Depression of lymphocyte responses to phytohaemagglutinin in lambs experimentally infected with bovine respiratory syncytial virus.

Eight lambs were experimentally infected with bovine respiratory syncytial virus (BRSV) and the responses of their peripheral blood lymphocytes to the mitogen phytohaemagglutinin and BRSV antigen compared with that of control lambs injected with tissue culture fluid. The lymphocyte transformation responses to phytohaemagglutinin were significantly reduced five and 10 days after experimental infection with BRSV (P less than 0.05). It appears that these reductions were associated with CD4+ lymphocytes because CD4-enriched lymphocytes obtained five days after infection had more significantly reduced responses to phytohaemagglutinin than those obtained from the same group before infection and from the control group five days after inoculation (P less than 0.01). There were no significant lymphocyte transformation responses to BRSV antigen in both groups of lambs up to 21 days after inoculation (P greater than 0.05).     

The effects of bovine respiratory syncytial on normal ovine lymphocyte responses to mitogens or antigens in vitro

In the present study peripheral blod mononuclear cells (MNC) obtained from normal uninfected lambs were used to study the possible effects of bovine respiratory syncytial virus (BRSV) on lymphocyte responses to the mitogens, phytohaemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) in vitro. Live BRSV had a depressive effect on the proliferative responses of normal MNC to PHA, Con A and PWM. Inactivated BRSV and a commercial preparation of prostaglandin E2 were also found to depress the proliferative responses of normal ovine MNC to PHA but recombinant tumour necrosis factor-alpha (TNF-alpha) had no such effect. Serum samples obtained from BRSV-infected lambs contained substances inhibitory to PHA-driven lymphocyte blastogenesis. Memory blastogenic responses to border disease virus (BDV) of lymyphocytes obtained from lambs previously primed with BDV were significantly reduced when lymphocytes were exposed to infectious BRSV.     

Lymphocyte subpopulations in peripheral blood of lambs experimentally infected with bovine respiratory syncytial virus

The lymphocyte subpopulations of peripheral blood of normal lambs and lambs experimentally infected with bovine respiratory syncytial virus (RSV) were analysed by flow cytometry, using a panel of monoclonal antibodies against specific lymphocyte epitopes. Experimental infection with bovine RSV was characterized by a significant rise in SBU-T8+ (CD8+ or cytotoxic) T cells and a significant reduction in SBU-T4+ (CD4+ or helper) T cells and B (LCA p220+) lymphocytes (P less than 0.05). The helper/suppressor (CD4/CD8) ratio was reduced from 3.91 on the day of experimental infection to 1.13 on 10 days after experimental infection (P less than 0.001). The total number of SBU-T4+ (CD4+) and B cells returned to pre-inoculation values 14 days after experimental infection but the helper/suppressor ratio remained depressed up to 21 days post-inoculation.     

Increased pulmonary secretion of tumor necrosis factor-alpha in calves experimentally infected with bovine respiratory syncytial virus

Bovine respiratory syncytial virus (BRSV) is an important cause of respiratory disease among calves in the Danish cattle industry. An experimental BRSV infection model was used to study the pathogenesis of the disease in calves. Broncho alveolar lung lavage (BAL) was performed on 28 Jersey calves, of which 23 were experimentally infected with BRSV and five were given a mock inoculum. The presence of the cytokine tumor necrosis factor alpha (TNF-alpha) in the BAL fluids was detected and quantified by a capture ELISA. TNF-alpha was detected in 21 of the infected animals. The amount of TNF-alpha in the BAL fluid of calves killed post inoculation day (PID) 2 and 4 was at the same very low level as in the uninfected control animals. Large amounts of TNF-alpha were detected on PID 6, maximum levels of TNF-alpha were reached on PID 7, and smaller amounts of TNF-alpha were seen on PID 8. The high levels of TNF-alpha appeared on the days where severe lung lesions and clinical signs were obvious and the amounts of BRSV-antigen were at their greatest. Although Pasteurellaceae were isolated from some of the BRSV-infected calves, calves treated with antibiotics before and through the whole period of the infection, as well as BRSV-infected calves free of bacteria reached the same level of TNF-alpha as animals from which bacteria were isolated from the lungs. It is concluded that significant quantities of TNF-alpha are produced in the lungs of the calves on PID 6-7 of BRSV infection. The involvement of TNF-alpha in the pathogenesis of, as well as the anti-viral immune response against, BRSV infection is discussed.     

Reinfection of lambs with bovine respiratory syncytial virus.

Eight lambs which were experimentally infected with bovine respiratory syncytial virus (RSV) when they were six to eight weeks old were challenged with the same virus seven months later. After reinfection, lambs developed mild clinical disease and the virus was isolated from nasal swabs from three lambs and peripheral blood from two lambs. Reinfection resulted in changes in peripheral blood cell populations. There was an early increase in the number of CD8+ T lymphocytes and B (LCA p220+) lymphocytes but the proportions of CD4+ and CD4-CD8- T lymphocytes were significantly reduced. Peripheral blood mononuclear cells obtained from lambs reinfected with bovine RSV showed significantly higher responses to bovine RSV antigen in vitro than those obtained from control lambs but their responses to the mitogen phytohaemagglutinin were significantly lower than in control lambs. RSV-specific IgG, IgM and IgA levels of serum samples obtained 10 days after challenge were significantly higher than those of serum samples obtained before challenge.     

Lymphocyte subpopulations in peripheral blood of lambs experimentally infected with Pasteurella haemolytica.

The lymphocyte subpopulations in peripheral blood obtained from eleven lambs experimentally infected with Pasteurella haemolytica were compared with those obtained from eight control lambs by flow cytometry, using a panel of monoclonal antibodies against specific lymphocyte epitopes. Experimental infection with P. haemolytica was characterized by a transient but significant reduction in SBU-T1+ (CD5+) T cells and SBU-T4+ (CD4+ or helper) T lymphocytes (P less than 0.05) and a significant rise in lymphocytes which did not express the LCA p220 epitope and the pan T cell surface marker (CD5-LCA p220-) ("null"). The reductions in CD5+ and CD4+ lymphocytes occurred 24 h after experimental infection, returning to preinoculation levels 5 days post inoculation (DPI). Five to 9 days after experimental infection, there was a significant increase in the number of lymphocytes, which expresses the pan T cell surface marker (CD5+) but which were CD4-CD8-. Lymphocyte transformation responses to the mitogen phytohaemagglutinin (PHA) were significantly reduced 24 h after experimental infection with P. haemolytica (P less than 0.05).     

Evaluation of ethylenediaminetetraacetic acid, tetrasodium salt dihydrate (EDTA)-Tween 20 treatment versus protease digestion of formalin-fixed tissue sections for detection of bovine respiratory syncytial virus antigen in infected ovine lung

The efficacy of protease and ethylenediaminetetraacetic acid, tetrasodium salt dihydrate (EDTA)-Tween 20 in unmasking bovine respiratory syncytial virus (BRSV) antigens in formalin-fixed lung tissue was compared using avidin-biotin immunoperoxidase procedure. Tissues were taken from experimentally infected lambs. BRSV antigen stained in both techniques. Treatment with EDTA-Tween 20 resulted in more intense staining of BRSV infected cells, more uniform cytoplasmic staining, less non-specific background, and superior cellular detail in comparison to protease digestion.     

Lymphocyte subpopulations in peripheral blood of sheep experimentally infected with tick-borne fever.

The lymphocyte subpopulations in the peripheral blood of normal sheep and sheep experimentally infected with Cytoecetes phagocytophila, the causative agent of tick-borne fever, were analysed by flow cytometry, using a panel of monoclonal antibodies against specific lymphocyte epitopes. Experimental infection with tick-borne fever was characterised by a significant reduction in the total number of circulating lymphocytes six days after experimental infection (P less than 0.001). This lymphocytopenia was associated with a significant reduction in the number of B (LCAp220+) and T (CD5+) lymphocytes (P less than 0.001) but there was a significant increase in the number of cells which were neither T nor B (CD5-LCAp220-) cells (P less than 0.01). The reduction in the number of T lymphocytes was due to reduced numbers of circulating CD4+ (helper) T cells, CD8+ (cytotoxic/suppressor) T cells and those with the pan T cell marker (CD5+) but without CD4 or CD8 epitopes (CD4-CD8-). All lymphocytes returned to preinoculation levels 13 to 16 days after experimental infection.     

Effect of infections with swine fever virus on immune functions I. Response of lymphocytes from blood and lymphoid organs from infected and normal pigs to anti-immunoglobulin serum and protein A

Pigs exposed to a low-virulent strain of swine fever virus (SFV) developed an inapparent infection. At times when a transient leucopenia occurred, the peripheral blood lymphocytes (PBL) were unresponsive to the mitogenic stimulus of anti-immunoglobulin serum (anti-Ig) and protein A.Pigs lethally infected with a virulent SFV showed leucopenia and unresponsiveness of PBL to anti-Ig and protein A from 2 days post infection until death.This suggests a defect in B lymphocyte function in pigs infected with SFV. The unresponsiveness to anti-Ig appeared not to be caused by a reduced ability of lymphocytes to redistribute their receptors into caps, the presence of suppressor cells or absence of surface immunoglobulin bearing lymphocytes in the peripheral blood. A direct action of the virus itself also seemed unlikely.Lymphocytes from spleen reacted as PBL. However, lymph node cells did not lose their capability to respond to anti-Ig.These data suggest that a change in the migration pattern of anti-Ig responsive lymphocytes could account for the observed unresponsiveness of PBL and spleen lymphocytes to anti-Ig.     

Cell mediated immune responses in ponies following infection with equine influenza virus (H3N8): the influence of induction culture conditions on the properties of cytotoxic effector cells

Cytotoxic cell precursors and/or cytotoxic memory cells were demonstrated in the peripheral blood of ponies after aerosol infection with influenza A/equine/Newmarket/79 (H3N8). In order to reveal their cytotoxic potential, peripheral blood mononuclear cells required a secondary antigenic stimulation. In vitro induced cytotoxic cells showed activity against influenza infected target cells in a 3-4 h 51Cr-release assay. The reactivity of cytotoxic cells was markedly influenced by the conditions of the secondary induction culture. If high concentrations of exogenous crude equine IL-2 were used, virus infected target cells were susceptible to lysis by autologous or allogeneic effector cells. However, if IL-2 concentration was reduced, cytotoxic cells were generated which showed features consistent with cytotoxic T cells in that target-cell killing was genetically restricted.     

Effects of allergen challenge on plasma concentrations of prostaglandins, thromboxane B2, and histamine in calves infected with bovine respiratory syncytial virus.

To examine the influence of allergen-induced type-1 hypersensitivity on the pathogenesis of bovine respiratory syncytial virus (BRSV) infection, we sensitized calves by aerosol to Micropolyspora faeni (MF) and challenge exposed them during infection with BRSV. The development of MF-specific IgE serum concentrations was confirmed by ELISA. The dynamics of arachidonic acid metabolism and histamine release during a type-1 hypersensitivity reaction in the bovine lung were studied by quantitating the concentrations of prostaglandin (PG)E2, PGF2 alpha, PGI2 as 6-keto-PGF1 alpha, thromboxane (TX) A2 as TXB2, and histamine in plasma of BRSV-infected and/or MF-sensitized/challenge-exposed calves. Four treatment groups were established: (1) BRSV infection only, (2) aerosol sensitization to MF followed by BRSV infection and aerosol challenge exposure to MF, (3) MF aerosol sensitization and challenge exposure without BRSV infection, and (4) aerosol sensitization to MF followed by BRSV infection without MF challenge exposure. Significantly increased concentrations of PGI2 were associated with MF aerosol exposure, particularly when combined with BRSV infection in group 2. After MF challenge exposure, TXB2 concentrations were significantly greater in the virus and MF challenge-exposed group 2. Individual calf data for the change in MF-specific IgE concentration between the first and second MF challenge exposures and the change in PGE2 concentration 30 minutes after the second MF challenge exposure had a highly significant direct correlation. Histamine concentrations were significantly greater in calves infected with BRSV than in uninfected controls regardless of MF exposure. These data further substantiate the thesis that implicates type-1 hypersensitivity as a pathogenic mechanism in BRSV-related disease.     

Experimental studies on Marek's disease in Japanese quail (Coturnix coturnix japonica)

Day-old quails experimentally infected with Marek's disease (MD) virus of quail origin developed lymphoid tumors. The severity of the disease increased considerably with serial passage. Tumor transplants could be made with cells derived from gross tumors in skeletal muscles, spleen cells, and blood from MD-affected quails. After five to six serial transplants, the tumor could not be transplanted further. Marek's disease tumor-associated surface antigen (MATSA) was demonstrated in lymphoid cells of spleen and peripheral blood lymphocytes of MD-affected quails. The MATSA of quail differed from the MATSA of chicken. Chickens were susceptible to MD virus isolated and propagated in quails.     

Effect of CD4+ T lymphocyte depletion on resistance of Gulf Coast Native lambs to Haemonchus contortus infection

It has been reported that CD4(+) T lymphocytes are important in acquired immunity to gastrointestinal nematode infection. Whether these lymphocytes are also involved in the immune response of naturally resistant Gulf Coast Native (GCN) sheep to Haemonchus contortus infection remains to be defined. The objective of this study was to determine the role of CD4(+) T lymphocytes in this resistance. Ten GCN lambs were randomly assigned to a control (n=5) or a treatment (n=5) group. The treatment consisted of a series of IV injections with mouse anti-ovine CD4(+) T lymphocyte monoclonal antibodies for a period of 3 weeks. After the second treatment, all lambs were experimentally infected with 10,000 H. contortus infective larvae by oral inoculation. All lambs were monitored for fecal egg counts, blood packed cell volumes, white blood cell differential counts and serum antibody responses on a weekly basis. Fluorescence-activated cell sorter (FACS) analysis was done biweekly to enumerate CD4(+) T lymphocytes in peripheral blood. Necropsies were performed at the end of the study and 10% of the contents of the gastrointestinal tract were preserved for nematode enumeration and identification. Also at necropsy, mesenteric lymph nodes were extracted and FACS analysis was run on lymphoid cells. Mean fecal egg counts on day 21 and 28 post-infection and nematode counts at necropsy of the treated group were significantly (p<0.05) higher than that of the control group. Percent CD4(+) T lymphocytes in peripheral blood was significantly (p<0.05) lower in the treatment group than in the control group from day 9 to the end of the study. No differences were found in blood packed cell volumes, white blood cell differential counts, antibody titer or lymph node CD4(+) lymphocytes between groups. Lambs depleted of their CD4(+) T lymphocytes were more susceptible to H. contortus infection than undepleted lambs. The results of this study suggest that CD4(+) T lymphocytes are associated with the natural resistance of GCN sheep to H. contortus infection.     

Induction of specific cytotoxic T-cell activity against xenogeneic target cells in carp (Cyprinus carpio)

OBJECTIVE: To investigate the induction of cytotoxic T cells in carp (Cyprinus carpio) after inoculation of fish with 2 xenogeneic line cells and to examine specificity of the cytotoxic activity. ANIMALS: 22 carp. PROCEDURE: Fish were inoculated with mouse myeloma line cells P3.NS-1/1Ag4.1 (NS-1) or chicken Marek's disease tumor-derived lymphoma line cells (MDCC MSB-1). Cytotoxic activity of immune lymphocytes was evaluated by incubating effector cells with homologous and heterologous target cells. Populations of effector cells were identified by blocking T-lymphocytes from effector cells, using anti-carp T-cell monoclonal antibody and complement. RESULTS: Lymphocytes in blood, spleen, and head kidney of carp inoculated with NS-1 cells or MDCC MSB-1 cells had dose-dependent cytotoxic effects against homologous target cells but not against heterologous target cells. Lymphocytes from noninoculated carp did not have cytotoxic effects. Depletion of T-lymphocytes in spleen cells from NS-1-inoculated carp resulted in a decrease of cytotoxic activity against NS-1 cells. Cytotoxic activity of spleen lymphocytes from NS-1-inoculated or noninoculated carp was not evident when cytotoxic tests were performed after addition of anti-NS-1 carp serum. CONCLUSIONS AND CLINICAL RELEVANCE: Inoculation with xenogeneic target cells induces a specific cytotoxic T-cell response in carp. Thus, cell-mediated immunity plays a role in defense against infection of parasitic organisms such as protozoa and helminths.     

Lymphosarcoma development in sheep experimentally infected with bovine leukaemia virus

Twelve sheep were experimentally infected with a phytohemagglutinin (PHA) treated short term culture of lymphocytes from a cow naturally infected with BLV at the PL stage. Five of 12 (42%) BLV infected sheep had histologically confirmed lymphosarcoma 10-16 months after infection. The PBL's were increased to leukemic levels 3-21 weeks before death due to lymphoblastic leukemia. Lymphocyte proliferation and appearance of immature lymphocytes and lymphoblastic cells in the blood were a characteristic feature of tumour development following inoculation with an Australian strain of BLV. In contrast to a number of previous studies the peripheral lymph nodes of all infected sheep were clinically normal throughout the experimental period but at death gross tumours were evident in the mesentric lymph nodes and the heart in all cases. All the other lymph nodes, liver, spleen, kidney and lung were histologically infiltrated with lymphoid tumour cells. Gross tumours were present in the abomasum (1 out of 5) in the urinary tract (2 out of 5) and in the uterus (1 out of 2). The majority of the tumour cells isolated from the various tissues were centroblastic demonstrating that the malignant leukemia in experimentally infected sheep was of a multicentric centroblastic type. The central nervous system was not involved in any case.     

Distribution pattern of bovine viral diarrhoea virus type 1 genome in lymphoid tissues of experimentally infected sheep

In this study, cellular localization and the distribution pattern of BVDV genome in lymphoid tissues during the course of experimental acute BVDV-1 infection of sheep was investigated. Tonsils, mesenteric lymph nodes (MLN) and spleen were collected on 3, 6, 9, 12 and 15 days post infection (dpi) from twenty 4-month-old lambs, experimentally inoculated intra-nasally with 5?×?10⁵ TCID₅₀ of a non-cytopathic (ncp) BVDV-1 isolate, Ind-17555. Tissues collected from ten mock-infected lambs served as controls. In situ hybridization (ISH) was carried out in paraformaldehyde fixed paraffin embedded tissue sections using digoxigenin labelled riboprobe targeting 5′-UTR of BVDV-1. BVDV genome was detected at all the intervals from 3 dpi to 15 dpi in the lymphoid tissues with variations between the intervals and also amongst the infected sheep. During the early phase of acute infection, presence of viral genome was more in tonsils than MLN and spleen, whereas the distribution was higher in MLN during later stages. BVDV-1 genome positive cells included lymphocytes, macrophages, plasma cells, reticular cells and sometimes crypt epithelial cells. Genome distribution was frequently observed in the lymphoid follicles of tonsils, MLN and spleen, besides the crypt epithelium in tonsils, paracortex and medullary sinus and cords of MLN. Most abundant and widespread distribution of BVDV-1 genome was observed on 6 dpi while there was a reduction in number and intensity of positive signals by 15 dpi in most of the infected animals. This is the first attempt made to study the localisation of BVDV-1 in lymphoid tissues of acutely infected sheep by in situ hybridization. The results show that the kinetics of BVDV-1 distribution in lymphoid tissues of experimentally infected non-pregnant sheep follows almost a similar pattern to that demonstrated in BVDV infected cattle.     

Replication of bovine respiratory syncytial virus in bovine and ovine peripheral blood lymphocytes and monocytes and monocytic cell lines

The present study compared the replication of bovine respiratory syncytial virus (BRSV) in bovine and ovine peripheral blood mononuclear cells, ovine and bovine monocytic cell lines and ovine alveolar macrophages. Low titres of virus were detected in ovine and bovine lymphocytes and monocytes 24–96 h post-exposure to the virus but there was no apparent replication of the virus in ovine alveolar macrophages during the culture period. The virus replicated to higher but statistically insignificant titres in ovine and bovine peripheral blood monocytes than in lymphocytes, with lymphocytes yielding peak titres significantly earlier. The secondary cell lines obtained from ovine liver and bone marrow also supported the replication of BRSV to high titres. The titres of BRSV in ovine and bovine lymphocytes and monocytes were significantly lower than in secondary cell lines. The addition of human recombinant tumour necrosis factor alpha after exposure to the virus or pre-incubation of ovine or bovine monocytic cells with either human recombinant interleukin 2 or phorbol myristate acetate before exposure to BRSV, did not significantly affect virus titre. Pre-incubation of cells with indomethacin or actinomycin significantly lowered virus titre (p<0.05).     

Demonstration of cytotoxic lymphocytes to virus-infected target cells in pigs inoculated with transmissible gastroenteritis virus.

Lymphocytes, cytotoxic to virus-infected target cells, were induced in pigs orally exposed to transmissible gastroenteritis virus. They were studied and experiments were carried out by using autochthonous testicle cells as target cells to avoid genetic incompatibility of effector lymphocytes and target cells. Cytotoxic lymphocytes were demonstrated in Peyer's patches, mesenteric lymph nodes, spleen, and peripheral blood on postinoculation day (PID) 7. Cytotoxic activity of lymphocytes increased thereafter and reached the maximal amount at PID 21. Lymphocyte cytotoxicity was somewhat greater in lymphocytes of peripheral blood and spleen than in those of Peyer's patches and mesenteric lymph nodes after PID 14. On the contrary, lymphocyte reactivity to the viral antigen measured by lymphocyte proliferative assay was higher in Peyer's patch and mesenteric lymph node cells than in peripheral blood and splenic cells. Lymphocyte cytotoxicity was depressed by treating effector cells with anti-porcine thymocyte serum and complement. However, lymphocyte suspensions treated with anti-porcine thymocyte serum and complement were still cytotoxic to some extent against virus-infected target cells, although T lymphocytes were completely excluded by the treatment. This suggests that cytotoxic mechanism other than the direct action of cytotoxic T lymphocytes may be involved in the cytotoxicity assay systems used in the present studies. In experiments in which allogenic cells (testicle cells of siblings) were used together with autochthonous cells as targets, lymphocyte cytotoxicity was equally expressed against both autochthonous and allogenic target cells in 2 of 3 experiments. However, lymphocyte cytotoxicity was greater against autochthonous cells than against allogenic target cells in 1 of 3 experiments.     

不同感染剂量MDRV对番鸭免疫反应和细胞毒性作用的影响

用不同剂量番鸭呼肠孤病毒(MDRV MW9710株)感染8日龄番鸭后,通过检测血液中淋巴细胞对ConA、LPS的反应和NK细胞、细胞毒T细胞(CTL)的细胞毒性作用,探讨MDRV感染对番鸭免疫细胞功能和细胞毒性作用的影响。结果显示,不同感染剂量MDRV均会抑制番鸭血液淋巴细胞对ConA、LPS的增殖反应,降低NK细胞和CTL细胞的杀伤活性,且影响程度与剂量相关;同时感染番鸭生长缓慢,脾脏肿大,胸腺和法氏囊缩小。上述结果表明,MDRV感染能导致番鸭免疫抑制,且细胞免疫抑制程度与感染病毒量有关。