Distribution and developmental change of lymphoid tissues in the chicken proventriculus

In the chicken proventricular mucosa, aggregations of lymphocytes were localized in three different sites of the lamina propria, namely, underneath the surface epithelium, near the duct orifice of the deep proventricular gland, and in the gland tissue itself. In the lymphoid masses underneath the surface epithelium and in those near the duct orifice, CD4+ T lymphocytes and TCR2+ T lymphocytes occupied their central part, and B lymphocytes were localized in the periphery. CD8+ T lymphocytes and TCR1+ lymphocytes were evenly distributed in the masses. Infiltration of lymphocytes into these sites was first observed on the 20th embryonic day. At 1 week after hatching, CD3+ lymphocytes began to occupy the central area of the masses and His-C1+ B lymphocytes tended to be located in the periphery. Ultrastructurally, M cells were found neither in the epithelium of the mucosa nor in that of the excretory duct close to the lymphoid masses. In the deep proventricular gland, the lymphoid masses had a germinal center consisting of B lymphocytes, surrounded by the T lymphocyte-rich periphery. These masses were first recognized at the 3rd post-hatching week, presumably being formed against possible antigens invading into the lumen of the proventricular gland. On the other hand, the lymphoid masses beneath the surface epithelium and those near the duct orifice existing before the hatching period were considered to be prepared to establish the local mucosal immune barriers against the expectant antigenic invasion.     

鸡贫血病雏鸡ND免疫后局部免疫组织抗体生成细胞变化

对感染鸡贫血病毒（ＣＡＶ）雏鸡新城疫（ＮＤ）疫苗免疫及其强毒攻击后局部免疫组织——盲肠扁桃体和哈德尔腺的ＩｇＡ、ＩｇＧ和ＩｇＭ抗体生成细胞数量的动态变化进行了检测。结果发现,ＣＡＶ感染雏鸡ＮＤ免疫后７、１４、２８ｄ及新城疫强毒（ｖＮＤＶ）攻击后,上述免疫组织的三种抗体生成细胞数量均不同程度地低于未感染ＣＡＶ的ＮＤ免疫对照雏鸡。其中盲肠扁桃体弥散区的ＩｇＡ减少最为明显。表明ＣＡＶ感染雏鸡消化道和呼吸道局部免疫组织对新城疫疫苗的体液免疫应答功能降低。新城疫强毒攻击后ＣＡＶ感染ＮＤ免疫雏鸡的免疫保护率也明显低于未感染ＣＡＶ的免疫对照雏鸡。     

Analysis of the susceptibility of the Minor inbred chicken strain to Rous sarcoma virus infection

The sensitivity and resistance of the Minor hen inbred line to two antigenic subgroups of the Rous sarcoma virus were determined by the method of the infection of chorioallantoic membranes. The Minor line is sensitive to infection with the virus of the antigenic B subgroup and, as demonstrated by the testing of the interline hybrids of F1 and B1 generations, the birds of the Minor line are dominant homozygotes of genotype bsbs. The testing with the virus of antigenic A subgroup revealed a heterogeneity of the inbred population as to sensitivity and resistance. A formula was derived for the determination of the frequency of the dominant allele as determining sensitivity.     

Development of Peyer's patch and cecal tonsil in gut-associated lymphoid tissues in the chicken embryo

It is well known that chicken B cells develop in the bursa of Fabricius (BF), which is categorized as gut-associated lymphoid tissue (GALT). Chicken GALT also includes Peyer's patch (PP) and cecal tonsil (CT). The relationship between these tissues in GALT during B cell development is currently unknown. In this study, we conducted comparative examination of PP, CT and BF development during embryogenesis using immunohistochemical staining. On day 13 of embryogenesis (E13), accumulation of MHC class II(+) cells was observed in the intestine. Thereafter, Bu-1(+) cells and IgM(+) cells appeared, and their number continuously increased at the same sites where MHC class II(+) cells were present. Similar results were obtained in the CT. The locations of embryonic PP were limited to two sites; near the Meckel's diverticulum and the ileocecal junction. Anlage of bursal follicles first appeared at E13 and developed thereafter. Immigration of Bu-1(+) cells to bursal follicles began at E13, and the number of Bu-1(+) cell subsequently increased. When the follicle of BF was eliminated from the embryo by treatment with testosterone, development of PP and CT were observed. We concluded therefore that the development of PP and CT start during late embryogenesis at the same time as the follicle of BF, and that appearance of surface IgM(+) cells in PP and CT is independent form the development of the follicle of BF.     

Effect of the VP3 gene of chicken anemia virus on canine mammary tumor cells

OBJECTIVE: To investigate the antitumor effect of the chicken anemia virus (CAV) VP3 gene in canine mammary tumor (CMT) cells. SAMPLE POPULATIONS: Established primary canine cell lines that originated from epithelial cells of resected CMTs and nonneoplastic mammary gland epithelial (MGE) cells. PROCEDURES: Expression vectors and lentiviral vectors encoding the VP3 gene from a Taiwan-Ilan isolate of CAV were used to deliver the VP3 gene into CMT cells and nonneoplastic MGE cells. Ectopic gene expression and the pro-apoptotic effect of the VP3 gene on CMT and nonneoplastic MGE cells by either transfection or viral infection were evaluated via immunofluorescence microscopy, western blot analysis, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling analysis. RESULTS: Overexpression of the enhanced green fluorescent protein-VP3 fusion protein was detected predominantly in the nuclei of CMT cells. In contrast, the VP3 protein was localized to the cytoplasm of nonneoplastic MGE cells. Among the fusion protein-expressing CMT cells, most underwent characteristic changes of apoptosis, whereas apoptosis was not detected in fusion protein-expressing, nonneoplastic MGE cells. Induction of apoptosis by VP3 gene overexpression in CMT cells was associated with the caspase-9-, but not the caspase-8-, mediated apoptosis pathway. CONCLUSIONS AND CLINICAL RELEVANCE: These data indicate that the VP3 gene of the CAV induces apoptosis in malignant CMT cells, but not in nonneoplastic canine MGE cells. On the basis of such tumor cell-specific killing, the VP3 gene may be a promising agent for the treatment of malignant mammary gland tumors in dogs.     

Segmented filamentous bacteria associated with lymphoid tissues in the ileum of horses

Segmented filamentous bacteria preferentially attached to the follicle-associated epithelium overlying the lymphoid tissue in samples of the terminal ileum from seven horses examined by scanning electron microscopy. The bacteria adhered to the apical membrane of the enterocytes by a holdfast segment. Each filament tended to be of uniform diameter, but the filaments ranged from 0·7 to 1·4 μm in diameter. The bacteria were usually absent from the adjacent villous epithelium.     

M cells and associated lymphoid tissue of the equine nasopharyngeal tonsil

The aim of this study was to characterise the morphological and histochemical features of equine nasopharyngeal tonsillar tissue. Nasal and oropharyngeal tonsillar tissue has been described as the gatekeeper to mucosal immunity because of its strategic location at the entrance to the respiratory and alimentary tracts. A combination of light, scanning and transmission electron microscopy has revealed the presence of follicle-associated epithelium (FAE) overlying lymphoid tissue of the equine nasopharyngeal tonsil caudal to the pharyngeal opening of the guttural pouch. Membranous microvillus (M) cells were identified in the FAE on the basis of short microvilli, an intimate association with lymphocytes, cytoplasmic vimentin filaments and epitopes on the apical surface reactive with lectin GS I-B4 specific for alpha-linked galactose. CD4-positive lymphocytes were scattered throughout the lamina propria mucosae as well as forming dense aggregates in the subepithelial part. The central follicular area was heavily populated with B lymphocytes and the dome and parafollicular areas contained both CD4- and CD8-positive lymphocytes. CD8-positive lymphocytes were also present in the epithelium and, together with B lymphocytes, in small numbers in the lamina propria mucosae. These observations indicate that the nasopharyngeal tonsil is potentially an important mucosal immune induction site in the horse and an appropriate target for intranasally administered vaccines.     

Detection of chicken anemia virus DNA in embryonal tissues and eggshell membranes

Chicken infectious anemia virus (CIAV) is a ubiquitous and highly resistant virus of chickens that causes anemia and death in chicks less than 3 wk of age and immunosuppression in chickens older than 3 wk of age. The production of specific-pathogen-free eggs free of CIAV is essential for research and vaccine production. Currently, flocks are screened for CIAV by antibody tests to ensure freedom from CIAV infection. Recent evidence, however, indicates that chickens may carry and vertically transmit CIAV DNA independently of their antibody status. In this study, we tested embryos and eggshell membrane residues by nested polymerase chain reaction (PCR) as a sensitive method of detecting CIAV DNA. CIAV DNA could be detected in the blastodisks and semen obtained from antibody-positive and -negative chickens. Examination of different tissues between 18 and 20 days of incubation indicated that many but not all organs of individual embryos were positive. The lymphoid organs and gonads had the highest incidence of CIAV DNA, which was significantly different (P < 0.05) from the incidence in the liver. Eggshell membrane samples from embryos or newly hatched chicks were an excellent noninvasive source for the detection of CIAV DNA, identifying significantly more positive embryos than did pooled lymphoid organs. The use of dexamethasone injections as a method to improve the detection of carrier birds did not result in an increase of vertical transmission or cause seroconversion in the treated hens. A combination of testing eggshell membrane residues at hatch and periodic testing of blood DNA by nested PCR can be used to identify chickens carrying CIAV DNA and may be used to eradicate carrier birds.     

Interferon-alpha-producing cells are localized in gut-associated lymphoid tissues in transmissible gastroenteritis virus (TGEV) infected piglets

Transmissible gastroenteritis virus (TGEV) infection of piglets results in a very rapid and massive release of IFN-alpha in serum and secretions. The objective of this work was to characterize the IFN-alpha-producing cells (IPC) in tissues of TGEV-infected piglets. Caesarean-derived colostrum-deprived piglets were infected orally with the TGEV virulent Miller strain and IPC were characterized in situ by immunohistochemistry, using a rabbit anti-pig IFN-alpha antiserum. IPC were almost exclusively detected in intestinal tissues and mesenteric lymph nodes (MLN), as early as 6 h post inoculation (p.i.), with a peak at 12-18 h. They disappeared by 24 h. IPC were localized between enterocytes in the small intestine epithelial layer, in the lamina propria, around the Peyer's patches and, at highest frequency, in MLN. Very few IPC were present in the spleen and popliteal lymph nodes of infected piglets. Double immunohistochemical staining for IFN-alpha and leukocyte markers on MLN cryosections showed that IPC were mainly Swine Leukocyte Antigen (SLA) class II positive, and were not stained by an anti-macrophage (SWC3a) MAb. In addition, double staining with anti-TGEV and anti-IFN-alpha MAbs showed that viral antigens were present in MLN, close to IPC. These results show for the first time the presence of IPC in gut mucosa and gut-associated lymphoid tissues in response to an enteropathogenic virus. Moreover, this work shows that IFN-alpha released in serum is likely to originate almost exclusively from gut IPC triggered locally by viral antigens to produce IFN-alpha, since there were very few IPC in spleen or peripheral lymph nodes. MHC class II molecule expression by gut-associated IPC suggests that these cells may be the in vivo mucosal counterparts of the dendritic cells recently shown to produce IFN-alpha after in vitro viral induction.     

Age-related changes in the number of mast cells in the avian lymphoid organs

The distribution of mast cells (MCs) was studied in the lymphoid organs (thymus, bursa of Fabricius and spleen) of 0-, 7-, 21-, 30- and 120-day-old chickens, using light microscopic histochemical techniques. Tissues samples were obtained under deep anaesthesia from animals in five groups. Tissues were fixed in Mota's fixative (basic lead acetate) for 24 h and embedded in paraffin. Six-micrometre-thick sections were stained with toluidine blue in 0.5% aqueous solution at pH 1.0 for 5 min and Alcian blue/Safranine at pH 1.42 for 30 min. MCs were found in the organs, mostly associated with sinuses and blood vessels. A large increase in MCs was observed in both thymus and spleen of 21-day-old chickens compared with 0-, 7-, 30- and 120-day-old chickens. However, in the bursa of Fabricius, numbers of MCs were significantly higher in the 7-day-old group compared with other age groups. Safranine-positive MCs were not observed in all organs and age groups. These results showed age-related changes in the number of MCs in avian lymphoid tissues.     

鸡贫血病雏鸡ND免疫后免疫器官抗体生成细胞动态变化

对感染鸡传染性贫血病毒（ＣＩＡＶ）雏鸡新城疫（ＮＤ）免疫及其强毒攻击后，其免疫器官ｋｋ法氏囊、脾脏和胸腺的ＩｇＧ、ＩｇＭ和ＩｇＡ抗体生成细胞的动态变化进行了检测。结果发现，感染雏鸡ＩｇＧ抗体生成细胞于免疫后７～２８ｄ，在法氏囊和胸腺髓质及脾脏红髓和白髓区明显低于未感染的免疫对照雏鸡；ＩｇＭ生成细胞分别于免疫后７～２８ｄ、７ｄ或１４ｄ明显降低；而ＩｇＡ生成细胞于免疫后２８ｄ，仅在法氏囊髓质区明显减少，表明ＣＩＡＶ感染雏鸡免疫器官对ＮＤ免疫的体液免疫应答降低。ＮＤ强毒攻击后，ＣＩＡＶ感染ＮＤ免疫雏鸡法氏囊、脾脏、胸腺的三种抗体生成细胞均程度不同地低于对照雏鸡，其中ＩｇＧ生成细胞减少最为明显。ＣＩＡＶ感染雏鸡免疫器官抗体生成细胞减少与免疫保护率降低密切相关     

Quantitation and localization of immunoglobulin E containing cells in bovine lymphoid tissues

Although recent studies have begun to describe and quantify IgE responses in bovine serum and secretions, little is known about the distribution and quantity of IgE containing cells in cattle. In the present study, cells with cytoplasmic IgE were quantitated in bovine lymphoid tissues, using immunoperoxidase staining and evaluation by an image analysing computer (Quantimet). Frozen sections from retropharyngeal, bronchial and mesenteric lymph nodes, tonsil and spleen were stained from 11 calves, some of which had been exposed to antigen by aerosol or injection. Although individual variability was considerable, bronchial and mesenteric lymph nodes generally contained the greatest percentage of IgE containing cells, while retropharyngeal lymph node, tonsil, and spleen had less. Parenteral immunization with ovalbumin appeared to increase the splenic percentage, while aerosol exposure to ovalbumin was associated with a greater percentage of IgE containing cells in bronchial lymph nodes. Comparison of the present results with those reported for other species shows some similar trends in IgE localization.     

Comparison of canine distemper virus strains in gnotobiotic dogs: effects on lymphoid tissues.

The effects of infection on various aspects of lymphoid function in gnotobiotic dogs with 2 virulent strains of canine distemper virus (CDV), Snyder-Hill CDV and R252-CDV, were compared. Both infections resulted in a viremia-related lymphopenia which was nonselective in that the percentages of B and T cells remained unchanged throughout the observation period. Nonfatal Snyder-Hill-CDV infection resulted in a transient depression of in vitro lymphocyte responses to phytohemagglutinin-P, whereas R252-CDV produced prolonged in vitro suppression of phytohemagglutinin-P stimulation. The differences observed are of minor significance and do not explain the differences in central nervous system demyelinating potential between these 1 strains of CVD.     

Isotype-specific antibody-secreting cells in systemic and mucosal associated lymphoid tissues and antibody responses in serum of conventional pigs inoculated with PEDV

An enzyme-linked immunospot (ELISPOT) has been developed to detect porcine epidemic diarrhea virus (PEDV)-specific antibody secreting cells (ASC) in gut associated lymphoid tissues (duodenum and ileum lamina propria and mesenteric lymph nodes) and systemic locations (spleen and blood) of conventional pigs so as to characterise the mucosal and systemic antibody response generated by the infection with PEDV. A total number of 28 eleven-day-old conventional pigs were orally inoculated with the field isolate of the PEDV strain CV-777. Diarrhea was observed in 32% of the pigs and virus shedding was demonstrated in 100% between postinoculation day (PID) 1 and 8. Serum IgG and IgA antibodies to PEDV were detected by isotype ELISA from PID 12 and 15, respectively, reaching maximum values at PID 32 (IgG) and 21 (IgA). PEDV specific IgM ASC occurred in all the tissues between PID 4 and 7, with the strongest response in the intestinal lamina propria. IgA and IgG ASC responses were evident in the intestinal lymphoid tissues from PID 21, the highest number of specific ASC corresponded to the duodenum lamina propria. In the systemic lymphoid tissues the number of IgG and IgA ASC detected were lower than in the mucosal tissues, however, in the blood, presence of IgA ASC was constantly detected from PID 14 until the end of the experiment. Memory antibody response to the PEDV was also studied by secondary in vitro stimulation of the mononuclear cells (MNC) isolated from mesenteric lymph nodes, spleen and blood. The memory B cell response was prominent at PID 21 and 25 and consisted in IgG and IgA ASC. To our knowledge, this is the first report to research into the presence and distribution of specific ASC in different locations of the systemic and the gut associated lymphoid tissues after a PEDV infection as well as the presence of memory B cells.     

犬瘟热病毒自然感染犬淋巴组织中T、B细胞变化的研究

为了调查患犬瘟热病犬淋巴组织中T、B细胞变化的特点及淋巴细胞减少的发病机制,试验通过免疫组织化学的方法观察了T细胞(用CD3和CD45RO检测T细胞)、B细胞(用IgG、IgM抗血清检测B细胞)和犬瘟热病毒(抗犬瘟热病毒抗体)在病犬淋巴组织中的分布。结果表明:在淋巴组织中的淋巴细胞、淋巴小结中树突状细胞和巨噬细胞中均检出了抗病毒阳性反应细胞。在骨髓组织的前髓细胞中也发现抗病毒阳性反应细胞和嗜酸性胞浆内及核内包涵体的存在。与对照组相比,CD3和CD45RO阳性细胞主要存在于T细胞的分布域;但CD3和CD45RO阳性T细胞的数量较少。位于淋巴组织中的巨噬细胞有的被CD45RO染成阳性。在B细胞分布的区域中,IgG、IgM阳性细胞的数量明显减少;一些位于淋巴组织的浆细胞也被IgG或IgM染成阳性。在淋巴组织中淋巴细胞减少的顺序为:IgG阳性细胞减少最明显,其次为IgM和CD45RO阳性细胞,再次为CD3阳性细胞。依据试验结果,作者认为病犬淋巴组织中淋巴细胞减少主要是由B细胞缺乏所引起的;淋巴细胞的增殖能力减弱是引起淋巴组织中淋巴细胞减少的重要原因。     

Bovine immunodeficiency virus in experimentally infected rabbit: tropism for lymphoid and nonlymphoid tissues

The bovine immunodeficiency virus (BIV)/New Zealand (Oryctolagus cuniculus) rabbit model was used to study events that underlie the early and chronic stages of viral replication, routes and time course of viral dissemination and the distribution of the virus in the lymphoid. nonlymphoid and mucosa associated tissues. The results indicated that BIV, a lentivirus with genetic relatedness to the HIV, induced changes of clinical (anorexia, weight loss, muscular wasting, diarrhea, hypoalgesia, torticollis), immunological (recurrent T- and B-cell dysfunctions) and histopathological (lymphadenopathy, splenomegaly) nature that closely parallels those described for cat (Fly), monkey (SIV) and human (HIV) lentiviral diseases. These findings showing that BIV induces both splenomegaly and lymphadenopathy syndromes with associated fatal immune dysfunctions and the ability of the virus to replicate productively at the mucosal surfaces in rabbits, emphasize the importance of the BIV/rabbit system as a good small-animal model for the study of retrovirus-induced AIDS and offers the opportunity to evaluate prophylactic and therapeutic anti-retroviral agents of relevance to HIV-1 as well as the opportunity to study mechanisms of drug resistance phenomena.     

Distribution of lymphoid leukosis virus and p27 group-specific antigen in tissues from laying hens

In order to gain insight into transmission and pathogenesis of infection, specimens from laying hens that had been naturally exposed to lymphoid leukosis virus (LLV) were tested for group-specific antigen (gsa) of the virus by immunofluorescence (IF), complement fixation (CF), and the enzyme-linked immunosorbant assay (ELISA). Electron microscopic examinations determined the distribution of C-type virus particles in tissues, and the phenotypic-mixing test served as a biological assay for exogenous LLV. The IF gsa was found in all organs tested, and fluorescence was usually found where virus particles were concentrated. In the oviduct and intestine, IF gsa was frequently at the border of the lumina and in the connective tissue associated with basal membranes of glands. In skin, the antigen was detected in smooth muscle, in feather pulp, and in basal epidermal cells of developing feathers. Results of various tests on Ottawa strains of chickens were usually in agreement. For example, among hens that shed gsa into egg albumen, only the viremic hens were consistently positive for IF gsa in both spleens and oviducts. Geometric mean CF titers of antigen were respectively five- and 23-fold higher in spleens and oviducts from viremic hens than in those from nonviremic hens. These findings suggest that the gsa was associated with exogenous virus infection. In Cornell S strain hens that had not been exposed to LLV, gsa was detected in splenic tissue by CF and ELISA but not by the IF test. This gsa was presumed to be of endogenous origin.     

Effect of methisoprinol and KLP-602 on virus replication in chicken embryos

The aim of the present study was to examine the effects of two immunomodulators (KLP-602 and Methisoprinol) on the proliferation of two strains of Newcastle disease virus in chick embryos. The effect of the maximum tolerable doses of both drugs (Methisoprinol--6 mg/embryo, KLP-602--5 mg/embryo) on lymphocyte reactivity were determined prior to the experiment. Both drugs inhibited the replication of the Roakin strain of NDV in various experimental designs, but neither of them affected the proliferation of the LaSota strain of NDV.     

Preliminary age-related histological studies of the lymphoid tissues of the respiratory tract and associated structures of the brushtail possum

Abstract Extract Lymphoepithelium was observed in palatine and pharyngeal tonsils in possums from 2 months of age, but was not seen in the lung. Morphological maturity of lymphoid tissue occurred at about 4 months of age, coinciding with first emergence of young from the pouch. Peribronchiolar and/or perivascular lymphoid aggregations were first observed in the lung of a 3.5-month-old possum, and subpleural lymphoid aggregations were first seen at 7 months of age.