In vitro production of antibodies to bovine virus diarrhoea virus by peripheral blood mononuclear cells from cattle immunized by infection

A method for in vitro production of antibodies to bovine virus diarrhoea virus (BVDV) by peripheral blood mononuclear cells (PBMC) was developed. The PBMC were cultured in microtitre plates coated with detergent-solubilized BVDV and the supernatants were tested in an enzyme-linked immunosorbent assay which detects IgG antibodies to BVDV. Following incubation of PBMC with an optimal concentration of pokeweed mitogen for 5 days, antibodies to BVDV were detected in culture supernatants of PBMC from immune cattle, but not in supernatants of PBMC from seronegative cattle, from persistently BVDV-infected cattle or from a 5-day-old calf that received BVDV antibodies via colostrum. This antibody-production assay may therefore be used as a tool to discriminate between passively acquired antibodies and those actively induced.     

In vitro interactions of cytokines and bovine viral diarrhea virus in phytohemagglutinin-stimulated bovine mononuclear cells

Bovine viral diarrhea (BVD) virus inhibited phytohemagglutinin (PHA)-stimulated bovine peripheral blood mononuclear cell (PBMC) proliferation and bovine interleukin-2 (IL-2) production. In the controls, the heat-inactivated BVD virus was not capable of suppressing the PHA-stimulated PBMC proliferation. Presence of exogenous cytokines, such as purified human IL-2, recombinant bovine interleukin-1 (rbovIL-1), recombinant bovine IL-2, and recombinant human IL-6 failed to reverse the BVD virus-induced immunosuppression. Also, we found that the BVD virus inhibited PHA and IL-2 induced proliferation of bovine PBMC in the early and late stages of activation. In summary, our data suggest that BVD virus induced immunosuppression was not due to destruction of the PBMC but may be inhibiting one or more of the important intracellular enzymes that may regulate PBMC proliferation.     

Interferon stimulated genes, CXCR4 and immune cell responses in peripheral blood mononuclear cells infected with bovine viral diarrhea virus

Non-cytopathic bovine viral diarrhea virus (ncpBVDV) induces immune responses mediated by chemokines and interferon (IFN) stimulated genes (ISGs). Cultured bovine peripheral blood mononuclear cells (PBMC) from ncpBVDV-naïve cattle were used herein to demonstrate that BVDV infection modulates chemokine receptor 4 (CXCR4), CXCL12, IFN-I, ISGs and selected immune cell marker (CD4, CD8, CD14) mRNAs, and that these acute responses to viral infection are reflected in PBMC cultured with serum from heifers carrying fetuses persistently infected (PI) with ncpBVDV. Infection of PBMC with ncpBVDV increased IFN-β, ISG15, RIG-I, CXCR4, CXCL12, and CD8 mRNA concentrations after 32 h. Culture of PBMC with uterine vein serum from acutely infected heifers, inoculated with ncpBVDV during early gestation to generate PI fetuses, also increased the concentration of CXCR4, RIG-I and ISG15 mRNAs. In vitro PBMC treatment with ncpBVDV or uterine vein serum from acutely infected pregnant heifers activates chemokine, ISG and immune cell responses.     

Bovine virus diarrhoea virus induces in vitro a proliferative response of peripheral blood mononuclear cells from cattle immunized by infection.

The ability of bovine peripheral blood mononuclear cells (PBMC) to mount a proliferative response to bovine virus diarrhoea virus (BVDV) in vitro was examined. After culturing PBMC in the presence of a non-cytopathic strain of BVDV for 6 days, the magnitude of PBMC proliferation was measured as incorporation of radiolabelled thymidine, present during the last 18 h. Live, but not heat-inactivated, BVDV evoked a proliferative response of PBMC obtained from cattle seropositive to the virus. However, PBMC from seronegative or persistently BVDV-infected animals were not stimulated by BVDV. The presence of live BVDV did not alter the proliferative response of PBMC to stimulation with concanavalin A.     

牛病毒性腹泻病毒感染牛外周血单核细胞对CD80和CD86 mRNA转录的影响

用非致细胞病变（noncytopathic,NCP）和致细胞病变（cytopathic,CP）型牛病毒性腹泻病毒（BVDV）感染临床健康BVDV检测阴性的荷斯坦奶牛外周血单核细胞（PBMC）,利用实时荧光定量PCR技术对感染后共刺激分子CD80和CD86mRNA转录水平的变化进行定量分析。结果表明,在NCP型BVDV感染牛PBMC后CD80在4h（P〈0.05）和12,24h（P〈0.01）出现2次转录高峰,CD86在6h（P〈0.05）出现转录高峰;CP型BVDV感染后,CD80在24h（P〈0.05）出现转录高峰,CD86在6h（P〈0.05）出现转录高峰。尽管CD80在NCP型BVDV感染后呈现较复杂的动态变化,但结果提示NCP型和CP型BVDV感染均可导致牛PBMC的共刺激分子CD80和CD86基因转录在感染早期明显受到抑制,PBMC的抗原呈递能力受到影响。     

2-Mercaptoethanol influences the in vitro function of bovine peripheral blood mononuclear cells.

The effects of 2-mercaptoethanol (2-ME) on some in vitro functions of bovine peripheral blood mononuclear cells (PBMC) were examined. It was shown that 2-ME enhanced, in a dose-dependent manner, the production of antibodies to bovine coronavirus. In this test the optimal concentration of 2-ME was 50 microM. This molarity of 2-ME was also optimal for the pokeweed mitogen (PWM)-induced proliferation of PBMC obtained from the 7 cattle tested. Similarly, the spontaneous proliferation of PBMC from 4 out of these cattle was enhanced. Thus, 2-ME evoked an increase (up to 2.5 times) or a decrease (at most 10 times) of the quota between the PWM-induced and the spontaneous proliferation (stimulation index). In general, the presence of 50 microM 2-ME enhanced the in vitro production of interferon by bovine PBMC. On the contrary, the highest proliferative response of PBMC to stimulation with bovine virus diarrhoea virus was achieved in cultures without 2-ME or in cultures with 0.5 or 5 microM 2-ME. Since the effects of 2-ME varied, for different tests as well as for cattle tested, attention should be paid to the use of 2-ME in cultures of bovine PBMC.     

牛病毒性腹泻病毒感染牛外周血单核细胞对IFN-α、β、γmRNA转录的影响

为了解牛病毒性腹泻病毒（BVDV）感染对干扰素（IFN）mRNA转录时相的影响,探讨宿主-病毒之间的相互关系,用非致细胞病变（noncytopathic,NCP）和致细胞病变（cytopathic,CP）型BVDV感染临床健康BVDV检测阴性的荷斯坦奶牛外周血单核细胞（PBMC）,利用实时荧光定量PCR技术对感染后IFN-α、β、γmRNA转录水平的变化进行定量分析。结果表明,CP型和NCP型BVDV感染PBMC后,Ⅰ型IFN（IFN-α、β）均呈现出不同程度的转录水平上调,且差异极显著（P〈0.01）;只有IFN-α在CP型BVDV感染后4,12h（P〈0.5）出现转录下调。IFN-γ在整个感染过程中均呈现出不同程度的转录水平上调,且差异显著（P〈0.05）。这表明2种生物型BVDV感染可引起PBMC中IFN mRNA转录水平升高。     

In vitro inhibition of 5-lipoxygenase metabolite, leukotriene B4, in bovine mononuclear cells by bovine viral diarrhea virus.

Bovine viral diarrhea (BVD) virus inhibited phytohemagglutinin (PHA)-, PHA plus phorbol-12-myristate-13 acetate (PMA)- or PHA plus calcium ionophore (A23187)-stimulated bovine peripheral blood mononuclear cell (PBMC) proliferation. Further, BVD-virus inhibited A23187-stimulated leukotriene B4 (LTB4) synthesis into the culture supernatants. Presence of exogenous LTB4 failed to reverse the BVD virus-induced immunosuppression. Our results suggest that BVD virus-induced immunosuppression is due to a factor that may be necessary to induce LTB4 synthesis for normal mononuclear cell proliferation.     

Genetic characterization of bovine viral diarrhea virus strains in Beijing,China and innate immune responses of peripheral blood mononuclear cells in persistently infected dairy cattle

To acquire epidemiological data on the bovine viral diarrhea virus (BVDV) and identify cattle persistently infected (PI) with this virus, 4,327 samples from Holstein dairy cows were screened over a four-year period in Beijing, China. Eighteen BVD viruses were isolated, 12 from PI cattle. Based on genetic analysis of their 5''-untranslated region (5''-UTR), the 18 isolates were assigned to subgenotype BVDV-1m, 1a, 1d, 1q, and 1b. To investigate the innate immune responses in the peripheral-blood mononuclear cells of PI cattle, the expression of Toll-like receptors (TLRs), RIG-I-like receptors, interferon-α (IFN-α), IFN-β, myxovirus (influenza virus) resistance 1 (MX1), and interferon stimulatory gene 15 (ISG15) was assessed by qPCR. When compared with healthy cattle, the expression of TLR-7, IFN-α, and IFN-β mRNA was downregulated, but the expression of MX1 and ISG-15 mRNA was upregulated in PI cattle. Immunoblotting analysis revealed that the expression of interferon regulatory factor 3 (IRF-3) and IRF-7 was lower in PI cattle than in healthy cattle. Thus, BVDV-1m and 1a are the predominant subgenotypes in the Beijing region, and the strains are highly divergent. Our findings also suggest that the TLR-7/IRF-7 signaling pathway plays a role in evasion of host restriction by BVDV.     

Cell-mediated cytotoxicity of peripheral blood mononuclear cells stimulated in vitro for infectious bovine rhinotracheitis virus-infected cells

Peripheral blood mononuclear cells (PBMC) from calves infected with and hyperimmunized to infectious bovine rhinotracheitis virus (IBRV) were stimulated in vitro with viral antigens to evaluate their cytotoxicity for a variety of cells. The 51-Cr release assay was used to measure cytotoxicity. Cytotoxicity was not present in fresh nonstimulated cells, but was detected in cultured, IBRV-stimulated cells at day 3, was maximal at day 7, and declined thereafter. PBMC stimulated in vitro with IBRV expressed a preference for killing IBRV-infected cells compared to pseudorabies virus (PRV)-infected cells. IBRV-infected, but not PRV-infected, cold target cells inhibited lysis of IBRV-labeled target cells. High concentrations of IBRV hyperimmune serum partially blocked cytotoxicity. Cells expressing a viral preference for cytotoxicity showed no preference for lysis of autologous compared to heterologous bovine cells. PBMC from calves that were either IBRV-immune or not immune were cultured without IBRV stimulation and had similar levels of cytotoxicity for IBRV-infected cells as cells from IBRV-infected cattle.     

In vitro effects of lactoferrin on lipopolysaccharide-induced proliferation, gene expression, and prostanoid production by bovine peripheral blood mononuclear cells

Objective-To evaluate the effect of lactoferrin on lipopolysaccharide (LPS)-induced proliferation of bovine peripheral blood mononuclear cells (PBMCs), gene expression of inflammatory mediators, and production of prostanoids in vitro. Sample Population-PBMCs isolated from 15 Holstein bull calves. Procedures-Mixed populations of PBMCs were isolated by differential centrifugation. Proliferation assays were conducted in 96-well plates designed to allow addition of lactoferrin (200 ng/mL) with and without LPS (1 mug/mL) in a checkerboard design. Incorporation of (3)H-thymidine was used to determine proliferation of PBMCs. Prostaglandin E(2) production was determined in culture-conditioned medium by use of enzyme immunoassay. Effects of lactoferrin on LPS-induced gene expression of cyclooxygenase (COX)-2 and matrix metalloproteinase (MMP)-9 were monitored by use of PCR assays. Results-Lactoferrin supplementation significantly reduced LPS-induced incorporation of (3)H-thymidine and production of prostaglandin E(2) by PBMCs. Lactoferrin reduced LPS-induced expression of COX-2 and MMP-9 mRNA. Conclusions and Clinical Relevance-Lactoferrin reduced LPS-induced cellular proliferation, inflammatory mediator gene expression, and prostaglandin E(2) production by bovine PBMCs in vitro. These effects may be beneficial in reducing the impact of endotoxemia in neonates.     

In vitro stimulation of antibody production to Mycoplasma hyopneumoniae by porcine peripheral blood mononuclear cells.

A method to stimulate and detect the in vitro production of antibodies to Mycoplasma hyopneumoniae by porcine peripheral blood mononuclear cells (PBMC) was established. PBMC were cultured in microtiter plates coated with a sonicated M. hyopneumoniae whole cell antigen and the amount of antibody bound to the coating antigen was determined by an enzyme linked immunosorbent assay (ELISA). In addition, the amount of non-bound antibody was determined by testing the culture supernatants in the ELISA which detects porcine antibodies to M. hyopneumoniae. The production of antibodies, in terms of total absorbance values, was enhanced by including 2.5 ng pokeweed mitogen (PWM) per ml growth medium without altering the specificity of the assay. In a pilot experiment, the applicability of the method to follow the development of antigen-reactive cells during primary and secondary immunizations with M. hyopneumoniae was evaluated. Antigen-reactive cells, identified by their ability to produce antibodies to M. hyopneumoniae in vitro, were detected seven days after the primary immunization and reached their highest antigen reactivity one week later. In comparison, antigen-reactive cells could be detected three days after the booster immunization and remained in the circulation for 2 weeks.     

Reproduction of mucosal disease with cytopathogenic bovine viral diarrhoea virus selected in vitro

Isolates of non-cytopathogenic bovine viral diarrhoea (BVD) virus from 18 persistently infected calves from one herd were compared by using monoclonal antibodies directed against the major viral glycoprotein gp53. All the isolates displayed an almost identical reaction pattern. Based on this antigenic analysis three cytopathogenic BVD and three non-cytopathogenic BVD viruses closely related to the non-cytopathogenic BVD herd isolate were selected. Six of the persistently infected calves were inoculated with a pool of the three closely related cytopathogenic BVD viruses and two with a pool of the three non-cytopathogenic BVD viruses. In addition three animals were infected with one closely related cytopathogenic BVD strain (Indiana) and two animals with the antigenetically different cytopathogenic BVD viral strain A1138/69. Regardless of the inoculation route all the animals superinfected with closely related cytopathogenic BVD viruses developed the characteristic lesions of mucosal disease within 14 days of infection. Animals which were inoculated with non-cytopathogenic BVD viruses which closely resembled the herd isolate, or with cytopathogenic BVD viruses which did not resemble the herd isolate did not develop any signs of disease. However, the latter group produced antibodies to the superinfecting virus.     

In vitro activation of bovine macrophage and peripheral blood mononuclear cells by Nocardia rubra cell wall skeleton (N-CWS)

Nocardia rubra cell wall skeleton (N-CWS) was used for immunotherapy to bovine leukemia virus (BLV)-positive cattle with enlarged subcutaneous lymphatic nodules. Electron microscopical observations showed the infiltration of macrophage and T cells around N-CWS treated lesions. Antitumor effect induced by N-CWS was examined in vitro. The maximum cytolytic activity of macrophage was observed, when the cells were incubated with 10 micrograms/ml of N-CWS. Chemiluminescence response of peripheral blood mononuclear cells (PBMC) using N-CWS as stimulant was observed with a high level of activity for a long period, 5 hr. Mitogenic effect of N-CWS to PBMC was observed in the presence of macrophages but not without macrophages. Furthermore, interleukin 2 activity was recognized in supernatant of PBMC cultured with N-CWS. Maximum cytotoxic T lymphocyte response was induced when PBMC were cultured with 0.1 micrograms/ml of N-CWS.     

Effects of mycotoxins on mitogen-stimulated proliferation of bovine peripheral blood mononuclear cells

The effects of mycotoxins on mitogen-stimulated proliferation of bovine peripheral blood mononuclear cells (PBMCs) were investigated. Aflatoxin B(1) (AFB(1)), deoxynivalenol (DON) and zearalenone (ZEN) were added to cultures of PBMCs, and the proliferation responses were measured using MTT bioassays. Suppression of the proliferation of calf PBMCs by AFB(1) and DON was significantly stronger than that of cow PBMCs, whereas there were no differences in suppressive effects on PBMCs from Holstein and Japanese Black calves and cows. The suppressive effect was greatest in the order of DON, AFB(1) and ZEN, and the effects of DON and AFB(1) seemed to be dose-dependent. The results suggest that some mycotoxins directly suppress proliferation of bovine PBMCs.     

In vitro production of specific antibody by equine peripheral blood mononuclear cells using tetanus toxoid as a recall antigen.

Anti-tetanus toxoid (TT) antibody (Ig) levels in the supernatant of cultured, pre-immunised equine peripheral blood mononuclear cells (PBMC) were measured by an indirect enzyme-linked immunoabsorbent assay (ELISA). Optimal anti-TT Ig production occurred at concentrations of stimulating, purified TT of between 0.001 and 0.1 micrograms ml-1, which varied depending on the cell concentration. Optimal anti-TT Ig production was most consistently produced when the cell concentration was 5 x 10(6) ml-1. At this cell concentration maximal anti-TT Ig was induced using 0.1 micrograms ml-1 TT. At a cell concentration of 5 x 10(6) ml-1 and a TT concentration of 0.1 micrograms ml-1 anti-TT Ig was first detectable in supernatant on day 5 of stimulation. Maximal levels of anti-TT Ig were present in the supernatant by day 10. No anti-TT Ig was produced in cultures of PBMC from non-immune animals.     

In vitro and in vivo immune responses in neonatally thymectomized holstein-friesian calves exposed to bovine viral diarrhea virus

Neonatally thymectomized and normal Holstein-Friesian calves were exposed to bovine viral diarrhea virus and challenged 22 days later. Baseline values in non-specific mitogen induced lymphocyte blastogenesis and serologic responses to tetanus toxoid and Brucella abortus strain 19 were established in neonatally thymectomized and normal calves.Neither viral recovery nor protective antibody production followed the very low level primary exposure to bovine viral diarrhea virus. The expected clinical response to an appropriate challenge inoculum did not occur, however, suggesting an immune response to bovine viral diarrhea virus had been initiated by the primary exposure. Five of eight calves developed protective serum levels of bovine viral diarrhea antibody. This may represent an example of immunologic priming. Exposure to low levels of bovine viral diarrhea virus also has potential implications in the pathogenesis of chronic bovine viral diarrhea wherein low level exposure may interfere with the development of protective levels of bovine viral diarrhea antibody as was observed in three calves.Circulating lymphocyte concentrations and results of non-specific mitogen stimulation of lymphocytes revealed a reduced phytohemagglutinin response in conjunction with a slightly increased response to bacterial lipopolysaccharide and pokeweed mitogen in the presence of moderate lymphopenia. The capacity of lymphocytes to respond to phytohemagglutinin was unchanged but an increased total response, as occurred in control thymectomized and intact calves, did not occur.The response to tetanus toxoid, a thymus-independent antigen, was increased following exposure to bovine viral diarrhea virus, perhaps reflecting an alteration in thymic-independent regulation. The lack of a differential response to Brucella abortus strain 19 implied competence in thymus-dependent helper cells.