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.     

mRNA from porcine colostral and milk cells coding for specific antibodies in response to orally administered Escherichia coli strains

The effect of feeding live $\text{Escherichia coli}$ bacteria to sows on the lymphocyte populations in colostrum and milk was studied by $\text{in ovo}$ translation of the mRNA of these cells (Kortbeek-Jacobs and van der Donk, 1978). Four $\text{E. coli}$ strains were tested: one wild type K88 producing strain, two conjugated K12 strains producing K88 antigen and one wild type K12 strain. Only after feeding the wild type K88 bacteria, colostral lymphocytes contained mRNA coding for anti-K88 antibodies, predominantly of the IgA and IgM isotypes. Milk lymphocytes of the sows receiving one of the other strains were capable of producing more anti-K88 antibodies than the sows that were fed the wild type K88 strain. Antibodies were predominantly of the IgM class. The reactivity of the milk lymphocytes is ascribed to the strain with which the piglets were challenged. The results support the phenomenon of sensitized gut lymphocytes that home to the mammary gland.     

Differential enhancement and distribution of antigen-specific cells in various lymph nodes in response to locally inoculated bacterial antigens.

The proliferation responses of antigen-specific lymphocytes from various anatomical sites were studied in dairy goats locally immunized with heat-killed Staphylococcus aureus (HKS). Animals were inoculated three times subcutaneously in the right udder with HKS at 1 month intervals. One week following the last inoculation, prescapular, mesenteric and ipsilateral (draining) and contralateral (non-draining) suprammammary lymph nodes were collected and the cells assayed in 3- and 6-day cultures to determine the immune proliferative responses of antigen-specific lymphocytes to HKS and the polyclonal T cell mitogen phytohemagglutinin (PHA). The cells from draining and non-draining supramammary lymph nodes responded to HKS in 3-day cultures. Peripheral lymph nodes, such as the prescapular, showed similar responses. In contrast, mesenteric lymph nodes responded optimally in 6-day cultures, notably to lower concentrations of the antigen. Cells from all lymph nodes tested showed increased responses to PHA in immunized animals, although non-draining lymph nodes demonstrated a greater response to the T cell mitogen than those of draining lymph nodes. These results suggest that unilateral introduction of Staphylococcus cell antigens to the supramammary region can induce an anamnestic response in ipsilateral as well as contralateral supramammary lymph nodes and other distant peripheral lymphoid organs. Furthermore, these data indicate that cells from intestinal lymph nodes respond differently from those of peripheral lymph nodes, suggesting the presence of a unique gastrointestinal lymphoid cell circulation in goats. Concomitant peripheral responses may be attributed to memory cell migration or to antigen leakage and relocation to distant sites from the inoculated region. Analysis with PHA suggests a difference in general responsiveness and perhaps, immunocompetence, by lymphocyte populations in various lymphoid tissues of immunized animals.     

Limiting dilution analyses for the quantification of cellular immune responses in bovine ostertagiasis

A sensitive limiting dilution analysis (LDA) was used to quantitate the local and systemic cellular immune response of cattle after immunization with keyhole limpet hemocyanin (KLH) and infection with Ostertagia ostertagi. The assay measures the proliferative response of bovine T-cells after in vitro stimulation with antigen. Interleukin 2 activity was supplied by supernates from mitogen-stimulated bovine peripheral blood lymphocytes (PBL) and accessory cell function was in the form of irradiated autologous PBL. The assay measures the response of a single cell and was most easily demonstrated in the lymph nodes draining the site of antigen inoculation. Comparison of cell frequencies and maximal responses generated in conventional proliferative assays showed several differences between the two assays. First, after antigen injection, the highest cell frequencies were seen in the draining lymph nodes within 3 days, and decreased by 10 days post-immunization. In contrast, in mass cultures maximal stimulation was not seen until 7-10 days after injection, but remained high up to 4 weeks after immunization. Second, at 17 days post-infection, a time of eruption of the parasite from the gastric glands, high frequencies of inducible cells were demonstrated by LDA in all lymphoid populations tested. In contrast, low levels of proliferation were seen in mass cultures. The reasons for these differences may include different sensitivities to suppression or more stringent requirements for specificity between the two assays. Finally, it was found that immunologically naive calves have relatively high frequencies of Ostertagia-specific cells in PBL, and that after infection these frequencies decrease. These results indicate either active suppression of the potential anti-Ostertagia response or an extra-vascularization of these cells to the site of infection.     

Characterization of adaptive immune responses induced by a new genetically inactivated Salmonella Enteritidis vaccine

The superior conservation of antigenic determinants on the surface of genetically inactivated bacterial ghosts makes them attractive immunogenic inactivated vaccine candidates. The efficacy of Salmonella Enteritidis (SE) ghost vaccination was evaluated in chickens by characterizing the nature of the adaptive immune response. Chickens from the immunized group demonstrated significant increases in SE-specific plasma IgG, intestinal secretory IgA, and lymphocyte proliferative response. The populations of CD4, CD8, and TCR γδ T-cells in immunized chickens were significantly greater than in the controls. Increased levels of IFN-γ, IL-2, IL-6 and IL-10 were observed in peripheral blood mononuclear cells stimulated with SE specific antigen. After virulent SE challenge, the immune system of immunized chickens was rapidly stimulated, as indicated by significantly increased population of CD4 and CD8 T-cells. Furthermore, the immunized group exhibited decreased challenge strain recovery of the internal organs compared to the non-immunized group. Together, these data indicate that the immunization induced humoral and cell-mediated immunity might be responsible for significant reduction of the virulent challenge strain load in the internal organs of immunized chickens.     

Immunohistochemical detection and localization of new type gosling viral enteritis virus in paraformaldehyde-fixed paraffin-embedded tissue

To determine the distribution and localization of new type gosling viral enteritis virus (NGVEV) in paraformaldehyde-fixed paraffin-embedded tissues of experimentally infected goslings, for the first time, an immunohistochemical (IHC) staining method was reported. Anti-NGVEV polyclonal serum was obtained from the rabbits immunized with purified NGVEV antigen, which was extracted by caprylic-ammonium sulphate method and purified through High-Q columns anion exchange chromatography. Three-day-old NGVEV-free goslings were orally inoculated with NGVEV-CN strain suspension as infection group and phosphate buffered saline solution (PBS) as control group, respectively. The tissues were collected at sequential time points between 0.5 and 720 h post inoculation (PI), and prepared for IHC staining and ultra-structural observation. The positive immunoreactivity could be readily detected in the lymphoid and gastrointestinal organs of infected goslings as early as 48 h PI, in the liver, kidney, pancreas and myocardium from 72 h, and in the cerebrum and cerebellum from 96 h, while it was hardly detected in the respiratory organs at any time. The positive staining reaction could be detected in NGVEV-infected goslings until 600 h PI, and no positive staining cell could be observed in the controls. The highest levels of viral antigen were found in the bursa of Fabricius (BF), thymus, proventriculus, gizzard and intestine tract, moreover, the liver, kidney, spleen, myocardium and pancreas were intensively and widely stained. The target cells had a ubiquitous distribution, especially included the epithelial cells, endothelial cells, superficial and crypt mucosal cells, glandular cells, fibrocytes, macrophages and lymphocytes, which served as the principal sites for antigen localization. The ultra-structural observation by transmission electron microscope (TEM) further indicated that NGVEV particles could be widely detected in the lymphoid and digestive organs of infected goslings from 72 h PI onwards. This work may be useful not only for offering a possibility of routine diagnosis of NGVE, but also for better understanding of the pathogenesis of the disease.     

Effects of oral supplementation with bovine colostrum on the immune system of weaned piglets

The aim of this study was to evaluate the influence of bovine colostrum supplementation on the immune system of weaned piglets in a context of a full ban of in-feed antibiotics. After weaning at 21 days, 24 outbred piglets were fed with a diet supplemented daily for three weeks with 0, 1 or 5 g of colostrum. Feed intake, growth performance, haematological parameters, and serum and local anti-colostrum immunoglobulin levels were examined. Lymphocytes from the blood, spleen, and gut-associated lymphoid were analysed for phenotype as well as for their ability to produce cytokines. The stimulation index (SI) of mononuclear cells from different organs was obtained after colostral or mitogenic stimulation. Feed intake, growth, and haematological parameters were not significantly affected by colostrum. Total serum IgA levels were increased after colostrum supplementation, with a transient decrease in total IgG. Local anti-colostrum immunization was observed in colostrum-fed piglets. The CD21+/CD3+ cells populations of the ileal Peyer's patch (iPP) were markedly affected. The SI of lymphocyte populations changed significantly whereas, naive blood lymphocytes were not stimulated in vitro in the presence of bovine colostrum, suggesting local anti-colostrum immunization and an absence of direct mitogenic effects of the colostrum. Both Th1 and Th2 cytokine production was present in the different organs of colostrum-fed piglets. Bovine colostrum especially stimulated iPP cells.     

Development of tertiary lymphoid structures in the kidneys of pigs with chronic leptospiral nephritis

Tertiary lymphoid organs (TLOs) are structures that are morphologically and functionally similar to secondary lymphoid organs. TLOs usually arise in a background of chronic inflammation. Several histological patterns of interstitial nephritis have been documented in porcine leptospirosis. Among them the lympho-follicular pattern is characterized by infiltrates of mononuclear cells organized in lymphoid follicle-like structures. Immunohistological analysis of 5 cases of porcine lympho-follicular nephritis associated with Leptospira Pomona infection demonstrated the presence of inflammatory cell populations, including B cells, T cells, macrophages and follicular dendritic cells (FDCs), which were compartmentalized as in TLOs. Immunohistochemistry for Leptospira Pomona revealed an intimate association between leptospiral antigen and FDCs. Overexpression of MHCII in different populations of both professional and non-professional antigen presenting cells was also demonstrated. FDCs play role during TLOs induction for their ability to retain non-self antigens in the form of immune complexes, thus causing persistent T cell activation, generation of a complex cytokine network and stimulation of humoral immunity. Sustained bacterial antigen presentation in the context of chronic leptospiral nephritis, may also lead to autoimmune mechanisms involved in the generation of TLOs. Whether lymphoid neogenesis and TLOs play a protective role in porcine leptospiral nephritis is still unclear.     

Delayed-type hypersensitivity in sheep induced by synthetic peptides of bovine leukemia virus encapsulated in mannan-coated liposome

Four sheep were immunized with synthesized peptides, derived from bovine leukemia virus (BLV) envelope glycoprotein, encapsulated in mannan coated liposomes as adjuvant. On the seventh day after the immunization, the sheep were intradermally challenged with BLV antigen, or synthesized peptides. The areas challenged with antigen were increased skin thickness and biopsied sequentially for immunohistological examinations. Strong delayed-type hypersensitivity was induced in sheep immunized and challenged with peptides encapsulated in mannan-coated liposomes. The major phenotype of the infiltrating lymphocytes was CD5(+). The ratio of CD4(+) to CD8(+) cells was about 1:1.     

Expression of the T200 family of glycoproteins in canine malignant lymphoma.

High molecular weight surface proteins were examined in lymph node lymphocytes from five control dogs and 27 dogs with malignant lymphoma. Polyclonal rabbit antiserum was raised against a 210,000-dalton (210 K) membrane protein which was purified from a canine lymphoid tumor by preparative slab gel electrophoresis. Three high MW proteins at 210, 195 and 170 K and a common proteolytic fragment at 95 K were detected in the electrophoretically separated plasma membrane preparations by immunoblotting with polyclonal anti-210 K antiserum. Two antigenically distinct patterns were evident: 1) Type 1 lymphomas expressed a major 210 K peptide (with or without a minor 195 K component), and 2) Type 2 lymphomas lacked the 210 K form but had a 170 K or 195 K peptide singly or in combination. Immunoperoxidase staining of formalin-fixed, paraffin-embedded tissue sections demonstrated that the antigen was localized predominantly to the surface membrane of lymphocytes, and that canine lymphoma cells expressed a greater amount of the antigen than normal lymph node lymphocytes. It was concluded that these structurally and antigenically related high molecular weight proteins, based on their antigenic patterns, limited peptide analysis and tissue distribution, represent the canine homologue of the lymphocyte differentiation antigen known as T200.     

Perinatal development of lymphoid tissue and its associated epithelium in the ovine pharyngeal tonsil: A morphological study

The perinatal development of lymphoid tissue and its associated epithelium in the pharyngeal tonsil of sheep was investigated by light and electron microscopy. The lymphoid cells first appeared in the subepithelium in a scattered form at about 92 days gestation. These cells proliferated rapidly during the last trimester of gestation, and had formed many dense aggregates at the time of parturition. At birth, the epithelium overlying the aggregates was extensively infiltrated with lymphocytes and showed early transformation of follicle-associated epithelium. The lymphoid tissue and its associated epithelium did not, however., fully develop until after birth, when well-differentiated follicle-associated epithelium and lymphoid follicles with vigorous lymphopoiesis were present. In l–2 week old lambs, these structures were ultrastructurally similar to those seen in adult sheep although their size was smaller. The results of this study suggest that the lymphoid tissue of the ovine pharyngeal tonsil and its associated epithelium are morphologically ready to cope with antigens in the extra-uterine environment at birth, but that their full development and maturation are dependent on postnatal antigen stimulation.     

Salmonid B lymphocytes demonstrate organ dependent functional heterogeneity

The passive hemolytic plaque assay was used to examine the functional heterogeneity of antibody producing cells in salmonid immune organs. In this study, the antibody response to Vibrio anguillarum antigens was induced by the injection of a somatic antigen extract. This antigen was also coated onto sheep red blood cells (SRBC) for plaque forming cell (PFC) determination. Previous studies have demonstrated that this response is antibody dependent and antigen specific (Kaattari and Irwin, 1985). The present study was focused upon the heterogeneity of antibody producing cells that arise in the spleen, anterior and posterior kidney of immunized coho salmon (Oncorhynchus kisutch). The functional heterogeneity of lymphocytes was assessed by histogram analysis of the antigen inhibition profiles of the plaque forming responses. These analyses have revealed that the anterior kidney lymphocytes possess a much more restricted profile of antibody specificities than do lymphocytes from the posterior kidney or spleen. These data suggest that B cell repetoires differ among the immune organs of salmonids.     

Perinatal development of lymphoid tissue and its associated epithelium in the ovine pharyngeal tonsil: a morphological study

The perinatal development of lymphoid tissue and its associated epithelium in the pharyngeal tonsil of sheep was investigated by light and electron microscopy. The lymphoid cells first appeared in the subepithelium in a scattered form at about 92 days gestation. These cells proliferated rapidly during the last trimester of gestation, and had formed many dense aggregates at the time of parturition. At birth, the epithelium overlying the aggregates was extensively infiltrated with lymphocytes and showed early transformation of follicle-associated epithelium. The lymphoid tissue and its associated epithelium did not, however, fully develop until after birth, when well-differentiated follicle-associated epithelium and lymphoid follicles with vigorous lymphopoiesis were present. In 1-2 week old lambs, these structures were ultrastructurally similar to those seen in adult sheep although their size was smaller. The results of this study suggest that the lymphoid tissue of the ovine pharyngeal tonsil and its associated epithelium are morphologically ready to cope with antigens in the extra-uterine environment at birth, but that their full development and maturation are dependent postnatal antigen stimulation.     

Intercellular junctions in the developing mammalian thymus.

Lymphopoiesis in fetal thymuses of mice and rabbits was studied to try to correlate heterogeneity of lymphocytes with heterogeneity of embryonic origin. The thymuses of fetal mice of 11 to 14 days gestation and of fetal rabbits of 18 and 19 days gestation were examined by electron microscopy. At the time of earliest appearance of lymphoid cells in both species desmosomes were seen on both epithelial and lymphoid cells. These desmosomes persisted for several days, then disappeared from completely differentiated lymphocytes in both species. The similarity between epithelial and lymphoid cells in their earliest developmental stages suggests a common precursor cell for both.     

Experimental fetal infection with bovine viral diarrhea virus. II. Morphological reactions and distribution of viral antigen.

The effect of an infection with bovine viral diarrhea virus on fetal bovine tissues as well as the tissue-localization of viral antigen are described. Four bovine fetuses, 120-165 days of gestation, were inoculated in utero with a second passage virus strain. Lymphoid tissues were studied by light and electron microscopy. The infection induced precocious development of the secondary lymphoid organs. Characteristic changes were seen in postcapillary venules, cells of the mononuclear phagocyte system and lymphoid follicles. In all four fetuses the thymus was hypoplastic. In three fetuses this implicated a morphological immaturity, but no actual pathological alterations. In the fourth fetus, the hypoplasia was caused by necrosis and depletion of lymphocytes, attended by infiltration of macrophages. Histopathological changes were also noted in the cerebellum of three fetuses, consisting of necrosis in and depletion of the external germ layer and in the skin and mucous membranes of all four fetuses. The viral antigen was present in cells of the mononuclear phagocyte system, primarily in the lymphoid tissues. The infection was further demonstrated in the affected cerebelli.     

Induction of lymphocyte responsiveness by the outer membrane-peptidoglycan complex of rough strains of Brucella abortus

The outer membrane-peptidoglycan complex (OM-PG) from rough strains of Brucella abortus was tested for its ability to induce lymphocyte responsiveness in cattle. Six groups of heifers were immunized with varying doses and administration schedules of rough OM-PG and assayed for responsiveness of their lymphocytes in proliferation assays in vitro. All OM-PG preparations were emulsified in a commercial adjuvant for administration. Two other groups of heifers were immunized with strain 19 vaccine or adjuvant alone. Three groups of heifers received two inoculations of OM-PG antigens from a naturally-occurring rough strain at a 57-day interval. The doses of OM-PG given in these three groups were 400 micrograms, 1200 micrograms, and 4000 micrograms at each inoculation. The frequency of cows that responded in lymphocyte proliferation assays increased with the dose of OM-PG given. Two groups received single inoculations of OM-PG, either 2400 micrograms or 8000 micrograms. Although there were responsive cows in these immunization groups, their frequency was lower than in the groups receiving the same total dose in two inoculations. A sixth group of cows was inoculated with OM-PG from a rough transposon mutant of B. abortus, and the frequency of responsive cows in this immunization group was comparable to that of responsive cows immunized with the same dose of OM-PG from the spontaneous rough mutant. In comparisons of cows inoculated with strain 19 to those inoculated with OM-PG preparations, differences were observed in the relative responsiveness of their lymphocytes to whole cells and OM-PG in the in vitro lymphocyte proliferation assays. These differences suggested that lymphocytes stimulated by strain 19 vaccination have different specificities than those stimulated by immunization with OM-PG of rough mutant strains of B. abortus.     

Enhanced resistance to cattle grub infestation (Hypoderma lineatum de Vill.) in calves immunized with purified hypodermin A, B and C plus monophosphoryl lipid A (MPL).

The influence of an antigen-specific cellular and humoral immune response, stimulated by immunization, on survival of a challenge infestation of Hypoderma lineatum was investigated. Calves immunized with a purified combination of hypodermin A, B and C plus monophosphoryl lipid A (MPL) developed a strong antigen-specific cellular immune response by completion of the immunization schedule which persisted to 12 weeks post-infestation. Responsiveness of peripheral blood lymphocytes to the mitogens concanavalin A and pokeweed was also elevated at 4 and 12 weeks post-infestation. Western blot analysis at the time of maximum grub counts demonstrated that immunized calves responded to hypodermin A, B and C while those receiving only MPL or infested controls responded only to hypodermin B and C. The antigen-specific antibody response as measured by ELISA at maximum grub count was significantly higher in vaccinated calves than in infested controls while the response in calves receiving only immunostimulator was also significantly elevated. Immunized (antigen plus MPL) calves produced 5.0 +/- 6.9 grubs per animal which successfully pupated while those receiving MPL alone produced 16.4 +/- 6.1 and infested controls produced 32.2 +/- 10.9 grubs per animal.     

In vitro adhesion of K88acEscherichia coli to Peyer's patch and peripheral blood lymphocytes, buccal and rectal epithelial cells or intestinal epithelial brush borders of weaned pigs

Escherichia coli adhesion assays were conducted using isolated porcine peripheral blood lymphocytes, Peyer's patch lymphocytes, rectal epithelial cells or brush borders, buccal epithelial cells and brush borders from small intestinal epithelial cells. The cells and brush borders were tested for their ability to bind K88-piliated exterotoxigenic E. coliStrain M1823B (K88ac) and E. coli Strain 1476 (K-12, K88ac). Comparison of adhesive phenotypes of 37 weaned pigs as determined by the adhesion assay with small intestinal brush borders and the adherence of K88ac⁺ enterotoxigenic E. coli to peripheral blood lymphocytes, Peyer's patch lymphocytes and rectal epithelial cells or brush borders, revealed no correlation. In vitro adhesion of K88ac-bearing E. coli was always negative with buccal epithelial cells. K88ac strains varied in their ability to adhere to lymphocytes and rectale pithelial cells or brush borders, indicating that the mechanism of adherence is unrelated to K88-mediated adhesion observed in animals that had the receptors on small-intestinal epithelial-cell brush borders. The non-piliated control E. coli Strain 123 adhered to fresh peripheral blood lymphocytes, and less intensively to frozen-thawed peripheral blood lymphocytes or Peyer's patch lymphocytes. It was concluded that none of the cell types or brush borders, except small-intestinal epithelial-cell brush borders, could be used as targets for phenotyping pigs for the presence of the K88 receptors that have been associated with adhesion and colonization of K88⁺ enterotoxigenic E. coli in the porcine small intestine.     

Antigen distribution in organs of mink with Aleutian disease parvovirus infection

On tissues from naturally infected non-Aleutian mink an immunohistological study was performed using monoclonal antibodies and the immunoperoxidase method. Structural proteins of ADV were demonstrated in cryosections and in ethanol-fixed and paraffin-embedded material which provide antigen detection in a similar amount together with good histological structure. In lymphoid organs viral antigen was restricted to B-cell areas, particularly lymphoid follicles. The pattern of antigen distribution was typical for follicular dendritic cells which are capable to retain immune complexes. Beside macrophages in the interior of lymphoid follicles most likely proliferating B-lymphoblasts reveal nuclear and cytoplasmatic presence of structural proteins indicating viral replication. Cells of the mononuclear phagocyte system such as cells of lymphatic sinuses and hepatic Kupffer cells harbor viral protein in the cytoplasm, probably resulting from phagocytosis of immune complexes. Renal glomeruli were consistently negative for virus antigen whereas in interstitial infiltrates cells resembling macrophages stained positive for ADV structural proteins.