Monitoring responses by use of five-color flow cytometry in subsets of peripheral T cells obtained from cattle inoculated with a killed Mycobacterium avium subsp paratuberculosis vaccine (original) (raw)
Related papers
Veterinary Immunology and Immunopathology, 2010
Vaccination against Johne's disease with an inactivated, oil-adjuvanted Mycobacterium avium ssp. paratuberculosis (MAP) bacterin can reduce clinical signs in infected herds; however, the development of indurated swelling at the injection site limits vaccine acceptability to producers. This study determined whether a reduced dose of vaccine antigen, with a full dose of adjuvant, would produce comparable T cell-mediated immune responses with smaller lesions. T cell responses induced by in vitro stimulation with MAP antigen from calves vaccinated with full, half, and quarter doses of antigen were evaluated 2, 4, and 9 months after vaccination by multi-parameter flow cytometry (FCM) and the whole blood interferon-g (WB IFN-g) assay. The WB IFN-g responses were significantly elevated in vaccinated animals, but did not differ significantly between doses. FCM demonstrated antigen-specific responses for both IFN-g and IL-4 in the CD4 T cell population from vaccinated animals, while CD8 T cells and gd T cells mainly responded with increased IFN-g. Dose may have affected some T cell subset parameters at some time points, but intradermal skin test responses, WB IFN-g production, IFN-g responses by T cell subsets in FCM were not significantly different between full, half, or quarter doses of antigen. Injection site lesions were smaller in animals vaccinated with a lower dose of antigen, but reached statistical significance (P < 0.05) in the half dose group only.
Mycobacterium bovis is the causative agent of bovine tuberculosis, a disease that is increasing in developing countries. In addition to increasing economic losses, the rise in bovine tuberculosis poses a human health risk. There is an urgent requirement for effective strategies for disease eradication; this will likely involve vaccination in conjunction with current test and slaughter policies. A policy involving vaccination would require an accurate diagnosis of M. bovis-infected animals and the potential to distinguish these animals from vaccinates. Currently used diagnostic tests, the skin test and gamma interferon (IFN-γ) blood test, have a sensitivity of up to 95%. A further complication is that M. bovis BCG-vaccinated animals are also scored positive by these tests. We assessed the possibility of using the quantification of IFN-γ-producing CD4+T lymphocytes by Cytokine Flow Cytometric analysis of intracellular IFN-γ expression for discrimination of M. bovis-infected animals from BCG-immunized in Egypt. Heparinized blood was collected from 2 infected dairy cattle, 2 BCG-immunized cattle and 2 control cattle. Blood was stimulated in the presence of anti-CD49d, anti-CD28 (1 µg/ml) and activated with PPDb (20 ug/ml) and PPDa (20 ug/ml). PMA (50 ng/ml) and ionomycin (1μg/mL) were used as a positive control. Blood was incubated at 37°C in 5% CO2 for 6 hours. After 2 hours, Brefeldin A (10μg/mL) was added. Cells were stained with CD3, CD4, CD45R0, CD69 and IFN-γ antibodies. Two flow cytometer (FC) gating strategy were designed for data acquisition and data were analyzed using the De Novo Software. Significant numbers of IFN-γ-expressing CD4+ T cells were detected following culture of heparinized blood from M. bovis-infected animals, but not from BCG-immunized with purified protein derived from M. bovis (PPDb). The finding suggested that this assay could allow the discrimination of BCG-immunized cattle from infected cattle. It is recommended to evaluate this discrimination technique on large number of animals to deduce dependable results.
Veterinary Immunology and Immunopathology, 2012
Vaccination of cattle against Mycobacterium avium subsp. paratuberculosis (MAP) provides partial protection by delayed shedding of MAP and reduced numbers of clinically affected animals. The duration of vaccine induced immune response is not known. The primary objective of this study was therefore to characterize the long-term effect of whole-cell based vaccination against MAP on the immune response. A secondary objective was to evaluate whether immunodiagnosis of MAP and Mycobacterium bovis infections is affected by MAP vaccination. Two studies were performed: (1) A retrospective longitudinal study including 895 vaccinated and 2526 non-vaccinated dairy cows in 9 Danish dairy herds aiming at characterizing the long-term antibody-response to vaccination; and (2) a cross-sectional study of responses in the IFN-␥ assay carried out in 140 vaccinated animals in two herds to evaluate the effect of vaccination on the cell-mediated immune response and to evaluate a possible interference with the diagnosis of M. bovis infections. The results showed that 37% of samples from vaccinated animals and 5% of samples from non-vaccinated animals, respectively, were test positive in the milk antibody ELISA. The prevalence of antibody responses of the vaccinated animals was relatively constant from 2 to 6 years of age, but decreased in older animals. Among the 140 vaccinated animals 88% tested positive with the IFN-␥ test to johnin PPD and 50% responded to PPDb with IFN-␥ production above a similar cutoff. Although Denmark is free of M. bovis, two of the vaccinated animals responded with higher IFN-␥ levels when cultured with PPDb compared to PPDa. In conclusion, immunization with whole-cell MAP vaccines elicits both humoral and cell-mediated immune reactions, which may interfere with surveillance and diagnosis of both MAP and M. bovis infections using currently available tests.
PLOS ONE
Conventional control and eradication strategies for bovine tuberculosis (BTB) face tremendous difficulties in developing countries; countries with wildlife reservoirs, a complex wildlifelivestock-human interface or a lack of veterinary and veterinary public health surveillance. Vaccination of cattle and other species might in some cases provide the only suitable control strategy for BTB, while in others it may supplement existing test-and-slaughter schemes. However, the use of live BCG has several limitations and the global rise of HIV/AIDS infections has furthermore warranted the exploration of inactivated vaccine preparations. The aim of this study was to compare the immune response profiles in response to parenteral vaccination with live BCG and two inactivated vaccine candidates in cattle. Twenty-four mixed breed calves (Bos taurus) aged 4-6 months, were allocated to one of four groups and vaccinated sub-cutaneously with live M. bovis BCG (Danish 1331), formalin-inactivated M. bovis BCG, heat-killed M. bovis or PBS/Montanide™ (control). Interferonγ responsiveness and antibody production were measured prior to vaccination and at weekly intervals thereafter for twelve weeks. At nine weeks post-priming, animals were skin tested using tuberculins and MTBC specific protein cocktails and subsequently challenged through intranodular injection of live M. bovis BCG. The animals in the heat-killed M. bovis group demonstrated strong and sustained cellmediated and humoral immune responses, significantly higher than the control group in response to vaccination, which may indicate a protective immune profile. Animals in this group showed reactivity to the skin test reagents, confirming good vaccine take. Lastly, although not statistically significant, recovery of BCG after challenge was lowest in the heatkilled M. bovis group. In conclusion, the parenteral heat-killed M. bovis vaccine proved to be clearly immunogenic in cattle in the present study, urging further evaluation of the vaccine in challenge studies using virulent M. bovis and assessment of vaccine efficacy in field conditions.
Veterinary Sciences
Flow cytometry (FC) is widely used in microbiology, immunology, hematology, and oncology. In the veterinary field, FC enabled the study of the immune response in cattle infected with different pathogens, as well as vaccine testing. However, few fluorochrome-conjugated antibodies recognize bovine antigens, limiting the possible benefits of FC and the implementation of multiparametric analysis for more complex studies. Two cytometry panels with five colors each were designed and implemented for the study and identification of populations and subpopulations of T cells derived from the peripheral blood mononuclear cells of dairy heifers. Both panels detected differences in T cell subpopulations between heifers positively and negatively tested for tuberculin; they detected overexpression of CD25+ and CD45RO+ in tuberculin-positive heifers after stimulation with a culture filtrate protein extract (CFPE) from Mycobacterium bovis (M. bovis). We identified subpopulations of T cells from peri...
Veterinary Immunology and Immunopathology, 2005
Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides SC (MmmSC), is one of the most significant cattle disease in Africa. The control measures, which led to eradication from numerous countries are not feasible in Africa where the only prophylaxis relies on vaccination. However, the attenuated vaccines, used up to now in Africa, are of low efficiency. The development of an improved vaccine is, therefore, a necessity. The purpose of this study was to compare some immunological parameters in MmmSC-infected cattle (endobronchial versus natural in-contact infection) and assess the response in correlation with the clinical outcome (death versus recovery). Characterization of the immune parameters elicited in recovered animals, known to be refractory to new infection, will be an important step towards development of new vaccines against CBPP. A significant outcome of this study was the demonstration that all MmmSC-infected cattle developed a MmmSC-specific cell-mediated immune response. A kinetic analysis of the MmmSC responsiveness showed that the main difference between endobronchially-and in-contact infected animals was the delay before the onset of the MmmSC-specific immune response. The first MmmSC-responding PBMC sample was selected from each animal for cell phenotyping. The phenotypic analysis of this early MmmSC-induced response revealed the predominant contribution of the CD4 T-cells in all animals whereas IFNg was only constantly produced in recovered animals. Evolution of this early MmmSC-specific immune response was then followed by a kinetic analysis of the MmmSC-induced CD4 T-cell response and IFNg released. The results demonstrated that in recovered animals, the MmmSC-specific CD4 Th1-like T-cell response was maintained until slaughtering whereas in animals with acute disease, progression of CBPP was associated with a decreased ability of the PBMC to produce IFNg.
PLoS ONE, 2013
Johnes disease (JD), caused by Mycobacterium avium subsp paratuberculosis (MAP), occurs worldwide as chronic granulomatous enteritis of domestic and wild ruminants. To develop a cost effective vaccine, in a previous study we constructed an attenuated Salmonella strain that expressed a fusion product made up of partial fragments of MAP antigens (Ag85A, Ag85B and SOD) that imparted protection against challenge in a mouse model. In the current study we evaluated the differential immune response and protective efficacy of the Sal-Ag vaccine against challenge in a goat model as compared to the live attenuated vaccine MAP316F. PBMCs from goats vaccinated with Sal-Ag and challenged with MAP generated significantly lower levels of IFN-c, following in vitro stimulation with either Antigen-mix or PPD jhonin, than PBMC from MAP316F vaccinated animals. Flow cytometric analysis showed the increase in IFN-c correlated with a significantly higher level of proliferation of CD4, CD8 and cdT cells and an increased expression of CD25 and CD45R0 in MAP316F vaccinated animals as compared to control animals. Evaluation of a range of cytokines involved in Th1, Th2, Treg, and Th17 immune responses by quantitative PCR showed low levels of expression of Th1 (IFN-c, IL-2, IL-12) and proinflammatory cytokines (IL-6, IL-8, IL-18, TNF-a) in the Sal-Ag immunized group. Significant levels of Th2 and antiinflammatory cytokines transcripts (IL-4, IL-10, IL-13, TGF-b) were expressed but their level was low and with a pattern similar to the control group. Over all, Sal-Ag vaccine imparted partial protection that limited colonization in tissues of some animals upon challenge with wild type MAP but not to the level achieved with MAP316F. In conclusion, the data indicates that Sal-Ag vaccine induced only a low level of protective immunity that failed to limit the colonization of MAP in infected animals. Hence the Sal-Ag vaccine needs further refinement to increase its efficacy.