Immunization and culture of rainbow trout organ sections in vitro

Splenic and anterior kidney sections or whole organs were excised from large (1 kg) or small (200 g) rainbow trout (Salmo gairdneri) and placed in sterile 60 mm plastic plates containing 10 ml of Eagle's minimal essential medium (EMEM) supplemented with normal or fetal calf serum for in vitro culture. The organ samples were immunized in vitro by direct injection or by mixing in the medium Yersinia ruckeri O-antigen or dinitrophenyl-Ficoll. The medium was changed once during the 10-day incubation at 15 C. The passive hemolytic plaque assay demonstrated antibody production from the plaque-forming cells (PFC); passive hemagglutination was used to measure antibody titers in the media. High numbers of PFC occurred in cultures of either kidney or spleen, demonstrating that these organs can function independently for antibody production. Splenic sections from large fish produced more PFC than comparable whole organs from small fish. EMEM supplemented with 2% normal calf serum was a satisfactory culture medium. 2-hydroxyethyl-mercaptan an ingredient used in mammalian cell culture, inhibited antibody production in trout cells. These techniques are being used in the culture of organs and cells to elucidate pathways and sequences of antigen uptake and delivery of the immunopoietic tissues in trout.     

Long term substitution and specific immune responses after transfer of bovine peripheral blood lymphocytes into severe combined immunodeficient mice.

The long term immune responsiveness of bovine peripheral blood lymphocytes engrafted into severe combined immunodeficient mice (bovine PBL SCID mice) was analyzed. After intraperitoneal transfer (i.p.) of 2x10(7) bovine PBL into SCID mice, FACS analysis revealed successful engraftment of bovine CD4 and CD8+ T cells in the peritoneal cavity, the peripheral blood, spleen, lymph nodes, bone marrow, and thymus of reconstituted mice for up to 13 weeks. As shown by immunocytochemistry in sections of spleens from SCID mice 16 weeks after substitution, bovine T and B cells were localized perivasculary forming pseudofollicular structures. Nevertheless, histopathology of spleen and liver from bovine PBL SCID mice revealed pathological alterations indicating rejection of xenogenic cells or graft versus host disease (GVHD). On the functional level, i.p. transfer of bovine PBL into SCID mice induced increasing levels of bovine IgM and IgG in the sera of recipients. Bovine Ig could be detected up to 20 weeks. Immunization of SCID mice reconstituted with PBL of normal donors with dinitrophenol (DNP)-edestin induced a weak specific bovine antibody response in recipient mice. In contrast, a secondary specific bovine IgG response was observed after antigen restimulation of SCID mice reconstituted with PBL from calves preimmunized either with DNP-edestin or keyhole limpet hemocyanin (KLH) showing functional T cell-independent and -dependent antibody responses of bovine PBL SCID mice. Our data demonstrate that transfer of bovine PBL into SCID mice leads to a long term engraftment of bovine cells in lymphatic and non-lymphatic organs inducing a functional substitution of T and B cell immune response of SCID mice. Therefore, bovine PBL SCID chimera can serve as a small animal model for the analysis of bovine lymphopoiesis and infectious diseases of cattle.     

Antibody-forming cells in the contralateral and ipsilateral lymph nodes draining the locally immunized supramammary/suprainguinal region

The Jerne hemolytic plaque assay was used to compare the number of antibody forming cells in the ipsilateral supramammary/suprainguinal lymph node which drains the udder, its counterpart area in males, of dairy goats inoculated with the antigen, sheep red blood cells, and in the contralateral lymph node which drains the corresponding non-inoculated area. Parenteral immunization was shown to have suppressing effects upon the local immune responses to the subsequently applied antigens. Three monthly intramammary inoculations of the antigen induced significant numbers of indirect plaque-forming cells (i.e. immunoglobulin G antibody producing cells) in both draining (ipsilateral) and non-draining (contralateral) nodes, suggesting antigen relocation and/or cell relocation from the inoculated side. However, the number of indirect plaque-forming cells in the ipsilateral node was far greater than that in the contralateral node, indicating that the majority of memory cells remained in the inoculated site.     

Influence of betulinic acid on lymphocyte subsets and humoral immune response in mice

Betulinic acid is a pentacyclic triterpene found in many plant species, among others, in the bark of white birch Betula alba. Betulinic acid was reported to display a wide range of biological effects, including antiviral, antiparasitic, antibacterial, anticancer and anti-inflammatory activities. The effects of betulinic acid (50, 5, 0.5 mg/kg) administered orally five times at 24 hours intervals to non-immunized and red blood cells (SRBC)-immunized mice were determined. The present study examined the total number of lymphocytes in the thymus, spleen and mesenteric lymph nodes, and the percentage of subsets of T cells (CD4+CD8+, CD4CD8, CD4+, CD8+) in thymus,T (CD3+, CD4+, CD8+) and B (CD19+) lymphocytes in the spleen and mesenteric lymph nodes, as well as white blood cell (WBC) and differential leukocyte counts in non-immunized mice, and humoral immune response in SRBC-immunized mice. SRBC was injected 24 hours after administration of the last dose of betulinic acid. It was found that betulinic acid administered orally five times at the dose of 0.5 mg/kg increased the total number of thymocytes, splenocytes, lymphocytes of mesenteric lymph node cells, and the weight ratio of the spleen and mesenteric lymph nodes in non-immunized mice. Betulinic acid also changed the percentage of T cell subsets in the thymus and T and B lymphocytes in peripheral lymphatic organs. The effects of betulinic acid on T and B cell subpopulations depended on the dose applied. The strongest stimulating effect of betulinic acid was observed when the drug was administered at the dose of 0.5 mg/kg. Five exposures to betulinic acid (0.5 mg/kg) decreased the percentage of immature CD4+CD8+ thymic cells with corresponding increases in the percentage and absolute count of mature, single-positive CD4+ thymocytes and decreased the percentage and total count of CD3+ splenocytes and mesenteric lymph node cells with corresponding decreases in the percentage and absolute count of CD4+ and CD8+ cells. Multiple administration of betulinic acid at the investigated doses augmented the percentage and absolute count of CD19+ cells in the peripheral lymphatic organs. Moreover, betulinic acid at the dose of 5 mg/kg administered prior to SRBC immunization increased the number of plaque forming cells (PFC) but decreased the production of anti-SRBC antibodies on day 4 after priming. Thus, betulinic acid is a potential biological response modifier and may strengthen the immune response of its host.     

The effect of oral immunization on the population of lymphocytes migrating to the mammary gland of the sow

Sows were immunized orally with live Escherichia coli according to various immunization schedules. Six pregnant gilts were used; 4 immunized at various intervals during the last month of gestation, 1 control immunized after parturition following suppression of lactation by weaning and 1 non-immunized control. The effect of oral vaccination on cell populations from lymphoid organs was studied. The in vitro proliferative responses of the cell populations to K88 antigen, anti-Ig sera and mitogens were used to demonstrate the distribution of sensitized lymphocytes over different lymphoid organs. The capacity of these cells to produce antigen-specific Ig was determined by in ovo translation of their mRNA.Oral administration of antigen resulted in the appearance of K88-positive cells in lymphoid organs. In lactating sows, sensitized cells preferentially occured in the mammary lymph nodes, whereas after suppression of lactation such a distribution was not seen. A possible route of migration of sensitized lymphocytes is discussed in relation to the local immune response. The antibody isotype produced by sensitized lymphocytes seemed to depend on the immunization schedule. The most effective schedule was one starting early in gestation and comprising frequent administration of antigen. This caused an optimal distribution of sensitized lymphocytes capable of IgA production.     

Local immune response in the chicken Harderian gland to antigen given by different ocular routes.

The effectiveness of three ocular routes of antigen administration to produce a local immune response in the Harderian gland was studied. The routes were by eyedrop, injection into the ocular conjunctiva and injection into the nictitating membrane. The antigen was observed in the cytoplasm of macrophages located within the lymphoid tissue only after the injection into the nictitating membrane. The numbers of germinal centres and plaque forming cells found in the gland after injection into the nictitating membrane was higher than the numbers observed following the other two ocular applications. These findings indicate that the injection of the antigen into the nictitating membrane is the most effective ocular route for producing a local immune response in the Harderian gland.     

CpG oligodeoxynucleotides stimulate immune cell proliferation but not specific antibody production in rainbow trout (Oncorhynchus mykiss)

Bacterial DNA and CpG ODN have both been shown to have immunostimulatory effects in mammals, activating APCs and inducing a potent Th1 type immune response. They have also been shown to have a strong adjuvant effect and up-regulate MHC class 2 expression in murine cells, augment human and murine NK cell lytic activity, activate human B cells and induce murine B cell proliferation. However, little work has been carried out with regard to their effects on the piscine immune system. Here it is shown that various CpG ODN induce proliferation of peripheral blood leucocytes, spleen and head kidney cells from rainbow trout although, at the range of concentrations tested CpG ODN 2133 lacked the ability to induce specific antibody production to a protein antigen.     

感染鸡传染性贫血病毒（CIAV）雏鸡免疫器官抗体生成细胞的动态变化

对１日龄雏鸡感染鸡传染性贫血病毒（ＣＩＡＶ）后免疫器官法氏囊、脾脏和胸腺的ＩｇＧ、ＩｇＭ、ＩｇＡ抗体生成细胞数量的动态变化进行了检测。结果发现,感染雏鸡法氏囊、脾脏和胸腺的３种抗体生成细胞数量均程度不同地低于未感染对照雏鸡,其中法氏囊的ＩｇＧ、ＩｇＭ抗体生成细胞和ＩｇＡ抗体生成细胞分别在感染后７～３５ｄ和１４～３５ｄ明显减少;脾脏红髓、白髓和淋巴小结的ＩｇＧ抗体生成细胞分别于７～３５ｄ、１４～３５ｄ和１４～２１ｄ明显减少,ＩｇＭ抗体生成细胞分别在７～４２ｄ、２８～３５ｄ和１４ｄ时明显减少,ＩｇＡ抗体生成细胞仅在红髓中（７～２８ｄ）明显减少;胸腺髓质的ＩｇＧ、ＩｇＭ、ＩｇＡ抗体生成细胞分别在１４～２８ｄ、７～２１ｄ和２１ｄ时明显减少。结果表明,ＣＩＡＶ感染雏鸡免疫器官的体液免疫功能明显降低。     

The immune response of the pig following oral immunisation with soluble protein

The immune response of pigs fed 200 mg per day of dinitrophenylated bovine gamma globulin has been evaluated in terms of the antibody and lymphocyte responses and of the induction of tolerance and immune exclusion. Although dosing for ten days resulted in a small IgA response as indicated by splenic plaque forming cells, serum antibody could only be detected when dosing was continued for 42 days. secretory antibody was detected at any time. Antigen feeding for two weeks rendered the animals hyporesponsive to subsequent parenteral antigenic challenge but had little effect upon their ability to exclude an oral dose of antigen from the circulation.     

Primary in vitro stimulation of antibody production by lizard splenocytes

Single-cell suspensions of adult lizard (Chalcides ocellatus) spleen have been induced, in vitro, to produce a primary immune response. Using rat red cells (RRBC) as antigen and the culture conditions normally used in most vertebrate species but new for reptilia, it has been found that, in vitro at 37 degrees C, lizard spleen cells produce an antibody-forming response optimal at day 10. The response depends on the number of cultured cells and the dose of antigen, and parallels that obtained in vivo. Leibovitz (L-15) medium supplemented with 10% normal adult lizard serum was a satisfactory culture medium. 2-mercaptoethanol (2-ME), an ingredient used in mammalian cell culture, enhanced antibody production in lizard cells.     

The effects of florfenicol on lymphocyte subsets and humoral immune response in mice

Florfenicol is a broad-spectrum bacteriostatic antibiotic used in domestic animals. The aim of the study was to determine the effect of florfenicol on the total number of lymphocytes in the thymus, spleen and mesenteric lymph nodes and the percentage and the absolute number of T cell subsets (CD4+CD8+, CD4-CD8-, CD4+, CD8+) in the thymus and T (CD3+, CD4+, CD8+) and B (CD19+) lymphocytes in the peripheral lymphatic organs in non-immunized mice and humoral immune response in sheep red blood cells (SRBC)-immunized mice. Florfenicol was administered orally at a dose of 30 mg/kg six times at 24 h intervals to non-immunized mice and four or seven times at 24 h intervals to SRBC-immunized mice. SRBC was injected 2 hours prior to the first dose of the drug. Florfenicol increased the percentage of CD4CD8- thymocytes and the absolute number of CD4+ and CD8+ thymocytes on day 7. The increased percentage and absolute number of CD3+, CD4+ and CD8+ lymphocytes in mesenteric lymph nodes and decreased percentage of lymphocytes B were also observed 24 hours from the last administration of florfenicol. Florfenicol administered after SRBC immunization reduced the number of plaque forming cells (PFC) and the production of anti-SRBC antibodies on days 4 and 7 after priming.     

The immune system of cyprinid fish. Oxytetracycline and the regulation of humoral immunity in carp (Cyprinus carpio)

The antibiotic oxytetracycline (oxyTC) was administered either by mixing with food or by intraperitoneal injections. In oxyTC treated animals decreased serum immunoglobulin levels were found. The primary anti-sheep red blood cell (SRBC) response was measured by enumerating plaque forming cells (PFC). It was observed that the PFC response was depressed by 80–95% in oxyTC treated animals. When an anti-SRBC serum was injected together with SRBC the immunosuppressive effect of oxyTC was absent. A secondary anti-SRBC response was not inhibited by oxyTC. On base of the results a model for antigen presentation and the interaction between macrophages, T- and B-like cells during primary and secondary responses in fish is proposed. In primary responses cellular interaction is needed to develop a proper immune response whereas after a high antigen dose challenge SRBC might behave as a T-independent antigen.     

鸡感染J亚群禽白血病病毒的免疫抑制机理

通过人工接种建立了雏鸡感染J亚群禽白血病病毒（ALV-J）后发生免疫抑制的病理模型。对免疫抑制的机理进行了初步研究。结果表明：ALV-J感染早期可诱导胸腺、法氏囊的淋巴细胞凋亡，这种早期的淋巴细胞凋亡是胸腺和法氏囊萎缩的重要原因之一。病理组织学动态观察表明。ALV-J主要引起骨髓的髓系细胞灶状或弥漫性增生，病变导致骨髓机能受损，使机体免疫机能下降。是引起免疫抑制的根本原因。病毒感染组免疫器官均发生了严重的实质萎缩性病变．这种病变的发生除与骨髓的病变及淋巴细胞凋亡有关外。还与中后期淋巴细胞的坏死有关．从而导致严重的免疫抑制。ALV-J感染可引起免疫器官及部分内脏器官中嗜酸性粒细胞样的瘤细胞浸润增生，这可以作为病毒感染早期的病理诊断指标。     

E．stiedai感染兔免疫器官内球虫抗原的免疫组化定位

用免疫组化SABC法，对斯氏艾关耳球虫感染后兔肝脏、脾脏和肝门淋巴结中球虫抗原的分布进行了观察。结果表明，在肝脏窦状隙、胆管上皮细胞、肝门淋巴结和脾脏内可见到明显的呈棕黄色阳性反应区，表明在这些区域和部位有相应的E．stiedai孢子囊、裂殖子或子孢子抗原存在，且抗原数量以肝脏内最多，其次为脾脏，最少者为肝门淋巴结；显微镜下还可见到大量坏死的肝细胞、淋巴细胞以及朗罕氏巨细胞，说明多核巨细胞也参与了对球虫的吞噬和处理过程。     

Changes in Numbers of Leukocytes in Immune Organs of Juvenile Coho Salmon after Acute Stress or Cortisol Treatment

Abstract

We examined the effects of acute stress and cortisol treatment on the number of leukocytes (normalized for fish body weight) in the blood, thymus, spleen, and anterior kidney of juvenile coho salmon Oncorhynchus kisutch. In acutely stressed or cortisol-fed fish, the numbers of leukocytes increased significantly in the thymus and anterior kidney, and decreased significantly in blood and spleen within 1 d after treatment. Numbers of cells in the anterior kidney, blood, and spleen returned to control levels by 3 d after treatment, but cell numbers in the thymus remained significantly greater than control values until 3–7 d after acute stress. Although dietary cortisol resulted in increased plasma cortisol titers and caused the same changes in leukocyte distribution as those caused by acute stress, the magnitude or duration of elevated cortisol levels and leukocyte numbers were not correlated. These results suggest that, although increased plasma cortisol titers induced by stress may be involved in the change in number of cells in various immune organs, factors other than cortisol are involved as well.     

Anterior chamber-associated immune deviation.

The eye possesses a critical method of self-preservation in response to intraocular antigen presentation. Instead of conventional immunity by means of delayed-type hypersensitivity (DTH), the eye participates in a systemic immune response involving the thymus and spleen, ultimately leading to suppression of cell-mediated (T helper 1) immunity. The immune response begins with intraocular capture of antigen by specialized ocular antigen-presenting cells (APCs). These activated APCs then migrate preferentially to the marginal zone of the spleen, where they become part of an intricate and highly specific cluster of immune cells. The end result is the emergence of a population of antigen-specific T-regulatory lymphocytes that return to the eye and suppress DTH.     

Interactions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with immune responses of rainbow trout

Chlorinated dioxins, as typified by the most potent isomer, TCDD, are immunosuppressive in mammalian species and can enhance the susceptibility to a number of diseases. In recent years chlorinated dioxins have been detected in fish in many freshwater and marine habitats. Thus far, the effects of these chemicals on the immune responses of fish have not been examined. We studied the influence of TCDD on the defense mechanisms of rainbow trout. Yearling trout were injected intraperitoneally with the vehicle, 0.1, 1.0 or 10 micrograms/kg of TCDD. Interactions with the humoral immune response to sheep red blood cells (SRBC) were assessed by the Jerne plaque assay using head kidney and spleen leukocytes. Serum antibody was measured by complement-mediated lysis of SRBC in a chromium release assay. Effects of TCDD on the cellular immune responses were evaluated by the response of thymic and splenic lymphocytes to Con A and PWM. In addition, the phagocytic activity of peritoneal macrophages was examined in vitro. Trout which received 0.1 or 1.0 micrograms/kg TCDD remained clinically normal, and defense mechanisms were unaltered in these fish. Trout which received 10 micrograms/kg of TCDD became hypophagic and exhibited fin necrosis, ascites and suppression of hematopoiesis. In this treatment group, Con A-induced blastogenesis of thymic and splenic lymphocytes was not significantly changed, however, suppression of the PWM-induced response of splenic lymphocytes occurred. No statistically significant alterations occurred in humoral immune responses, and phagocytic activity of peritoneal macrophages was not decreased. The dose-response curve for various biologic effects of TCDD in the rainbow trout appears different from that in sensitive mouse strains. The 30-day, single-dose, parenteral LD50 for TCDD in the C57BL mouse is 100 micrograms/kg, and TCDD suppresses both cell-mediated and humoral immune responses at 1-2 micrograms/kg in this mouse strain. In the rainbow trout, however, immunosuppression was evident only at doses of TCDD approaching the 80-day, single-dose, parenteral LD50 of 20 micrograms/kg.     

多糖作为兽用疫苗佐剂作用机制的研究进展

多糖可促进免疫调节,具有多靶点、多功能和多因子效应,因此可广泛用作兽用疫苗免疫佐剂。多糖作为疫苗佐剂可提高免疫动物脾脏、胸腺和法氏囊等免疫器官指数,增强巨噬细胞吞噬活性及抗原递呈能力,增强NK细胞杀伤能力,促进树突细胞的成熟和分化,增强机体对抗原的摄取能力,影响T细胞亚群的种类和数量,改变Th1、Th2型免疫应答反应的类型,促进B淋巴细胞增殖,刺激细胞因子及抗体的产生等。而多糖作为配体通过与Toll样受体(Toll-like receptor, TLR)、甘露糖受体(mannose receptor, MR)、C型凝集素受体(C-type lectins receptor, CLR)、清道夫受体(scavenger receptor, SR)等受体分子结合,激活下游信号通路,进而发挥上述免疫活性。文章主要从上述几方面对多糖作为兽用疫苗佐剂的作用机制进行阐述,以期为多糖的进一步开发和应用提供参考。     

Presence of antigen sensitized leukocytes in carp (Cyprinus carpio L) following bath immunization against Flexibacter columnaris

Bath immunization of carp (Cyprinus carpio L) resulted in protection of fish at natural challenge. Stimulation of leukocytes derived from thymus, spleen, anterior kidney and mid-kidney of fish immunized with Flexibacter columnaris bacterin revealed the presence of antigen sensitized cells in all lymphoid tissues except the anterior kidney. After 28 days a response was obtained in thymus and spleen leukocyte cultures.     

Ontogeny of channel catfish lymphoid organs

Previously, we showed that catfish could not mount a detectable antibody response after bacterial exposure until 21 days post-hatch (ph). In order to evaluate the changes associated with the development of a functional humoral response, we evaluated the temporal and spatial distribution of immune cell populations in developing catfish. Cells functioning in nonspecific immunity were present in the renal hematopoietic tissue (rht) and thymus at hatch and in the spleen by day 3 ph. Immunoglobulin (Ig) positive lymphocytes were first detected on day 7, 10, and 14 in the rht, thymus and spleen, respectively. Mature thymocytes were first detected on day 10 ph. Distinct thymic regionalization and splenic lymphoid tissue organization were not observed until day 21 ph. We suggest that the reason for a lack of antibody production until day 21 ph is the poor organization of secondary lymphoid tissue until that age.