The use of CD4+ T-cell lines to screen for immunogenic proteins of Cowdria ruminantium.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
A Bensaid
Publications and source records attributed to A Bensaid.
Inhibition of MHC class I and class II cell surface expression on bovine endothelial cells upon infection with Cowdria ruminantium.
Endothelial cells constitute a main target for Cowdria ruminantium (CR) and can potentially play a role as antigen presenting cells (APC). Therefore, we measured, in vitro, the effect of CR infections on the expression of MHC class I and class II molecules on bovine umbilical endothelial cells (BUEC) and on bovine brain endothelial cells (BBEC). A dramatic inhibition of the expression of IFNgamma induced MHC class II molecules was observed on BUEC and to a lesser extent on BBEC upon CR infection. This inhibitory effect was also observed on constitutively expressed MHC class I molecules. Part of the reduction of cell surface MHC molecules could be ascribed to their accumulation in intracellular compartments pinpointing a disruption in the transit of these molecules to the surface of the cells. The exact mechanisms of inhibition are not yet known but, as opposed to what is described in other models, the involvement of prostaglandin E2 can be excluded. The results obtained in this study show that endothelial cells have a decreased capacity to express both MHC class I and class II molecules on their surface upon CR infection, thus favouring the escape of this pathogen from the host immune system.
Animals↗
Characterization of variable immunodominant antigens of Cowdria ruminantium by ELISA and immunoblots.
Cross-immunization experiments have revealed a significant antigenic diversity of the isolate of Cowdria ruminantium which needs to be characterized for the development of vaccines. We identified polymorphic immunodominant antigens by ELISA and immunoblot. Using serum from a goat immune to the Gardel stock of Cowdria (isolated in Guadeloupe) adsorbed on antigen of the Senegal stock of this pathogen, distinct serogroups were revealed by ELISA among six isolates from different geographical origins. Furthermore, a goat serum directed against the Senegal stock and adsorbed on Gardel antigens was shown to be specific for the Senegal stock, thus confirming the existence of serotypes in Cowdria. The Major Antigenic Protein 1 (MAP1) of Cowdria was shown to have variable antigenic determinants. Also in a group of variable proteins ranging from 23 to 29 kDa, one antigen of 26-27 kDa had a determinant specific for the Gardel isolate. These polymorphic antigens may be relevant components of Cowdria ruminantium for a vaccine as the sera revealing these antigens originated from a goal surviving a lethal challenge. However, the presence of T-cell epitopes and the ability of the these antigens to confer protection to ruminants remain to be investigated. The production of a rabbit antiserum against this group of polypeptides will be of great use for their purification and for the screening of expression libraries.
Animals↗
Detection of genomic polymorphisms among isolates of the intracellular bacterium Cowdria ruminantium by random amplified polymorphic DNA and Southern blotting.
Sixteen primers were successfully used in a RAPD assay to generate reproducible fingerprints for six isolates of Cowdria ruminantium, a tick-transmitted rickettsia of ruminants. Distinction between stocks was possible by using one or at most two primers. Two stocks were very similar although originating from widely distant geographical regions. A genetic distance tree was constructed by analysing 108 fragments in pairwise comparison between stocks. Three amplification fragments probed with C. ruminantium genomic DNA determined a restriction fragment length polymorphism which allowed the distinction between stocks except for the two stocks that had similar RAPD patterns. The potential of RAPD to determine the extent of genetic diversity of C. ruminantium and to develop probes or PCR primers for diagnostic purposes is discussed.
Blotting, Southern↗
Development of an in vitro cloning method for Cowdria ruminantium.
Cowdria ruminantium is a tick-borne rickettsia which causes severe disease in ruminants. All studies with C. ruminantium reported so far were carried out with stocks consisting of infective blood collected from reacting animals or from the same stocks propagated in vitro. Cloned isolates are needed to conduct studies on immune response of the host, on genetic diversity of the parasite, and on mechanisms of attenuation and the development of vaccines. A method of cloning based on the particular chlamydia life cycle of Cowdria was developed. Instead of cloning extracellular elementary bodies, it appeared more convenient to clone endothelial cells infected by one morula resulting from the infection of the cell by one elementary body of Cowdria. Two hundred and sixteen clones were obtained by limiting dilution of infected cells. The method was experimentally validated by comparing randomly amplified polymorphic DNA fingerprints from individual clones obtained from endothelial cell cultures coinfected with two different stocks of C. ruminantium.
Animals↗
Analysis of T-cell responses in cattle immunized against heartwater by vaccination with killed elementary bodies of Cowdria ruminantium.
Cattle were successfully immunized against heartwater with a lysate of Cowdria ruminantium formulated in Freund's adjuvant. Vaccinated animals proved fully resistant to virulent challenge 3 and 10 months after vaccination. For the first time a helper T lymphocyte response to Cowdria antigens was observed and characterized. Cowdria-specific T-cell lines generated from vaccinated animals by in vitro restimulation with Cowdria lysates are 95 to 100% CD4+, are MHC class II restricted, and produce gamma interferon. They proliferate in response to autologous monocytes infected with live Cowdria but not in response to uninfected monocytes. These T-cell lines will facilitate the search for Cowdria antigens that are immunogenic for T cells and will therefore be of relevance in the development of a subunit vaccine against the disease.
Animals↗
Comparative efficacy of Freund's and Montanide ISA50 adjuvants for the immunisation of goats against heartwater with inactivated Cowdria ruminantium.
Two vaccines, based on inactivated elementary bodies of Cowdria ruminantium, one formulated in Montanide ISA50, the other in Freund's adjuvant, were compared in goats. Administered twice subcutaneously with an interval of 81 days, both protected three out of five goats against a very severe challenge, lethal for all 14 control goats, 3.5 months after the second injection. Both vaccines elicited similar antibody levels. The protection afforded by the Montanide ISA50 vaccine was tested 15 and 17 months after the second injection of the vaccine. Three out of six and five out of six goats, respectively, survived a challenge which killed all four control goats used on each occasion. Antibodies were still detectable in the immunised goats. The level of protection appears to be influenced by the dose of virulent C. ruminantium used for the challenge. As any stock of C. ruminantium can be incorporated in order to cover the antigenic repertoire of the organism, this kind of inactivated vaccine can now be tested in the field.
Adjuvants, Immunologic↗
[How effective is glucose control in insulin-dependent diabetics followed-up in specialized hospital consultation?].
Explore the source record for details and available documents.
Adult↗
An amino acid sequence coded by the exon 2 of the BoLA DRB3 gene associated with a BoLA class I specificity constitutes a likely genetic marker of resistance to dermatophilosis in Brahman zebu cattle of Martinique (FWI).
One hundred and twenty-seven Brahman cattle from several locations in Martinique (FWI), reared under different environmental conditions, were followed over three years and checked for clinical signs of dermatophilosis. To confirm that these animals had been in contact with the pathogen Dermatophilus congolensis, their sera were tested by ELISA. On the basis of this epidemiological study, 12 animals were classified as resistant (seropositive without clinical signs), belonging to herds in which the prevalence of the disease ranged from 25 to nearly 98%. Eighteen animals classified as highly susceptible displayed severe characteristic skin lesions. These 30 selected animals were typed for class I antigens of the major histocompatibility complex (MHC). MHC class II genes were analyzed using the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques, on the exon 2 of the bovine leucocyte antigen (BoLA) DRB3 gene. Several alleles were found, according to patterns provided by the restriction enzymes used: Fnu 4HI, Dpn II, Hae III, and Rsa I. A particular sequence "EIAY" at amino acid positions 66/67/74/78 located in the antigen recognition sites (ARS) was found in the 12 animals classified as resistant, and 10 of them displayed also class I BoLA-A8 specificity. On the other hand, only 3 out of the 18 susceptible animals showed simultaneously the BoLA-DRB3 "EIAY" sequence and BoLA-A8 specificity. Interestingly, a serine residue at position 30 of the ARS was found in 8 of the susceptible animals and was completely absent from all resistant animals. Furthermore, in a same animal, the serine at position 30 and the EIAY sequence were never found simultaneously on the same haplotype. These results show a strong correlation between the resistant character to dermatophilosis and the association of MHC haplotypes: the BoLA-A8 specificity and the BoLA-DRB3 "EIAY" sequence at ARS positions 66/67/74/78 with the lack of serine in position 30. To confirm these results, family segregation studies are in progress and some interesting observations have been obtained.
Actinomycetales Infections↗
Cross-talk between topoisomerase I and HU in Escherichia coli.
In Escherichia coli about one half of the negative supercoiling of DNA is constrained by proteins, in contrast to the situation in eukaryotic cells where most of the DNA is constrained by histones. The level of supercoiling in the unrestrained portion is controlled by a balance between the supercoiling activity of gyrase and the relaxing activity of DNA topoisomerase I. In the present work we show, by disrupting one or both genes encoding the heterodimeric protein HU, that an interplay exists in bacteria between HU and topoisomerase I activity: a decrease in the intracellular concentration of HU was accompanied by an increase in relaxing activity as measured in cell extracts. Conversely, a topA10 mutant of topoisomerase I, which has low levels of relaxing activity, was unable to accept an HU deficiency introduced by transduction. Thus it appears that the ability to increase relaxing activity, or to decrease an excess of supercoiling, is important for cells to survive in the absence of HU. These data can be explained in terms of HU constraining supercoiling in vivo as it does in vitro: the absence of HU would generate more unconstrained supercoiling, which in turn would require an increase in relaxing activity to maintain physiological levels.
Bacterial Proteins↗
Histone-like protein HU and bacterial DNA topology: suppression of an HU deficiency by gyrase mutations.
The abundant bacterial protein called HU has the ability to wrap and bend DNA in vitro, and thus it has long been thought to play a role in DNA supercoiling. In the absence of HU, Escherichia coli formed tiny colonies on agar, rapidly accumulated suppressor mutations, and was hypersensitive to novobiocin. Three types of evidence implicated gyrase in the suppression of an HU deficiency. First, spontaneous suppressors that restored normal growth and reduced sensitivity to novobiocin mapped in gyrB, one of the genes encoding DNA gyrase. Second, a pair of known gyrB mutations (gyrB-203 Ts gyrB-221 NovR) allowed normal growth at permissive (30 degrees C) but not at intermediate (37 degrees C) conditions. Third, introduction of a gyrB-expressing plasmid restored normal colony size. DNA supercoiling comparisons showed that chromosomal supercoiling decreased in the absence of HU and increased toward wild-type levels in the presence of a spontaneous gyrB suppressor. Taken together, these data establish that HU has a physiological role in chromosomal DNA topology, probably by facilitating the action of gyrase.
Alleles↗
Recombinant bovine interferon gamma inhibits the growth of Cowdria ruminantium but fails to induce major histocompatibility complex class II following infection of endothelial cells.
Recombinant bovine IFN gamma is a potent inhibitor of Cowdria ruminantium growth in vitro irrespective of the rickettsial stock, or the origin of the endothelial cells. These results suggest an important role for IFN gamma in protective immune responses against C. ruminantium infections. Here we also show that IFN gamma can induce the expression of MHC class II molecules on the surface of endothelial cells. However, treatment of endothelial cells with IFN gamma following infection with Cowdria fails to induce MHC class II expression. The implications of this pathogen-specific effect on class II expression by endothelial cells with regard to its recognition by the host immune system are discussed.
Animals↗
Contagious bovine pleuropneumonia vaccines: the current situation and the need for improvement.
The control of contagious bovine pleuropneumonia (CBPP) has been clearly identified by the Organisation of African Unity/Inter-African Bureau of Animal Resources as a priority. In the first part of this article, the authors introduce the past and present vaccines, based on the two classic strains, T1, and KH3J. They describe the guidelines for vaccine production technology, and the quality control requirements for CBPP vaccines of the Office International des Epizooties. The failure of the currently used T1-SR vaccine to provoke satisfactory immunity in cattle, particularly in the newly infected areas of Africa, is pointed out. Other shortcomings of the current CBPP vaccines are also highlighted. Thus, there is a need to improve CBPP vaccines and the authors propose detailed emergency measures to address this problem. In the second part of the article, a subunit approach using immunostimulating complex technology is outlined. The authors emphasise the importance of current research in cell-mediated immunity and immunopathology, which is aimed at improving the efficacy of CBPP vaccines.
Animals↗
Infection of bovine brain microvessel endothelial cells with Cowdria ruminantium elicits IL-1 beta, -6, and -8 mRNA production and expression of an unusual MHC class II DQ alpha transcript.
Cowdria ruminantium is a bacterial parasite that infects ruminants, causing an acute and often fatal disease. These obligate intracellular Gram-negative bacteria preferentially infect neutrophils and vascular endothelial cells, especially in the brain. The present study was performed with bovine brain microvessel endothelial cells in culture, infected by C. ruminantium in the presence or absence of IFN-gamma. Infection induced the production of IL-1 beta, -6, and -8 mRNAs, and this effect was potentiated by IFN-gamma. A semi-quantitative PCR analysis indicated that similar amounts of IL-1 beta and IL-6 mRNAs were produced in response to C. ruminantium infection and to treatment with 30 to 40 ng/ml LPS. In addition, although IFN-gamma induced the synthesis of an MHC class II DQ alpha transcript (1.3 kb), an unusual transcript (1.5 kb) was induced by infection and not after LPS treatment. Infection did not affect MHC class I, class II DQ beta, and invariant chain mRNA levels. The present results suggest that C. ruminantium infection raises the immune activity of brain endothelial cells in vitro and that only part of this response can be attributed to LPS. One can hypothesize that cerebral endothelium in vivo efficiently contributes, by MHC Ag expression and production of ILs, to the activation and/or recruitment of leukocytes to the brain and thus plays an active role in the pathogenesis of cowdriosis and in the immune response to this pathogen.
Animals↗
Sequence conservation of microsatellites between Bos taurus (cattle), Capra hircus (goat) and related species. Examples of use in parentage testing and phylogeny analysis.
A panel of 70 bovine microsatellites was tested for amplification from goat DNA. Forty-three could be successfully amplified by PCR, 20 of which were tested for polymorphism. Three were applied for parentage testing in goat families and their exclusion probability evaluated. Fourteen were cloned and sequenced from goat DNA, and goat and bovine sequences were compared to evaluate interspecific conservation. Correlation between the structure of the dinucleotide repeat and the number of alleles was studied and indicated that interruption(s) in the repeat could explain the difference in the levels of polymorphism between the two species. This study provides a valuable in vivo clue to the mechanism generating polymorphism in microsatellites. Sequence conservation was also observed for several microsatellites with two wild species of Bovidae, Nilgaï (Boselaphus tragocamelus) and Himalayan Tur (Capra cylindricornis), and with one species of Cervidae, the fallow deer (Cervus dama). This study showed that an estimated 40 per cent of the microsatellites isolated from cattle will prove useful to study the caprine genome and to characterize economically important genetic loci in this species. Moreover, bovine microsatellites were shown to constitute very useful tools for the study of genetic diversity of the Artiodactyla.
Animals↗
[Fear of hypoglycemia and/or pseudo-acceptance: what are the obstacles to control of insulin-dependent diabetes in the adult?].
Explore the source record for details and available documents.
Adult↗
Members of the novel WC1 gene family are differentially expressed on subsets of bovine CD4-CD8- gamma delta T lymphocytes.
CD4-CD8- gamma delta T cells of ruminants uniquely express a 220-kDa surface Ag recognized by several mAbs clustered as WC1. We recently reported the isolation of a cDNA clone encoding a WC1 Ag. Southern blotting suggested that the bovine genome contains multiple sequences highly related to the isolated WC1 cDNA. Here, we demonstrate that some of the clustered WC1 mAbs stain predominantly nonoverlapping subsets of bovine CD4-CD8- gamma delta T cells. By the isolation of two additional cDNA clones encoding molecules highly related to the original WC1 Ag, we provide a molecular basis for this phenomenon. Cells transfected with cDNAs encoding individual WC1 Ags are differentially recognized by various WC1 mAbs. Thus, expression of members of the WC1 gene family divides bovine CD4-CD8- gamma delta T cells into phenotypical subsets. Field inversion gel electrophoresis revealed that all WC1 genes map to a single, large (> 1 Mbp) Notl fragment. Although the function of WC1 remains unknown, it likely involves interaction with ligands that originate from a similarly complex genetic system.
Amino Acid Sequence↗
Protection of goats against heartwater acquired by immunisation with inactivated elementary bodies of Cowdria ruminantium.
In two experiments, four and five goats were vaccinated by giving two subcutaneous injections of a preparation of inactivated elementary bodies of Cowdria ruminantium (Gardel stock) mixed with Freund's adjuvant. All vaccinated animals together with four naive controls were challenged intravenously with 5 ml of supernatant of a culture of bovine endothelial cells infected with the same stock of Cowdria. All goats developed a high temperature. Two out of four, and four out of five vaccinated goats survived the challenge whereas all naive control animals died within 7-12 days. Vaccinated goats which died survived longer than the controls. No difference in antibody titres was observed between protected and non-protected vaccinated goats. Moreover, immune sera from surviving goats, whether heat inactivated or not, were unable to neutralize the infection of bovine endothelial cells by Cowdria in vitro. Mechanisms conferring protection on the immunized goats are unknown at the moment but the hypothesis that T-helper lymphocyte populations have been elicited seems to be likely. This method of immunization with dead organisms will help in the search for protective antigens against cowdriosis.
Animals↗