胃腺;在隐窝固有层中,抗体生成细胞以IgM^＋和IgA^＋细胞为主,并主要分布在生发中心中,而CD3^＋、CD4^＋和CD8^＋T淋巴细胞也有大量分布,主要存在于生发中心周围的滤泡间区域;在皱襞固有层中,各种淋巴细胞都分布在上皮下固有层中,数量明显少于隐窝固有层中的相应阳性细胞,抗体生成细胞以IgA^＋细胞为主,T淋巴细胞则以CD3和CD4为主。研究表明：食管扁桃体位置特殊,将接触到大量的外界未消化抗原,并且它具有参与粘膜免疫的组织基础和细胞基础,此研究结果对食物过敏、免疫耐受和肠道感染的研究具有重要意义。     

鸡十二指肠中T淋巴细胞及其亚群发育的研究

通过免疫组织化学法,应用CD3、CD4和CD8单克隆抗体研究了CD3+T淋巴细胞及CD4+和CD8+T淋巴细胞亚群在鸡盲肠扁桃体中的出现、迁移、组织定位分布及数量变化规律等一系列发育过程。结果显示:CD3+、CD8+T淋巴细胞最初于11胚龄时出现,而CD4+T淋巴细胞在15胚龄时出现。在定位分布变化上,CD3+主要分布在黏膜上皮中,CD4+主要分布在黏膜固有层中,CD8+在黏膜固有层和黏膜上皮内都有大量分布。在数量变化上,1～7日龄雏鸡各种阳性细胞的数量都骤然增加;而到7日龄时,雏鸡CD3+淋巴细胞的数量明显减少,CD4+、CD8+T细胞的数量无明显变化;从21日龄开始直到35日龄,雏鸡CD3+、CD4+和CD8+T淋巴细胞的数量持续增加。试验证明,鸡在出壳后初期十二指肠的细胞免疫功能增强。     

鸡回肠中T淋巴细胞及其亚群发育的试验

为探寻鸡回肠中T淋巴细胞及其亚群的发育规律,本试验通过免疫组织化学方法,应用CD3、CD4和CD8单克隆抗体研究鸡回肠中T淋巴细胞及其亚群出现、迁移、定位分布及数量变化过程.结果显示,CD3+、CD8+T淋巴细胞最初于18胚龄时出现,CD4+T淋巴细胞于出壳后1日龄时出现.在定位分布上,CD3+细胞在黏膜上皮内以及固有层中均匀分布,CD4+细胞以固有层中的分布为主,黏膜上皮内的分布较少.CD8+细胞最初主要分布在黏膜固有层中;随后,CD8+细胞逐渐向上皮内迁移;最终,黏膜上皮内出现广泛的CD8+细胞浸润.在数量变化上,CD3+、CD4+及CD8+整体呈逐渐增加趋势,第2周时阳性细胞数量稍有下降,21日龄时显著增加到达较高水平后保持稳定.结果表明,鸡出壳后,回肠的细胞免疫功能逐渐增强,并在21日龄时到达成熟水平.     

不同日龄鸡喉黏膜淋巴组织中的T和B淋巴细胞分布检测

为了解鸡喉黏膜不同时期的免疫状态,采集不同日龄海兰白鸡的喉组织,利用免疫组织化学方法研究CD3+ T淋巴细胞和Bu-1+ B淋巴细胞的出现、定位分布及数量变化过程。结果显示：4日龄时,CD3+ T淋巴细胞和Bu-1+ B淋巴细胞都首次出现在喉黏膜中,而且都主要分布在喉腔底壁的正中黏膜嵴中;7日龄时喉黏膜中CD3+ T淋巴细胞和Bu-1+ B淋巴细胞急剧增多,并形成淋巴聚集物,CD3+细胞占据淋巴聚集物的中下部区域,而Bu-1+细胞主要位于淋巴聚集物外周;14日龄时Bu-1+细胞虽仍主要分布于CD3+细胞的外周,但分界不如7日龄时明显,有部分Bu-1+细胞穿插分布于CD3+细胞之间;21日龄和35日龄时,喉黏膜固有层中淋巴细胞更为集中;56日龄时,黏膜固有层底端出现主要由Bu-1+ B淋巴细胞构成的生发中心,而CD3+ T淋巴细胞则分布在生发中心周围的滤泡间区域。在数量变化上,随着日龄增长,CD3+ T淋巴细胞和Bu-1+ B淋巴细胞数量均逐渐增加,且14日龄之前CD3+细胞数量多于Bu-1+细胞,而此后各日龄Bu-1+细胞的数量均显著多于CD3+细胞（P＜0.05）。结果表明,鸡喉黏膜中T、B淋巴细胞的分布和数量均呈现日龄相关性变化,并且其变化可以反映出14日龄之前喉黏膜以细胞免疫为主,而之后则倾向于体液免疫。本试验为进一步研究家禽喉黏膜的免疫机制奠定了基础。     

鸡直肠中T淋巴细胞的发育研究

通过免疫组织化学方法,应用CD3、CD4和CD8单克隆抗体研究了CD3＋、CD4＋和CD8＋B淋巴细胞在鸡直肠中的出现、迁移、组织定位分布以及数量变化规律等一系列发育过程。T淋巴细胞均在雏鸡出壳后7日龄时出现。在定位分布变化上,出壳后1～4日龄时,T淋巴细胞数量没有明显增加,7日龄时,CD4＋大量分布在肠绒毛皮下固有层中,少量分布在黏膜肌层和黏膜固有层中,CD3＋分散分布在黏膜肌层和黏膜固有层中,极少数的CD8＋出现在黏膜肌层中。随着日龄的增长,T淋巴细胞的数量持续增多,多数位于黏膜固有层,雏鸡生长至21日龄时,T淋巴细胞的数量维持稳定,其中数量以CD4＋细胞最多,CD3＋细胞次之,CD8＋细胞最少。鸡出壳后初期直肠的体液免疫逐渐增强,并在21日龄时达到成熟水平。     

鸡嗉囊组织结构及其淋巴组织分布的观察

本研究旨在对鸡嗉囊组织学形态及其淋巴组织的分布进行观察并与食管进行比较。选用27日龄海兰白鸡,无菌操作分离嗉囊及食管,制作冰冻切片,进行常规HE染色。结果显示,鸡嗉囊黏膜上皮为复层扁平上皮,相比于食管,角质化明显,黏膜皱襞数量更多,伸缩性大;同食管相似,嗉囊固有层中分布着大量的食管腺,但分布不均一,由腺细胞围成的管泡状腺直接开口于黏膜上皮,分泌大量的黏液;另外发现,在皱襞和隐窝的固有层中,大量淋巴细胞密集聚在黏液腺周围,甚至突入腺体内,从而使黏液腺上皮转化为淋巴上皮。结果显示,嗉囊在对摄入食物的贮存和浸润起着非常重要的作用,同时嗉囊位于消化道上段,其中的淋巴组织将对刚刚进入消化道的病原微生物起着重要的免疫防御作用。     

猪呼吸道淋巴组织发育的亚显微结构变化

应用组织学和电镜技术研究猪呼吸道发育过程中淋巴组织的变化。结果表明：扁桃体和咽部是呼吸道进入机体的第一个淋巴组织集中的部位，弥散淋巴组织在出生时就存在，淋巴小结不明显；20日龄时扁桃体中淋巴组织增生，淋巴小结清晰可见；120日龄淋巴小结数量增加，紧靠鳞状上皮密集排列，淋巴小结发育很好，并出现生发中心。扁桃体复层鳞状上皮中含有大量的上皮内淋巴细胞。气管叉是呼吸道进入机体的第二个淋巴组织集中的部位，出生时气管叉外膜中淋巴组织直接与气管支气管淋巴结相连，淋巴组织明显可见。20日龄时气管叉外膜中淋巴组织已分开，形成气管叉外膜密集的淋巴组织和气管支气管淋巴结两个部分。120日龄时气管叉处淋巴组织特别发达，黏膜上皮中上皮内淋巴细胞数量也显著增加。肺内气管和细支气管固有膜中均有较多的淋巴细胞，其中浆细胞数量增加，上皮中仍存在少量的上皮内淋巴细胞。本试验结果提示猪呼吸道是黏膜免疫较理想的诱导位点和效应位点，新生仔猪通过鼻腔免疫可提高呼吸道局部黏膜免疫力。     

鸡脾脏中B淋巴细胞的发育及其定位分布

应用IgM、IgG和IgA单克隆抗体的免疫组织化学方法,研究IgM+、IgG+和IgA+B淋巴细胞在鸡脾脏中的出现、迁移、数量变化规律等一系列的发育过程以及组织定位分布特点。结果显示,IgM+和IgA+细胞在胚胎15日龄时开始出现,IgG+细胞在胚胎18日龄时出现,21日龄雏鸡脾脏的红髓和生发中心中出现以IgG型为主的浆细胞。各日龄IgM+、IgG+和IgA+细胞主要分布在脾脏的特征性组织结构—椭球周围淋巴鞘和生发中心中,这些结构也随着B淋巴细胞的增多而不断发育成熟。B淋巴细胞的数量随着日龄增长持续增加,直至21日龄时趋于稳定,各日龄B淋巴细胞数量始终以IgG+细胞最多,IgM+细胞次之,IgA+细胞最少。结果表明,鸡出壳初期,脾脏的体液免疫功能逐渐增强,并在21日龄时达到成熟水平。     

感染传染性法氏囊病病毒雏鸡免疫器官组织的免疫学变化

一日龄雏鸡感染传染性发法氏囊病病毒(IBDV)后,免疫器官组织(法氏囊、胸腺、脾脏、盲肠扁桃体、丕氏斑、十二指肠粘膜固有层及支气官粘膜固有层淋巴组织、哈德尔腺)和局部组织中浆细胞、酯酶阳性T细胞(TANAE)和淋巴细胞数显著降低。表明一日龄感染IBDV雏鸡、其中枢及外周免疫器官以及呼吸道、消化道、局部免疫组织的体液免疫和细胞免疫均呈现抑制。     

板蓝根多糖对缺乳仔鼠免疫器官发育及T淋巴细胞亚群的影响

应用流式细胞仪检测缺乳仔鼠CD3+、CD4+和CD8+T淋巴细胞含量,研究添加不同剂量的板蓝根多糖(RIP)对缺乳仔鼠免疫器官及T淋巴细胞亚群的影响。结果表明,RIP可以提高缺乳仔鼠胸腺指数和脾脏指数,对胸腺指数和脾脏指数效果最佳的RIP剂量随日龄增大而减小;RIP可以增加缺乳仔鼠外周血CD3+、CD4+、CD8+T淋巴细胞数量,CD3+T淋巴细胞数量在7-28日龄时,A、C、D组与B组差异显著(P0.05);CD4+T淋巴细胞数量在7-21日龄时,A、C、D组与B组差异显著(P0.05);各处理组CD3+、CD4+、CD8+T淋巴细胞数量以及CD4+/CD8+比值随着日龄的增加有所下降。结果表明,一定剂量的RIP可以促进缺乳仔鼠免疫器官发育,提高T淋巴细胞亚群的水平。     

A morphologic and immunohistochemical study of the bronchus-associated lymphoid tissue of pigs naturally infected with Mycoplasma hyopneumoniae

Porcine enzootic pneumonia (PEN), caused by Mycoplasma hyopneumoniae (Mh), has been described in pigs in all geographic areas. The disease is characterized by high morbidity and low mortality rates in intensive swine production systems. A morphologic and immunohistochemical study was done to determine the cellular populations present in lung parenchyma of infected pigs, with special attention to the bronchus-associated lymphoid tissue (BALT). Polyclonal and monoclonal antibodies were used for the detection of antigens of Mh, T lymphocytes (CD3+, CD4+, and CD8+), IgG+ or IgA+ lymphocytes, and cells containing lysozyme, S-100 protein, major histocompatibility complex class II antigen or myeloid-histiocyte antigen. Findings in lung tissues associated with Mh infection were catarrhal bronchointerstitial pneumonia, with infiltration of inflammatory cells in the lamina propria of bronchi and bronchioles and alveolar septa. Hyperplasia of mononuclear cells in the BALT areas was the most significant histologic change. The BALT showed a high morphologic and cellular organization. Macrophages and B lymphocytes were the main cellular components of germinal centers. T lymphocytes were primarily located in perifollicular areas of the BALT, lamina propria and within the airway epithelium, and plasma cells containing IgG or IgA at the periphery of the BALT, in the lamina propria of bronchi and bronchioles, in alveolar septa, and around bronchial submucosal glands. The hyperplastic BALT in PEN cases consisted of macrophages, dendritic cells, T and B lymphocytes, and IgG+ and IgA+ plasma cells. CD4+ cells predominated over CD8+ cells. Local humoral immunity appears to play an important role in the infection.     

The effect of Lactobacillus acidophilus and Bifidobacterium spp. administration on the morphology of the gastrointestinal tract, liver and pancreas in piglets

The aim of the study was to investigate the influence of administration of probiotic bacteria on morphology of the gastrointestinal tract, liver and pancreas. The experiment was performed on 15 piglets at the age from 3 to 35 days, intragastrically administered with Bifidobacterium breve, B. animalis, and Lactobacillus acidophilus bacteria. In all piglets examined, the development of the gastrointestinal tract, liver and pancreas was observed to progress normally. After microflora administration to the piglets, an increase in the number of fibrocytes and fibroblasts was observed in the mucosa lamina propria of stomach and intestines. An increase was also reported in the number and activation of endocrine cells in the stomach and small intestines. The activity of alkaline and acid phosphatases, as well as succinic (SDH) and lactic (LDH) dehydrogenases, was found to be higher after the administration of probiotics. The administration of bacteria, especially of Lactobacillus acidophilus, caused an increase in the number of lymphocytes and lymphoid cells in lamina propria and intraepithelial lymphocytes in the small intestine. Enhanced proliferation of crypt cells was observed in the crypts of intestinal glands; however, there were no statistically significant differences in the PCNA index between the control and probiotic-administered groups. The performed study showed that the administration of probiotic bacteria has no negative impact on the morphology of the gastrointestinal tract, liver and pancreas and is found beneficial to its functioning and immune processes.     

Proventriculitis in broiler chickens: immunohistochemical characterization of the lymphocytes infiltrating the proventricular glands

Broiler chickens with transmissible proventriculitis have severe lymphocytic infiltration of the proventricular glands. The distribution of T cells and B cells in these infiltrates was studied histopathologically, and their identity was confirmed immunohistochemically (CD3, CD4, CD8, and B cells). To reproduce this disease, 1-day-old commercial boilers were orally gavaged with homogenized proventriculi from broilers with proventriculitis. Resulting lesions were examined at both acute (7 days postinoculation [i]) and chronic (14 and 21 dpi) time points. Lymphocytic infiltrates in the proventricular glands and the mucosal lamina propria were present at all time points and were most prominent and demarcated at 14 dpi. T and B lymphocytes were present during acute and chronic proventriculitis, but their distribution varied within the glands. Lymphocytic infiltrates in the proventricular glands and in the lamina propria were predominantly CD3+T cells, and most of these were also CD8+. B cells and CD4+ T cells formed aggregates in chronic proventriculitis. Thus, both cell-mediated and humoral immune responses are induced during transmissible proventriculitis, and the cell-mediated immune response is morphologically greater.     

Flow cytometric analysis of canine colonic mucosal lymphocytes from endoscopically obtained biopsy specimens

OBJECTIVE: To validate use of canine colonic biopsy specimens obtained via endoscopy as a source of mucosal lymphocytes (ML) for flow cytometric analysis. SAMPLE POPULATION: Mucosal biopsy specimens from 10 adult dogs. PROCEDURE: Mucosal lymphocyte subsets obtained from excised colon were compared with ML subsets obtained from biopsy specimens obtained by use of an endoscopic forceps (6 dogs). Endoscopic colonic biopsy specimens from 4 other dogs were used to define whether obtained ML were predominantly of intraepithelial or lamina propria origin. Mucosal lymphocytes were isolated and labeled, using commercially available monoclonal antibodies directed against canine cell surface antigens. Lymphocyte subsets (cytotoxic or helper T cells; B cells) were determined by use of flow cytometric analysis. RESULTS: A large number of viable ML was obtained after dissociation of the colonic epithelium from excised colon (45.5 + 21.5 X 10(6)) and endoscopic (7.2+/-3.4 X 10(6)) biopsy specimens. Lymphocyte subsets obtained with both methods were identical for each dog and consisted predominantly of intraepithelial lymphocytes, with some lymphocytes from the lamina propria. Collagenase digestion of excised colon also yielded a large number of viable lymphocytes from the lamina propria (56.7+/-20.4 X 10(6)), but collagenase digestion of endoscopic biopsy specimens was less rewarding. CONCLUSION AND CLINICAL RELEVANCE: A representative sample of viable intraepithelial ML is obtainable from endoscopic biopsy specimens. Flow cytometric analysis, a minimally invasive technique, can be used to study ML of client-owned animals.     

Feline gastrointestinal lymphoma: mucosal architecture, immunophenotype, and molecular clonality

Gastrointestinal lymphomas were identified in 120 cats between 1995 and 2006. Lymphomas were classified according to the World Health Organization (WHO) scheme. Cats with mucosal T-cell lymphoma (n = 84) predominated and had a median survival of 29 months. Mucosal T-cell lymphoma matched WHO enteropathy-associated T-cell lymphoma (EATCL) type II. Epitheliotropic T-cell infiltrates were present in 62% of cats and occurred as clusters or diffuse infiltrates of small to intermediate-sized T cells in villous and/or crypt epithelium. Similar lymphocytes infiltrated the lamina propria in distinctive patterns. Cats with transmural T-cell lymphoma (n = 19) had a median survival of 1.5 months. Transmural T-cell lymphoma matched WHO EATCL type I. Epitheliotropic T-cell infiltrates were present in 58% of cats. Large lymphocytes (n = 11), mostly with cytoplasmic granules (LGL-granzyme B+) (n = 9) predominated. Transmural extension across the muscularis propria characterized the lesion. Both mucosal and transmural T-cell lymphomas were largely confined to the small intestine, and molecular clonality analysis revealed clonal or oligoclonal rearrangements of T-cell receptor-γ in 90% of cats. Cats with B-cell lymphoma (n = 19) had a median survival of 3.5 months. B-cell lymphomas occurred as transmural lesions in stomach, jejunum, and ileo-cecal-colic junction. The majority were diffuse, large B-cell lymphomas of centroblastic type. In conclusion, T-cell lymphomas characterized by distinctive mucosal architecture, CD3 expression, and clonal expansion predominated in the feline gastrointestinal tract.     

Mycotoxicosis caused by aerosolized T-2 toxin administered to female mice

Thymus, spleen, adrenal glands, and small intestine of female mice exposed to aerosolized T-2 mycotoxin were examined at postexposure hours (PEH) 0.25, 1, 2, 4, 6, 9, 12, and 24. Lymphocyte necrosis was observed at PEH 1 in the thymus, spleen, and lamina propria and Peyer patches of the small intestine. Necrosis of small intestinal crypt epithelial cells was observed at PEH 2, and necrosis of parenchymal cells and increased number of neutrophils were seen in sinusoids of the adrenal cortex at PEH 4. These results indicated that the earliest microscopic evidence of T-2 mycotoxicosis after aerosol exposure was necrosis of lymphocytes in the thymus, spleen, and lamina propria and Peyer patches of the small intestine.     

A comparison of the effects of dietary spray-dried bovine colostrum and animal plasma on growth and intestinal histology in weaner pigs

An experiment was conducted to evaluate the effects of dietary spray-dried bovine and porcine plasma and spray-dried bovine colostrum on growth performance and intestinal histology in weaner pigs. Thirty-two 21-day-old piglets (6.65 ± 0.14 kg) were allocated to receive one of four dietary treatments: control, bovine plasma, porcine plasma, and bovine colostrum at weaning and another 8 piglets were killed at weaning to provide baseline data. The experimental diets were offered ad libitum for one week, after which animals were killed and post-mortem measurements obtained. No differences in average daily feed intake and growth rate were observed among dietary treatment groups (P > 0.05). Baseline piglets had taller villi and shallower crypts (P < 0.05) in the proximal jejunum, mid jejunum and distal ileum than those observed a week after weaning, irrespective of dietary treatment. Weaning-related expansion of intestinal lamina propria CD4+ and CD8+ T lymphocyte populations was observed (P < 0.05). Complex and variable treatment effects on villus height, crypt depth, villus and crypt goblet cell density, and lamina propria T cell density were observed, suggesting that the tested protein sources do not share a common or simple mode of action.     

Effects of spray-dried porcine plasma and plant extracts on intestinal morphology and on leukocyte cell subsets of weaned pigs

We evaluated the effects of a 6% spray-dried porcine plasma (SDPP) and a plant extracts mixture (XT; 5% carvacrol, 3% cinnamaldehyde, and 2% capsicum oleoresin) on the productive performance, intestinal morphology, and leukocyte cell subsets of early-weaned pigs compared with a control group. Morphometry of the jejunum, ileum, and colon, and immune cell analysis of blood, ileocolic lymph node (LN), and ileal Peyer's patches were done in 24 weaned pigs (20 +/- 2 d) at 19 or 21 d postweaning. Although SDPP and XT treatments did not increase ADG or ADFI, SDPP improved the G:F ratio (P = 0.024) compared with the control group. Dietary SDPP reduced the percentages of blood monocytes (P = 0.006) and macrophages in ileal Peyer's patches and LN (P = 0.04), of B lymphocytes (P = 0.04) and gammadelta+ T cells in LN (P = 0.009), and of intraepithelial lymphocytes (P = 0.026) as well as the density of lamina propria cells in the colon (P < 0.01). Dietary XT reduced intraepithelial lymphocyte numbers in jejunum (P = 0.034) and the percentages of blood cytotoxic cells (P = 0.07) and B lymphocytes in LN (P = 0.03); however, XT increased blood monocytes (P = 0.038) and the density of lamina propria lymphocytes in the colon (P = 0.003). These results indicate that dietary SDPP and plant extracts can affect intestinal morphology and immune cell subsets of gut tissues and blood in weaned pigs. Furthermore, the effects of SDPP suggest lower activation of the immune system of the piglets.     

Experimentally induced porcine proliferative enteritis in specific-pathogen-free pigs

Thirty-three, 10-week-old, specific-pathogen-free pigs were randomly allotted to 3 treatment groups: group 1--intragastrically given homogenized intestinal mucosa (crude inoculum) from pigs with naturally occurring proliferative enteritis; group 2--given cultures of Campylobacter sputorum subsp mucosalis; and group 3--controls. One pig from each group was killed 4, 7, 10, 14, 18, 21, 24, 28, 31, 36, and 38 days after inoculation. The earliest intestinal lesion observed in groups 1 and 2 was leukocytic exudate within crypt lumina and focal inflammation of the surrounding lamina propria. The lesions occurred primarily over ileal aggregated lymphoid nodules (Peyer's patches). These changes were followed by focal proliferation of immature crypt epithelial cells and infiltration of increasing numbers of macrophages into the lamina propria. Campylobacter sp-like organisms were observed within the cytoplasm of affected epithelial cells by light and electron microscopies. Lesions progressed to diffuse crypt cell proliferation, elongation of crypts, and loss of villi. Mucosal necrosis was not a prominent feature.