Design and evaluation of a unique SYBR Green real-time RT-PCR assay for quantification of five major cytokines in cattle, sheep and goats.

BACKGROUND: Today, when more than 60% of animal diseases are zoonotic, understanding their origin and development and identifying protective immune responses in ruminants are major challenges. Robust, efficient and cost-effective tools are preconditions to solve these challenges. Cytokines play a key role in the main mechanisms by which the immune system is balanced in response to infectious pathogens. The cytokine balance has thus become the focus of research to characterize immune response in ruminants. Currently, SYBR Green reverse transcriptase quantitative PCR (RT-qPCR) is the most widely method used to investigate cytokine gene expression in ruminants, but the conditions in which the many assays are carried out vary considerably and need to be properly evaluated. Accordingly, the quantification of gene expression by RT-qPCR requires normalization by multiple reference genes. The objective of the present study was thus to develop an RT-qPCR assay to simultaneously quantify the expression of several cytokines and reference genes in three ruminant species. In this paper, we detail each stage of the experimental protocol, check validation parameters and report assay performances, following MIQE guidelines. RESULTS: Ten novel primer sets were designed to quantify five cytokine genes (IL-4, IL-10, IL-12B, IFN-γ and TNF-α) and five reference genes (ACTB, GAPDH, H3F3A, PPIA and YWHAZ) in cattle, sheep, and goats. All the primer sets were designed to span exon-exon boundaries and use the same hybridization temperature. Each stage of the RT-qPCR method was detailed; their specificity and efficiency checked, proved and are reported here, demonstrating the reproducibility of our method, which is capable of detecting low levels of cytokine mRNA up to one copy whatever the species. Finally, we checked the stability of candidate reference gene expression, performed absolute quantification of cytokine and reference gene mRNA in whole blood samples and relative expression of cytokine mRNA in stimulated PBMC samples. CONCLUSIONS: We have developed a novel RT-qPCR assay for the simultaneous relative quantification of five major cytokines in cattle, sheep and goats, and their accurate normalization by five reference genes. This accurate and easily reproducible tool can be used to investigate ruminant immune responses and is widely accessible to the veterinary research community.

Expression and in situ localization of two major PR proteins of grapevine berries during development and after UV-C exposition.

In grapevine Vitis vinifera L. cv Pinot noir, the Pathogenesis-Related (PR) proteins CHI4D and TL3 are among the most abundant extractable PR proteins of ripe berries and accumulate during berry ripening from véraison until full maturation. Evidence was supplied in favor of the involvement of these two protein families in plant defense mechanisms and plant development. In order to better understand CHI4D and TL3 function in grapevine, we analyzed their temporal and spatial pattern of expression during maturation and after an abiotic stress (UV-C) by in situ hybridization (ISH) and immunohistolocalization. In ripening berries, CHI4D and TL3 genes were mainly expressed in the exocarp and around vascular bundles of the mesocarp. In UV-C exposed berries, CHI4D and TL3 gene expression was strongly induced before véraison. Corresponding proteins localized in the exocarp and, to a lesser extent, around vascular bundles of the mesocarp. The spatial and temporal accumulation of the two PR proteins during berry maturation and after an abiotic stress is discussed in relation to their putative roles in plant defense.

Adjuvants and delivery systems in veterinary vaccinology: current state and future developments.

Modern adjuvants should induce strong and balanced immune responses, and it is often desirable to induce specific types of immunity. As an example, efficient Th1-immunity-inducing adjuvants are highly in demand. Such adjuvants promote good cell-mediated immunity against subunit vaccines that have low immunogenicity themselves. The development of such adjuvants may take advantage of the increased knowledge of the molecular mechanisms and factors controlling these responses. However, knowledge of such molecular details of immune mechanisms is relatively scarce for species other than humans and laboratory rodents, and in addition, there are special considerations pertaining to the use of adjuvants in veterinary animals, such as production and companion animals. With a focus on veterinary animals, this review highlights a number of approaches being pursued, including cytokines, CpG oligonucleotides, microparticles and liposomes.

Identification of Mycoplasma mycoides subsp. mycoides small colony genes coding for T-cell antigens.

Genes of the Mycoplasma mycoides subsp. mycoides small colony biotype (MmmSC) coding for proteins capable of eliciting protective T-cell memory responses have potential for incorporation into a recombinant subunit vaccine against contagious bovine pleuropneumonia (CBPP). Here we used lymphocytes from cattle that had completely recovered from infection to screen products of MmmSC genes for recognition by CD4(+) effector memory (Tem) and central memory (Tcm) T lymphocytes. Six MmmSC genes (abc, gapN, glpO, lppA, lppB, and ptsG) were expressed as histidine-tagged recombinant polypeptides, or synthetic overlapping peptides, before inclusion in proliferation and gamma interferon (IFN-gamma) assays. Only two MmmSC antigens, LppA and PtsG, consistently induced recall proliferation from immune CD4(+) T cells and IFN-gamma production in all animals tested. Moreover, LppA and PtsG were shown to possess epitopes recognized by both short-lived CD4(+) Tem and long-lived CD4(+) Tcm cells.

Expression and immunogenicity of the mycobacterial Ag85B/ESAT-6 antigens produced in transgenic plants by elastin-like peptide fusion strategy.

This study explored a novel system combining plant-based production and the elastin-like peptide (ELP) fusion strategy to produce vaccinal antigens against tuberculosis. Transgenic tobacco plants expressing the mycobacterial antigens Ag85B and ESAT-6 fused to ELP (TBAg-ELP) were generated. Purified TBAg-ELP was obtained by the highly efficient, cost-effective, inverse transition cycling (ICT) method and tested in mice. Furthermore, safety and immunogenicity of the crude tobacco leaf extracts were assessed in piglets. Antibodies recognizing mycobacterial antigens were produced in mice and piglets. A T-cell immune response able to recognize the native mycobacterial antigens was detected in mice. These findings showed that the native Ag85B and ESAT-6 mycobacterial B- and T-cell epitopes were conserved in the plant-expressed TBAg-ELP. This study presents the first results of an efficient plant-expression system, relying on the elastin-like peptide fusion strategy, to produce a safe and immunogenic mycobacterial Ag85B-ESAT-6 fusion protein as a potential vaccine candidate against tuberculosis.

Comparative analysis of four lipoproteins from Mycoplasma mycoides subsp. mycoides Small Colony identifies LppA as a major T-cell antigen.

Control of contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC), remains an important goal in Africa. Subunit vaccines triggering B and T-cell responses could represent a promising approach. To this aim, the T-cell immunogenicity of four MmmSC lipoproteins (LppA, LppB, LppC and LppQ), present in African strains and able to elicit humoral response, was evaluated. In vitro assays revealed that only LppA was recognized by lymph node lymphocytes taken from three cattle, 3 weeks after MmmSC exposure. Maintenance of the LppA-specific response, relying on CD4 T-cells and IFN gamma production, was then demonstrated 1 year after infection. LppA is thus an important target for the CD4 T-cells generated early after MmmSC infection and persisting in the lymph nodes of recovered cattle. Its role as a protective antigen and ability to in vivo trigger both arms of the host immune response remain to be evaluated.

CD62L defines a subset of pathogen-specific bovine CD4 with central memory cell characteristics.

Central memory T cells (Tcm) have not previously been characterized in cattle and any other ruminant species. Here we described two phenotypically and functionally different subsets of pathogen-specific memory CD4(+) T cells in cattle that survived infection with Mycoplasma mycoides subsp. mycoides small colony (MmmSC). The first subset is CD45RO(+)CD45R(-)CD62L(-) and comprises two thirds of IFN-gamma producing CD4(+) T cells after MmmSC recall stimulation. The second is CD45RO(+)CD45R(-)CD62L(+) and represents the majority of proliferating CD4(+) T cells after 7 days of stimulation. Cell sorting experiments confirmed that both CD4(+)CD62L(+) and CD4(+)CD62L(-) subsets are present in vivo and proliferate independently in recall responses to MmmSC. In addition, MmmSC stimulation strongly decreased CCR7 and increased CCR5 transcripts levels in CD4(+)CD62L(-) cells whereas CD4(+)CD62L(+) were only slightly affected. High levels of recall proliferation but low IFN-gamma production, together with the capacity to preferentially migrate through the lymph nodes (i.e., expression of CD62L and CCR7), are characteristics of Tcm, in humans and mice. Tcm are associated with long-term protective immunity and a privileged target for vaccine development. Our results demonstrate the existence of Tcm in cattle and suggest that CD62L may serve as a marker to monitor Tcm in infections and vaccine development studies in ruminant.

Analysis of cellular responses to Mycoplasma mycoides subsp. mycoides small colony biotype associated with control of contagious bovine pleuropneumonia.

A better understanding of protective immune memory against contagious bovine pleuropneumonia (CBPP) is needed in order to facilitate the development of safer vaccines based on selected components of the pathogen. For this purpose, cells collected from lymph nodes draining the lungs of Mycoplasma mycoides subsp. mycoides small colony biotype (MmmSC)-infected cattle were stimulated with the pathogen in vitro and evaluated concurrently for proliferation (CFSE based method), expression of activation, memory markers and cytokine production. Direct evidence is presented for a major contribution of CD4+ T cells to the vigorous proliferative and T1 biased cytokine recall responses observed in cattle that have recovered from infection but not in animals developing the acute form of the disease. Two different phenotypes of MmmSC-specific memory CD4 were observed based on CD62L expression and proliferative capacities. Furthermore, recall proliferation of B cells also occurred but was strictly dependent on the presence of CD4. The information provided in this study will facilitate the search for MmmSC antigens that have potential for the development of subunit vaccines against CBPP.

Pulmonary and serum antibody responses elicited in zebu cattle experimentally infected with Mycoplasma mycoides subsp. mycoides SC by contact exposure.

The purpose of the present study was to characterize the Mycoplasma mycoides subsp. mycoides small colony (MmmSC)-specific humoral immune response at both systemic and local levels in cattle experimentally infected with MmmSC, for a better understanding of the protective immune mechanisms against the disease. The disease was experimentally reproduced in zebu cattle by contact. Clinical signs, postmortem and microbiological findings were used to evaluate the degree of infection. Serum and bronchial lavage fluids (BAL) were collected sequentially, before contact and over a period of one year after contact. The kinetics of the different antibody isotypes to MmmSC was established. Based on the severity of the clinical signs, post mortem and microbiological findings, the animals were classified into three groups as acute form with deaths, sub-acute to chronic form and resistant animals. Seroconversion was never observed for the control animals throughout the duration of the experiment, nor for those classified as resistant. Instead, seroconversion was measured for all other cattle either with acute or sub-acute to chronic forms of the disease. For these animals, IgM, IgG1, IgG2 and IgA responses were detected in the serum and BAL samples. The kinetics of the IgM, IgG1 and IgG2 responses was nearly similar between both groups of animals. No evident correlation could thus be established between the levels of these isotypes and the severity of the disease. Levels of IgA were high in both BAL and serum samples of animals with sub-acute to chronic forms of the disease, and tended to persist throughout the entire experimental period. In contrast, animals with acute forms of the disease showed low levels of IgA in their BAL samples with none or very transient but low levels of IgA in the serum samples. Our results thus demonstrated that IgA is produced locally in MmmSC experimentally infected cattle by contact and may play a role in protection against contagious bovine pleuropneumonia.

Characterisation of the lymph node immune response following Mycoplasma mycoides subsp. Mycoides SC infection in cattle.

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides biotype Small Colony (MmmSC), is still a major cattle disease in Africa. Development of long-term protective vaccines, the only relevant strategy to achieve CBPP eradication, requires the characterisation of the protective immune mechanism. To this aim, the present study investigated the cellular immune response persisting in the lymph nodes of cattle infected naturally and experimentally by contact, one year post exposure. The lymph node cell composition, MmmSC responsiveness and phenotype of the MmmSC-responding lymphocytes were compared between animals according to the different outcomes of the infection. To unravel the protective mechanism, the study focussed on the MmmSC-specific memory immune response generated in recovered cattle, known to develop long-term immunity and to be resistant to reinfection. An MmmSC-specific immune response, mediated by IFNgamma-secreting CD4 T-cells, was detected in the lymph nodes of all recovered cattle. Furthermore, the magnitude of this immune response was significantly higher in animals with complete recovery than in recovered animals presenting lung sequestra. The findings suggest that, in recovered cattle, a subset of MmmSC-primed IFNgamma-secreting CD4 T-cells homed to the regional lymph nodes as MmmSC-specific memory T-cells, likely responsible for the protective anamnestic response. Induction and expansion of this subset of MmmSC-specific CD4 memory T-cells might be a major goal to develop efficient long term protective vaccines against CBPP.

Detection of transposable elements in Drosophila salivary gland polytene chromosomes by in situ hybridization.

In situ hybridization is particularly appropriate for mapping specific DNA sequences on polytene chromosomes of Drosophila and other dipterans. This technique is based on the recognition and binding of one labeled sequence (the probe) to homologous sequences on chromosomes fixed on a microscope slide. The probes are labeled with biotin or other nonradioactive products, and the probe signal can be detected as a thin line on the chromosomes, following the shape of the classical Giemsa-stained chromosome bands, thus allowing the detection of TE insertions within the range of 50 to 200 kb. In our laboratory we work on many individuals from natural populations, and as a result we process high numbers of slides hybridized with various DNA probes of transposable elements every day. Therefore, the in situ hybridization technique we use is a simplification of earlier published protocols. This chapter presents our simplified standard in situ hybridization protocol for labeling polytene chromosomes of Drosophila with biotin and a fluorescence stain (FISH).