Mycobacterial lipolytic enzymes: a gold mine for tuberculosis research.

Tuberculosis (TB) is one of the deadliest infectious diseases worldwide with a strong impact in developing countries. Mycobacterium tuberculosis, the etiological agent of TB, has a high capacity to evade the host immune system and establish a chronic, asymptomatic and latent infection. In a latent TB infection, persistent bacilli are present in a non-replicating dormant state within host granulomas. During reactivation, bacilli start replicating again leading to an active TB infection that can be highly contagious. Mycobacterial lipids and lipolytic enzymes are thought to play important physiological roles during dormancy and reactivation. The role of lipolytic enzymes in the physiology of M. tuberculosis and physiopathology of the disease will be discussed in this review, with an emphasis on the secreted or cell wall-associated, surface exposed lipolytic enzymes characterized to date. Studies on the localization, enzymatic activity and immunological properties of these enzymes highlighted their possible usefulness as new diagnostic markers in the fight against TB.

Viable Mycoplasma mycoides ssp. mycoides small colony-mediated depression of the bovine cell responsiveness to the mitogen concanavalin A.

Mycoplasma mycoides ssp. mycoides biotype Small Colony (MmmSC) is the causative agent of contagious bovine pleuropneumonia (CBPP), which is still a major tropical cattle disease. Development of an efficient vaccine requires an understanding of the immunopathology of CBPP as MmmSC presents a strong ability to escape the host immune response. The objective of this study was to determine whether the presence of MmmSC can modulate the immune response induced by the mitogen Concanavalin A (ConA) on bovine immune cells [peripheral blood mononuclear cells (PBMC) and lymph node (LN) cells]. Comparative analysis of the immunomodulating properties of viable versus heat-killed MmmSC on ConA-stimulated immune cells revealed that while heat-killed MmmSC had no effect, viable MmmSC strongly depressed, in a concentration-dependent manner, the ConA mitogenic activity (blastogenesis and interferon-gamma production). Both B-cell and T-cell activation were affected with the highest impact on the CD4 T cells. The phenotypic analysis showed that the ConA-induced proliferation of CD25(+) cells was strongly reduced when co-exposed to viable MmmSC, confirming that events associated with ConA-induced cell activation were suppressed by the pathogen. This study thus demonstrated that viable MmmSC is able to inhibit the polyclonal mitogenic activity of the ConA on bovine PBMC and LN cells. This finding strongly suggests that the persistence of viable MmmSC may also thus inhibit the bovine immune response directed towards inactivated MmmSC, whether dead or in the form of antigens, also present during infection. This study confirmed that MmmSC has evolved an efficient mechanism to prevent its elimination from the host.

Mycoplasma mycoides ssp. mycoides biotype small colony-secreted components induce apoptotic cell death in bovine leucocytes.

Contagious bovine pleuropneumonia, caused by Mycoplasma mycoides ssp. mycoides biotype small colony (MmmSC), is one of the most serious cattle diseases in Africa. Several observations suggested that MmmSC had evolved an efficient way to escape the bovine immune responses by triggering host-cell cytotoxicity. This study was implemented to determine whether the cytotoxic effect was due to apoptotic cell death. To that end, bovine blood cells were cultured for up to 3 days in the presence of viable or heat-killed MmmSC compared to unstimulated cultures. The findings provided evidence for a viable MmmSC-induced, time-dependent apoptosis in bovine blood leucocytes, whereas heat-killed MmmSC had no effect. Morphological and physiological changes (evidenced by TUNEL and annexin V staining) typical of apoptosis were observed in response to viable MmmSC. All the lymphocyte subsets as well as the monocyte/granulocyte subset exhibited extensive apoptosis after exposure to viable MmmSC. Our results demonstrated a potential role for MmmSC-secreted components as pathogenic factors able to induce programmed cell death in bovine blood leucocytes.

Gamma interferon-producing CD4 T-cells correlate with resistance to Mycoplasma mycoides subsp. mycoides S.C. infection in cattle.

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 IFNgamma 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 IFNgamma 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 IFNgamma. The results led to the identification of immune parameters, which correlate with protection against CBPP and to a relevant strategy for the development of improved vaccines against this disease.

Evolution of the chromosomal location of rDNA genes in two Drosophila species subgroups: ananassae and melanogaster.

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 (11 of which were being examined for the first time) belonging to the melanogaster and ananassae subgroups suggests that the ancestral configuration consists of one nucleolus organizer (NOR) on each sex chromosome. This pattern, which is conserved throughout the melanogaster subgroup, except in D. simulans and D. sechellia, was observed only in the ercepeae complex within the ananassae subgroup. Both sex-linked NORs must have been lost in the lineage leading to D. varians and in the ananassae and bipectinata complexes, whereas new sites, characterized by intra-species variation in hybridization signal size, appeared on the fourth chromosome related to heterochromatic rearrangements. Nucleolar material is thought to be required for sex chromosome pairing and disjunction in a variety of organisms including Drosophila. Thus, either remnant sequences, possibly intergenic spacer repeats, are still present in the sex chromosomes which have lost their NORs (as observed in D. simulans and D. sechellia), or an alternative mechanism has evolved.

Contagious bovine pleuropneumonia vaccines, historic highlights, present situation and hopes.

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.

A new cytochrome P450 from Drosophila melanogaster, CYP4G15, expressed in the nervous system.

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.

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.

[Diagnosis of contagious bovine pleuropneumonia: problems and recent developments]. / Diagnostic de la péripneumonie contagieuse bovine: problèmes et nouveaux développements.

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.

Development of two PCR assays for the identification of mycoplasmas causing contagious agalactia.

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.

Development of a selective polymerase chain reaction assay for the detection of Mycoplasma mycoides subsp. Mycoides S.C. (contagious bovine pleuropneumonia agent).

A new selective assay for the detection of Mycoplasma mycoides subsp. mycoides SC (MmmSC) via the polymerase chain reaction (PCR) has been developed. This test used two PCR assays: a control-PCR (MYC-PCR) identifying the pathogen as a member of the mycoides cluster and the MSC-PCR which is specific for MmmSC. The MYC primers targeted a DNA sequence of about 460 bp from all the 59 mycoides cluster-strains tested. No amplification occurred with bovine genomic DNA or with the 11 other bacterial species assayed. The MSC primers selectively amplified a 275 bp sequence from the 27 MmmSC strains tested, with three specific internal restriction sites allowing confirmation of the identification. The sensitivity assessed by direct agarose gel analysis for both PCR assays was 100 CFU. The sensitivity of the MSC-PCR was increased to 1 CFU by a dot-blot hybridization step using, as a probe, the entire 275 bp sequence digoxigenin-labeled by PCR. These two PCR assays were successfully used to detect MmmSC in pleural fluids from naturally-infected cattle. We conclude that these two PCR assays may be valuable tools for the diagnosis of contagious bovine pleuropneumonia.

Field diagnostic kits: a solution for developing countries?

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.

Development of a species-specific DNA probe for Mycoplasma capricolum.

A specific DNA probe for the detection and identification of Mycoplasma capricolum, one of the causative agents of contagious agalactia syndrome, was selected from a genomic library. It consists of a 900bp RsaI genomic fragment of M. capricolum (reference strain), cloned into the EcoRV site of the plasmid Bluescript. By using the appropriate stringency this radiolabelled probe reacts specifically with M. capricolum when tested by dot blot hybridization against various mycoplasmal DNAs. The current level of sensitivity of the 32P-labelled 900bp RsaI probe is 500 pg of homologous DNA, corresponding to 5 x 10(4) mycoplasmas. A non radioactive labelling method, using the digoxigenin-11-dUTP, was also tested. The specificity of the digoxigenin-labelled probe was equivalent to that obtained with the radioactive probe. However the sensitivity of detection decreased to 1 ng of homologous DNA detected, corresponding to 1 x 10(5) mycoplasmas. Tests performed with milk samples have demonstrated that the radioactive 900 bp RsaI probe indeed detected M. capricolum contained in milk. A positive signal was obtained when 10(5) M. capricolum were present in the spot.