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The evolution of the chromosomal location of ribosomal RNA gene clusters and the organization of heterochromatin in the Drosophila melanogaster group were investigated using fluorescence in situ hybridization and DAPI staining to mitotic chromosomes. The investigation of 18 species belonging to the melanogaster and ananassae subgroups suggest that the ancestral configuration consists of one nucleolus organizer (NOR) on each sex chromosome.

Abstract Background The multicellular tumor spheroid (MCTS) is an in vitro model associating malignant-cell microenvironment and 3D organization as currently observed in avascular tumors. Methods In order to evaluate the relevance of this model for pre-clinical studies of drug combinations, we analyzed the effect of gemcitabine alone and in combination with the CHIR-124 CHK1 inhibitor in a Capan-2 pancreatic cell MCTS model. Results Compared to monolayer cultures, Capan-2 MCTS exhibited resistance to gemcitabine cytotoxic effect. This resistance was amplified in EGF-deprived quiescent spheroid suggesting that quiescent cells are playing a role in gemcitabine multicellular resistance. After a prolonged incubation with gemcitabine, DNA damages and massive apoptosis were observed throughout the spheroid while cell cycle arrest was restricted to the outer cell layer, indicating that gemcitabine-induced apoptosis is directly correlated to DNA damages. The combination of gemcitabine and CHIR-124 in this MCTS model, enhanced the sensitivity to the gemcitabine antiproliferative effect in correlation with an increase in DNA damage and apoptosis. Conclusions These results demonstrate that our pancreatic MCTS model, suitable for both screening and imaging analysis, is a valuable advanced tool for evaluating the spatio-temporal effect of drugs and drug combinations in a chemoresistant and microenvironment-depending tumor model.

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. [ABSTRACT FROM AUTHOR]

Background: Bacillus cereus is a notorious foodborne pathogen, which can grow under anoxic conditions. Anoxic growth is supported by endogenous redox metabolism, for which the thiol redox proteome serves as an interface. Here, we studied the cysteine (Cys) proteome dynamics of B. cereus ATCC 14579 cells grown under fermentative anoxic conditions. We used a quantitative thiol trapping method combined with proteomics profiling., Results: In total, we identified 153 reactive Cys residues in 117 proteins participating in various cellular processes and metabolic pathways, including translation, carbohydrate metabolism, and stress response. Of these reactive Cys, 72 were detected as reduced Cys. The B. cereus Cys proteome evolved during growth both in terms of the number of reduced Cys and the Cys-containing proteins identified, reflecting its growth-phase-dependence. Interestingly, the reduced status of the B. cereus thiol proteome increased during growth, concomitantly to the decrease of extracellular oxidoreduction potential., Conclusions: Taken together, our data show that the B. cereus Cys proteome during unstressed fermentative anaerobic growth is a dynamic entity and provide an important foundation for future redox proteomic studies in B. cereus and other organisms., (© 2021. The Author(s).)

At the end of exponential growth, aerobic bacteria have to cope with the accumulation of endogenous reactive oxygen species (ROS). One of the main targets of these ROS is cysteine residues in proteins. This study uses liquid chromatography coupled to high-resolution tandem mass spectrometry to detect significant changes in protein abundance and thiol status for cysteine-containing proteins from Bacillus cereus during aerobic exponential growth. The proteomic profiles of cultures at early-, middle-, and late-exponential growth phases reveals that (i) enrichment in proteins dedicated to fighting ROS as growth progressed, (ii) a decrease in both overall proteome cysteine content and thiol proteome redox status, and (iii) changes to the reduced thiol status of some key proteins, such as the transition state transcriptional regulator AbrB. Taken together, our data indicate that growth under oxic conditions requires increased allocation of protein resources to attenuate the negative effects of ROS. Our data also provide a strong basis to understand the response mechanisms used by B. cereus to deal with endogenous oxidative stress.

Background: Better understanding of the immune response directed against Mycobacterium tuberculosis (Mtb) is critical for development of vaccine strategies and diagnosis tests. Previous studies suggested that Mtb enzymes involved in lipid metabolism, are associated with persistence and/or reactivation of dormant bacilli., Methods: Circulating antibodies secreting cells (ASCs), memory B cells, and antibodies directed against Cut4 (Rv3452) and CFP21 (Rv1984c) antigens were explored in subjects with either active- or latent-tuberculosis (LTB), and in Mtb-uninfected individuals., Results: Circulating anti-Cut4 ASCs were detected in 11/14 (78.6%) subjects from the active TB group vs. 4/17 (23.5%) from the LTB group (p = 0.001). Anti-CFP21 ASCs were found in 11/14 (78.6%) active TB vs. in 5/17 (29.4%) LTB cases (p = 0.01). Circulating anti-Cut4 and anti-CFP21 ASCs were not detected in 38 Mtb uninfected controls. Memory B cells directed against either Cut4 or CFP21 were identified in 8/11 (72.7%) and in 9/11 (81.8%) subjects with LTB infection, respectively, and in 2/6 Mtb uninfected individuals (33.3%). High level of anti-Cut4 and anti-CFP21 IgG were observed in active TB cases., Conclusion: Circulating IgG SCs directed against Cut4 or CFP21 were mostly detected in patients presenting an active form of the disease, suggesting that TB reactivation triggers an immune response against these two antigens.

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.

The enzymes belonging to the cutinase family are serine enzymes active on a large panel of substrates such as cutin, triacylglycerols, and phospholipids. In the M. tuberculosis H37Rv genome, seven genes coding for cutinase-like proteins have been identified with strong immunogenic properties suggesting a potential role as vaccine candidates. Two of these enzymes which are secreted and highly homologous, possess distinct substrates specificities. Cfp21 is a lipase and Cut4 is a phospholipase A2, which has cytotoxic effects on macrophages. Structural overlay of their three-dimensional models allowed us to identify three areas involved in the substrate binding process and to shed light on this substrate specificity. By site-directed mutagenesis, residues present in these Cfp21 areas were replaced by residues occurring in Cut4 at the same location. Three mutants acquired phospholipase A1 and A2 activities and the lipase activities of two mutants were 3 and 15 fold greater than the Cfp21 wild type enzyme. In addition, contrary to mutants with enhanced lipase activity, mutants that acquired phospholipase B activities induced macrophage lysis as efficiently as Cut4 which emphasizes the relationship between apparent phospholipase A2 activity and cytotoxicity. Modification of areas involved in substrate specificity, generate recombinant enzymes with higher activity, which may be more immunogenic than the wild type enzymes and could therefore constitute promising candidates for antituberculous vaccine production.

Transcription and expression regulation of some individual cel genes (cel5A, cel5I, cel5D and cel44O) of Clostridium cellulolyticum were investigated. Unlike the cip-cel operon, these genes are transcribed as monocistronic units of transcription, except cel5D. The location of the transcription initiation sites was determined using RT-PCR and the mRNA 5'-end extremities were detected using primer extension experiments. Similarly to the cip-cel operon, cel5A and cel5I expressions are regulated by a carbon catabolite repression mechanism, whereas cel44O and cel5D expressions do not seem to be submitted to this regulation. The role of the putative transcriptional regulator GlyR2 in the regulation of cel5D expression was investigated. The recombinant protein GlyR2 was produced and was shown to bind in vitro to the cel5D and glyR2 promoter regions, suggesting that besides regulating its own expression, GlyR2 may regulate cel5D expression. To test this hypothesis in vivo, an insertional glyR2 mutant was generated and the effect of this disruption on cel5D expression was evaluated. Levels of cel5D mRNAs in the mutant were 16 fold lower than that of the wild-type strain suggesting that GlyR2 acts as an activator of cel5D expression.

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.

Lipid metabolism plays an important role during the lifetime of Mycobacterium tuberculosis, the causative agent of tuberculosis. Although M. tuberculosis possesses numerous lipolytic enzymes, very few have been characterized yet at a biochemical/pharmacological level. This study was devoted to the M. tuberculosis lipolytic enzymes belonging to the Hormone-Sensitive Lipase (HSL) family, which encompasses twelve serine hydrolases closely related to the human HSL. Among them, nine were expressed, purified and biochemically characterized using a broad range of substrates. In vitro enzymatic inhibition studies using the recombinant HSL proteins, combined with mass spectrometry analyses, revealed the potent inhibitory activity of an oxadiazolone compound, named MmPPOX. In addition, we provide evidence that MmPPOX alters mycobacterial growth. Overall, these findings suggest that the M. tuberculosis HSL family displays important metabolic functions, thus opening the way to further investigations linking the involvement of these enzymes in mycobacterial growth.

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.

Background: New diagnosis tests are urgently needed to address the global tuberculosis (TB) burden and to improve control programs especially in resource-limited settings. An effective in vitro diagnostic of TB based on serological methods would be regarded as an attractive progress because immunoassays are simple, rapid, inexpensive, and may offer the possibility to detect cases missed by standard sputum smear microscopy. However, currently available serology tests for TB are highly variable in sensitivity and specificity. Lipolytic enzymes have recently emerged as key factors in lipid metabolization during dormancy and/or exit of the non-replicating growth phase, a prerequisite step of TB reactivation. The focus of this study was to analyze and compare the potential of four Mycobacterium tuberculosis lipolytic enzymes (LipY, Rv0183, Rv1984c and Rv3452) as new markers in the serodiagnosis of active TB., Methods: Recombinant proteins were produced and used in optimized ELISA aimed to detect IgG and IgM serum antibodies against the four lipolytic enzymes. The capacity of the assays to identify infection was evaluated in patients with either active TB or latent TB and compared with two distinct control groups consisting of BCG-vaccinated blood donors and hospitalized non-TB individuals., Results: A robust humoral response was detected in patients with active TB whereas antibodies against lipolytic enzymes were infrequently detected in either uninfected groups or in subjects with latent infection. High specifity levels, ranging from 93.9% to 97.5%, were obtained for all four antigens with sensitivity values ranging from 73.4% to 90.5%, with Rv3452 displaying the highest performances. Patients with active TB usually exhibited strong IgG responses but poor IgM responses., Conclusion: These results clearly indicate that the lipolytic enzymes tested are strongly immunogenic allowing to distinguish active from latent TB infections. They appear as potent biomarkers providing high sensitivity and specificity levels for the immunodiagnosis of active TB.

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.

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., ((c) 2009 Elsevier Ltd. All rights reserved.)

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.

The thermophilic Campylobacters are enteropathogenic for humans. We recently showed that Omp50 is a Campylobacter species-specific porin produced in Campylobacter jejuni and Campylobacter lari but not in Campylobacter coli. In the present study, we investigated regulation of the omp50 gene and found that its expression in C. jejuni was temperature-dependent, but independent of growth phase or medium viscosity. The use of RT-PCR and omp50::lacZ fusions showed that growth temperature control occurred at the transcriptional level. The promoter and the coding sequence were cloned in an Escherichia coli-Campylobacter shuttle plasmid and transferred to E. coli and to a C. jejuni Omp50-deficient strain. Regulation of omp50 gene expression by growth temperature was observed in the recombinant C. jejuni strain, but not in E. coli. The same regulation was also observed in wild-type C. lari strains and in a C. coli strain supplemented by the plasmid, suggesting that omp50 expression is controlled by a mechanism conserved among Campylobacter species.

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.

Membrane proteins are of keen interest to structural biologists, as they are known to act as receptors, adhesins, sensors, transporters, and signal-transducers of living cells. During the past few decades, the efforts made to study the bacterial membrane proteins have been impaired by the problems encountered during the production and purification of native proteins. Herein we demonstrate that the Campylobacter jejuni CadF protein, which was isolated using a novel purification strategy, exhibits biological activity as evidenced by channel activity in lipid bilayers. CadF, an E. coli OmpA-like protein, facilitates the binding of C. jejuni to the extracellular matrix component, fibronectin.

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.

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.

We studied the prevalence of the omp50 gene and the Omp50 protein in Campylobacter strains. Immunodetection assays and DNA-DNA hybridizations showed that most C. coli strains tested were negative and most C. jejuni and C. lari strains tested were positive. A PCR assay was developed, using the omp50 gene as a species-specific target. We propose a combination of a hippurate test and an omp50 assay to perform identification of Campylobacter species.

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).

Contagious bovine pleuropneumonia (CBPP) is a contagious infection of cattle caused by a mycoplasma, M. mycoides subsp. mycoides SC (MmmSC). It induces lesions of pleuropneumonia in acute cases and the formation of pulmonary "sequestra" in chronic cases. The disease is prevalent mostly in Africa, where it is responsible for high losses, but it has also been sporadically present in Southern Europe until 1999. Vaccination is now prohibited in most countries except in Africa. An empirical "inoculation" procedure was developed as early as 1852 in Europe but it may have been used even earlier in Africa. The inoculation of pleural fluid was performed at the tip of the tail in Europe and on the bridge of the nose in Africa. It conferred good protection but induced a high number of fatal cases. Various inactivated preparations have been tested in the past with inconclusive results leading sometime to some protection and some other time to a sensitisation of the immunised animals. Such preparations have never been used in the field. Attenuated MmmSC strains have been developed in the 1950s and used extensively in the field both in Africa and Australia. The best known vaccine strains are KH3J, T1/44 and T1sr. Vaccination campaigns have succeeded in reducing considerably the CBPP prevalence in these two continents but eradication was achieved in Australia only by switching to strict measures of animal movement control and a stamping-out policy. The search for new CBPP vaccines has become a major issue for African countries that are facing an increase in outbreaks. The rationale for this search is based on a better understanding of the mycoplasma virulence mechanisms that could lead to a targeted attenuation of MmmSC strains. It is also based on a better understanding of the bovine immune response that may be driven to a pathogenic inflammatory response or conversely to a better balanced response leading to protection.

Porins allow exchanges between bacteria and their environment. In the gram-negative food-borne pathogen Campylobacter jejuni two porins, major outer membrane protein (MOMP) and Omp50, have been identified. MOMP is synthesized at a very high level under laboratory culture conditions, suggesting that its promoter functions very efficiently under these conditions. In Campylobacter samples, we observed that MOMP porin expression increased at a high temperature (42 degrees C) or a high pH (pH 8.5) compared to expression at a low temperature (31 degrees C) or an acidic pH (pH 5.5). To study the regulation of MOMP expression at the transcriptional level, we constructed an momp-gfp fusion in which gfp expression was put under the control of the momp promoter. Interestingly, we observed the same pattern of regulation in Escherichia coli, as monitored by green fluorescent protein production, that was found in CAMPYLOBACTER: The ranges of pH and temperature tested are physiologically relevant, because they can be found in the digestive tracts of both birds and humans, which are both colonized by CAMPYLOBACTER: Our results suggest that a component of the regulatory mechanism is conserved in C. jejuni and E. coli. However, medium osmolarity and sodium salicylate did not have a significant effect on C. jejuni momp promoter activity in E. coli, suggesting that major regulatory elements of E. coli porin expression do not participate in MOMP regulation. In contrast, mechanisms involving DNA supercoiling may be involved, as shown by DNA gyrase inhibition assays. These findings are a step towards determining the role of outer membrane proteins in the adaptation of C. jejuni to its environment.

A novel cytochrome P450 was isolated from Drosophila melanogaster by PCR strategy with primers deduced from the crayfish Orconectes limosus CYP4C15 sequence, which is supposed to be involved in ecdysteroid biosynthesis. The full-length cDNA contains a 1980 bp open reading frame encoding a predicted protein of 574 amino acids and was designated CYP4G15. The corresponding gene is located at 10C1 on the X chromosome. The presence of a N-terminal segment mainly hydrophobic indicated that the corresponding enzyme is probably microsomal. In situ hybridization demonstrated predominant expression of CYP4G15 in the brain of third larval instar and Northern-blots showed no overexpression in insecticide resistant strain. This is the first indication of a specific P450 expressed in the central nervous system of Drosophila, and the putative function of the corresponding enzyme is discussed., (Copyright 2000 Academic Press.)

While it is easy to diagnose contagious bovine pleuropneumonia (CBPP) in an animal in the acute clinical stage, subacute and chronic forms are more difficult to diagnose. Recourse to laboratory tests is essential to confirm any suspicion of CBPP. As standard diagnostic procedures (isolation, culture, biochemical tests, serological tests) are lacking in specificity and sensitivity, improvements are needed. Progress in molecular biology techniques has led to new tests, among which are the polymerase chain reaction (PCR) and the enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies. Application of these techniques to CBPP offers a number of advantages, and has considerably enhanced the specificity and sensitivity of diagnosis.

A new detection test for the mycoplasmas causing contagious agalactia, Mycoplasma agalactiae, M. capricolum subsp. capricolum and M. mycoides subsp. mycoides L. C., was developed. It was based on two polymerase chain reaction assays: the Ma-PCR for the detection of M. agalactiae and the MYC-PCR for the 'mycoides cluster' thus including M. capricolum subsp. capricolum and M. mycoides subsp. mycoides L. C. An M. agalactiae strain was identified by a 933-bp Ma-PCR product and no amplification with the MYC-PCR. In contrast, a 460-bp MYC-PCR product and a negative or a 350-bp Ma-PCR product characterized a 'mycoides cluster' strain. M. capricolum subsp. capricolum and M. mycoides subsp. mycoides L. C. were identified by their species-specific AseI pattern of the 460-bp MYC-PCR product.

An exact assessment of the animal health situation in a country is an essential element in formulating eradication and control programmes, and in regulating international trade in animals and animal products from that country. Due to a lack of human and technical resources, Veterinary Services in developing countries often lack precise knowledge on disease occurrence. Since the collection and transmission of reliable information on animal diseases in developing countries are major concerns of the Office International des Epizooties (OIE), a project aimed at improving this situation was implemented with international financial support. This project involved the development by the Centre for the Application of Methodology for the Diagnosis of Animal Diseases (CAMDA) of field kits for the diagnosis of the main diseases present in tropical Africa: rinderpest, peste des petits ruminants (PPR), contagious bovine pleuropneumonia (CBPP) and contagious caprine pleuropneumonia (CCPP). Several tests already exist, such as complement deoxyribonucleic acid (cDNA)-specific probes and polymerase chain reaction (PCR) for rinderpest and PPR, DNA probes and PCR for CBPP, capture enzyme-linked immunosorbent assay, the agglutination test and the immunobinding peroxidase test for CCPP, etc. With specific reference to these examples, the various problems faced by the OIE and CAMDA are reviewed.