Isothermal loop-mediated amplification (LAMP) for diagnosis of contagious bovine pleuro-pneumonia.

BACKGROUND Contagious Bovine Pleuropneumonia (CBPP) is the most important chronic pulmonary disease of cattle on the African continent causing severe economic losses. The disease, caused by infection with Mycoplasma mycoides subsp. mycoides is transmitted by animal contact and develops slowly into a chronic form preventing an early clinical diagnosis. Because available vaccines confer a low protection rate and short-lived immunity, the rapid diagnosis of infected animals combined with traditional curbing measures is seen as the best way to control the disease. While traditional labour-intensive bacteriological methods for the detection of M. mycoides subsp. mycoides have been replaced by molecular genetic techniques in the last two decades, these latter approaches require well-equipped laboratories and specialized personnel for the diagnosis. This is a handicap in areas where CBPP is endemic and early diagnosis is essential. RESULTS We present a rapid, sensitive and specific diagnostic tool for M. mycoides subsp. mycoides detection based on isothermal loop-mediated amplification (LAMP) that is applicable to field conditions. The primer set developed is highly specific and sensitive enough to diagnose clinical cases without prior cultivation of the organism. The LAMP assay detects M. mycoides subsp. mycoides DNA directly from crude samples of pulmonary/pleural fluids and serum/plasma within an hour using a simple dilution protocol. A photometric detection of LAMP products allows the real-time visualisation of the amplification curve and the application of a melting curve/re-association analysis presents a means of quality assurance based on the predetermined strand-inherent temperature profile supporting the diagnosis. CONCLUSION The CBPP LAMP developed in a robust kit format can be run on a battery-driven mobile device to rapidly detect M. mycoides subsp. mycoides infections from clinical or post mortem samples. The stringent innate quality control allows a conclusive on-site diagnosis of CBPP such as during farm or slaughter house inspections.

Beta-D-glucoside utilization by Mycoplasma mycoides subsp. mycoides SC: possible involvement in the control of cytotoxicity towards bovine lung cells

BACKGROUND: Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small-colony type (SC) is among the most serious threats for livestock producers in Africa. Glycerol metabolism-associated H2O2 production seems to play a crucial role in virulence of this mycoplasma. A wide number of attenuated strains of M. mycoides subsp. mycoides SC are currently used in Africa as live vaccines. Glycerol metabolism is not affected in these vaccine strains and therefore it does not seem to be the determinant of their attenuation. A non-synonymous single nucleotide polymorphism (SNP) in the bgl gene coding for the 6-phospho-beta-glucosidase (Bgl) has been described recently. The SNP differentiates virulent African strains isolated from outbreaks with severe CBPP, which express the Bgl isoform Val204, from strains to be considered less virulent isolated from CBPP outbreaks with low mortality and vaccine strains, which express the Bgl isoform Ala204. RESULTS: Strains of M. mycoides subsp. mycoides SC considered virulent and possessing the Bgl isoform Val204, but not strains with the Bgl isoform Ala204, do trigger elevated levels of damage to embryonic bovine lung (EBL) cells upon incubation with the disaccharides (i.e., beta-D-glucosides) sucrose and lactose. However, strains expressing the Bgl isoform Val204 show a lower hydrolysing activity on the chromogenic substrate p-nitrophenyl-beta-D-glucopyranoside (pNPbG) when compared to strains that possess the Bgl isoform Ala204. Defective activity of Bgl in M. mycoides subsp. mycoides SC does not lead to H2O2 production. Rather, the viability during addition of beta-D-glucosides in medium-free buffers is higher for strains harbouring the Bgl isoform Val204 than for those with the isoform Ala204. CONCLUSION: Our results indicate that the studied SNP in the bgl gene is one possible cause of the difference in bacterial virulence among strains of M. mycoides subsp. mycoides SC. Bgl does not act as a direct virulence factor, but strains possessing the Bgl isoform Val204 with low hydrolysing activity are more prone to survive in environments that contain high levels of beta-D-glucosides, thus contributing in some extent to mycoplasmaemia.

Mycoplasma leachii sp. nov. as a new species designation for Mycoplasma sp. bovine group 7 of Leach, and reclassification of Mycoplasma mycoides subsp. mycoides LC as a serovar of Mycoplasma mycoides subsp. capri

The Mycoplasma mycoides cluster consists of six pathogenic mycoplasmas causing disease in ruminants, which share many genotypic and phenotypic traits. The M. mycoides cluster comprises five recognized taxa: Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC), M. mycoides subsp. mycoides Large Colony (MmmLC), M. mycoides subsp. capri (Mmc), Mycoplasma capricolum subsp. capricolum (Mcc) and M. capricolum subsp. capripneumoniae (Mccp). The group of strains known as Mycoplasma sp. bovine group 7 of Leach (MBG7) has remained unassigned, due to conflicting data obtained by different classification methods. In the present paper, all available data, including recent phylogenetic analyses, have been reviewed, resulting in a proposal for an emended taxonomy of this cluster: (i) the MBG7 strains, although related phylogenetically to M. capricolum, hold sufficient characteristic traits to be assigned as a separate species, i.e. Mycoplasma leachii sp. nov. (type strain, PG50(T) = N29(T) = NCTC 10133(T) = DSM 21131(T)); (ii) MmmLC and Mmc, which can only be distinguished by serological methods and are related more distantly to MmmSC, should be combined into a single subspecies, i.e. Mycoplasma mycoides subsp. capri, leaving M. mycoides subsp. mycoides (MmmSC) as the exclusive designation for the agent of contagious bovine pleuropneumonia. A taxonomic description of M. leachii sp. nov. and emended descriptions of M. mycoides subsp. mycoides and M. mycoides subsp. capri are presented. As a result of these emendments, the M. mycoides cluster will hereafter be composed of five taxa comprising three subclusters, which correspond to the M. mycoides subspecies, the M. capricolum subspecies and the novel species M. leachii.

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.

Detection of Mycoplasma mycoides subsp. mycoides SC in bronchoalveolar lavage fluids of cows based on a TaqMan real-time PCR discriminating wild type strains from an lppQ(-) mutant vaccine strain used for DIVA-strategies

Contagious bovine pleuropneumonia (CBPP) is the most serious cattle disease in Africa, caused by Mycoplasma mycoides subsp. mycoides small-colony type (SC). CBPP control strategies currently rely on vaccination with a vaccine based on live attenuated strains of the organism. Recently, an lppQ(-) mutant of the existing vaccine strain T1/44 has been developed (Janis et al., 2008). This T1lppQ(-) mutant strain is devoid of lipoprotein LppQ, a potential virulence attribute of M. mycoides subsp. mycoides SC. It is designated as a potential live DIVA (Differentiating Infected from Vaccinated Animals) vaccine strain allowing both serological and etiological differentiation. The present paper reports on the validation of a control strategy for CBPP in cattle, whereby a TaqMan real-time PCR based on the lppQ gene has been developed for the direct detection of M. mycoides subsp. mycoides SC in ex vivo bronchoalveolar lavage fluids of cows and for the discrimination of wild type strains from the lppQ(-) mutant vaccine strain.

Molecular mechanisms of pathogenicity of Mycoplasma mycoides subsp. mycoides SC

Mycoplasma mycoides subsp. mycoides SC, the aetiological agent of contagious bovine pleuropneumonia (CBPP), is considered the most pathogenic of the Mycoplasma species. Its virulence is probably the result of a coordinated action of various components of an antigenically and functionally dynamic surface architecture. The different virulence attributes allow the pathogen to evade the host's immune defence, adhere tightly to the host cell surface, persist and disseminate in the host causing mycoplasmaemia, efficiently import energetically valuable nutrients present in the environment, and release and simultaneously translocate toxic metabolic pathway products to the host cell where they cause cytotoxic effects that are known to induce inflammatory processes and disease. This strategy enables the mycoplasma to exploit the minimal genetic information in its small genome, not only to fulfil the basic functions for its replication but also to damage host cells in intimate proximity thereby acquiring the necessary bio-molecules, such as amino acids and nucleic acid precursors, for its own biosynthesis and survival.

Domain analysis of lipoprotein LppQ in Mycoplasma mycoides subsp. mycoides SC

The lipoprotein LppQ is the most prominent antigen of Mycoplasma mycoides subsp. mycoides small colony type (SC) during infection of cattle. This pathogen causes contagious bovine pleuropneumonia (CBPP), a devastating disease of considerable socio-economic importance in many countries worldwide. The dominant antigenicity and high specificity for M. mycoides subsp. mycoides SC of lipoprotein LppQ have been exploited for serological diagnosis and for epidemiological investigations of CBPP. Scanning electron microscopy and immunogold labelling were used to provide ultrastructural evidence that LppQ is located to the cell membrane at the outer surface of M. mycoides subsp. mycoides SC. The selectivity and specificity of this method were demonstrated through discriminating localization of extracellular (i.e., in the zone of contact with host cells) vs. integral membrane domains of LppQ. Thus, our findings support the suggestion that the accessible N-terminal domain of LppQ is surface exposed and such surface localization may be implicated in the pathogenesis of CBPP.

Functional and antigenic properties of GlpO from Mycoplasma mycoides subsp. mycoides SC: characterization of a flavin adenine dinucleotide-binding site deletion mutant

L-alpha-glycerophosphate oxidase (GlpO) plays a central role in virulence of Mycoplasma mycoides subsp. mycoides SC, a severe bacterial pathogen causing contagious bovine pleuropneumonia (CBPP). It is involved in production and translocation of toxic H(2)O(2) into the host cell, causing inflammation and cell death. The binding site on GlpO for the cofactor flavin adenine dinucleotide (FAD) has been identified as Gly(12)-Gly(13)-Gly(14)-Ile(15)-Ile(16)-Gly(17). Recombinant GlpO lacking these six amino acids (GlpODeltaFAD) was unable to bind FAD and was also devoid of glycerophosphate oxidase activity, in contrast to non-modified recombinant GlpO that binds FAD and is enzymatically active. Polyclonal monospecific antibodies directed against GlpODeltaFAD, similarly to anti-GlpO antibodies, neutralised H(2)O(2) production of M. mycoides subsp. mycoides SC grown in the presence of glycerol, as well as cytotoxicity towards embryonic calf nasal epithelial (ECaNEp) cells. The FAD-binding site of GlpO is therefore suggested as a valuable target site for the future construction of deletion mutants to yield attenuated live vaccines of M. mycoides subsp. mycoides SC necessary to efficiently combat CBPP.

Cattle immunized against the pathogenic L-α-glycerol-3-phosphate oxidase of Mycoplasma mycoides subs. mycoides fail to generate neutralizing antibodies and succumb to disease on challenge

The membrane-associated enzyme L-α-glycerol-3-phosphate oxidase (GlpO) of Mycoplasma mycoides subs. mycoides (Mmm), the causal agent of contagious bovine pleuropneumonia (CBPP) has been identified as a virulence factor responsible for the release of toxic by-products such as H2O2 that mediate host cell injury. Since CBPP pathogenesis is based on host inflammatory reactions, we have determined the capacity of recombinant GlpO to generate in vivo protective responses against challenge in immunized cattle. We also investigated whether sera raised against recombinant GlpO in cattle and mice inhibit production of H2O2 by Mmm. Immunization of cattle with recombinant GlpO did not protect against challenge with a virulent strain of Mmm. Further, although both murine and bovine antisera raised against recombinant GlpO detected recombinant and native forms of GlpO in immunoblot assays with similar titres, only murine antibodies could neutralize GlpO enzymatic function. The data raise the possibility that Mmm has adapted to evade potential detrimental antibody responses in its definitive host.

The origin of the 'Mycoplasma mycoides cluster' coincides with domestication of ruminants.

The 'Mycoplasma mycoides cluster' comprises the ruminant pathogens Mycoplasma mycoides subsp. mycoides the causative agent of contagious bovine pleuropneumonia (CBPP), Mycoplasma capricolum subsp. capripneumoniae the agent of contagious caprine pleuropneumonia (CCPP), Mycoplasma capricolum subsp. capricolum, Mycoplasma leachii and Mycoplasma mycoides subsp. capri. CBPP and CCPP are major livestock diseases and impact the agricultural sector especially in developing countries through reduced food-supply and international trade restrictions. In addition, these diseases are a threat to disease-free countries. We used a multilocus sequence typing (MLST) approach to gain insights into the demographic history of and phylogenetic relationships among the members of the 'M. mycoides cluster'. We collected partial sequences from seven housekeeping genes representing a total of 3,816 base pairs from 118 strains within this cluster, and five strains isolated from wild Caprinae. Strikingly, the origin of the 'M. mycoides cluster' dates to about 10,000 years ago, suggesting that the establishment and spread of the cluster coincided with livestock domestication. In addition, we show that hybridization and recombination may be important factors in the evolutionary history of the cluster.

Characterization of the gene for an immunodominant 72 kDa lipoprotein of Mycoplasma mycoides subsp. mycoides small colony type.

With the aim of characterizing specific immunogenic proteins of Mycoplasma mycoides subsp. mycoides small colony (SC) type, the aetiological agent of contagious bovine pleuropneumonia, a gene encoding a major immunogenic protein of 72 kDa named P72 was cloned and expressed in Escherichia coli. The expressed protein was of the same apparent molecular mass as that produced by the parent strain. The predicted molecular mass of P72, based on the DNA-deduced amino acid sequence, was 61.118 kDa, significantly lower than the apparent molecular mass of endogenous or recombinant P72 on SDS-PAGE. Analysis of the amino acid sequence revealed a typical prokaryotic signal peptidase II-membrane lipoprotein lipid attachment site and a transmembrane structure domain in the leader sequence at the amino-terminal end of the protein. P72 was shown to be a lipoprotein and its surface location was confirmed by trypsin treatment of whole cells. An unassigned gene encoding a peptide with some similarity to P72 was found on the genome sequence of M. capricolum subsp. capricolum but not on that of Mycoplasma genitalium. The P72 gene was detected in 11/11 M. mycoides subsp. mycoides SC strains. Antiserum against recombinant P72 reacted strongly with 12/12 strains of M. mycoides subsp. mycoides SC, weakly with Mycoplasma bovine group 7 strain PG50, but not with other members of the 'mycoides cluster' or closely related mycoplasmas. Cows experimentally contact-infected with M. mycoides subsp. mycoides SC developed a humoral response against P72 within 35 d. P72 is a specific antigenic membrane lipoprotein of M. mycoides subsp. mycoides SC with potential for use in development of diagnostic reagents. It seems to belong to a family of lipoproteins of the "mycoides cluster'.

Identification and characterization of IS1296 in Mycoplasma mycoides subsp. mycoides SC and presence in related mycoplasmas

IS1296, a new insertion sequence belonging to the IS3 family of insertion elements has been identified in Mycoplasma mycoides subsp. mycoides (Mmm) biotype small colony (SC), the agent of contagious bovine pleuropneumonia (CBPP). IS1296 is 1485-bp long and has 30-bp inverted repeats. It contains two open reading frames, ORFA and ORFB, which show significant similarities to the ORFs which encode the transposase function of IS elements of the IS3 family, in particular IS150 of Escherichia coli. IS1296 is present in 19 copies in Mmm SC-type strain PG1 and in 18 copies in a recently isolated field strain L2. It seems to transpose at low frequency in Mmm SC. IS1296 is also present in 5 copies in Mmm biotype large colony (LC)-type strain Y-goat, and in two copies in Mycoplasma sp. 'bovine group 7' reference strain PG50. It is, however, not present in other species of the 'mycoides cluster' or other closely related Mycoplasma sp. of ruminants.

High quality draft genomes of the Mycoplasma mycoides subsp. mycoides challenge strains Afadé and B237.

Members of the Mycoplasma mycoides cluster' represent important livestock pathogens worldwide. Mycoplasma mycoides subsp. mycoides is the etiologic agent of contagious bovine pleuropneumonia (CBPP), which is still endemic in many parts of Africa. We report the genome sequences and annotation of two frequently used challenge strains of Mycoplasma mycoides subsp. mycoides, Afadé and B237. The information provided will enable downstream 'omics' applications such as proteomics, transcriptomics and reverse vaccinology approaches. Despite the absence of Mycoplasma pneumoniae like cyto-adhesion encoding genes, the two strains showed the presence of protrusions. This phenotype is likely encoded by another set of genes.

Vaccination of cattle with the N terminus of LppQ of Mycoplasma mycoides subsp. mycoides results in type III immune complex disease upon experimental infection.

Contagious bovine pleuropneumonia (CBPP) is a serious respiratory disease of cattle caused by Mycoplasma mycoides subsp. mycoides. Current vaccines against CBPP induce short-lived immunity and can cause severe postvaccine reactions. Previous studies have identified the N terminus of the transmembrane lipoprotein Q (LppQ-N') of M. mycoides subsp. mycoides as the major antigen and a possible virulence factor. We therefore immunized cattle with purified recombinant LppQ-N' formulated in Freund's adjuvant and challenged them with M. mycoides subsp. mycoides. Vaccinated animals showed a strong seroconversion to LppQ, but they exhibited significantly enhanced postchallenge glomerulonephritis compared to the placebo group (P = 0.021). Glomerulonephritis was characterized by features that suggested the development of antigen-antibody immune complexes. Clinical signs and gross pathological scores did not significantly differ between vaccinated and placebo groups. These findings reveal for the first time the pathogenesis of enhanced disease as a result of antibodies against LppQ during challenge and also argue against inclusion of LppQ-N' in a future subunit vaccine for CBPP.

Complete Genome Sequences of Virulent Mycoplasma capricolum subsp. capripneumoniae Strains F38 and ILRI181

Contagious caprine pleuropneumonia (CCPP) caused by Mycoplasma capricolum subsp. capripneumoniae is a severe epidemic affecting mainly domestic Caprinae species but also affects wild Caprinae species. M. capricolum subsp. capripneumoniae belongs to the "Mycoplasma mycoides cluster." The disease features prominently in East Africa, in particular Kenya, Tanzania, and Ethiopia. CCPP also endangers wildlife and thus affects not only basic nutritional resources of large populations but also expensively built-up game resorts in affected countries. Here, we report the complete sequences of two M. capricolum subsp. capripneumoniae strains: the type strain F38 and strain ILRI181 isolated druing a recent outbreak in Kenya. Both genomes have a G+C content of 24% with sizes of 1,016,760 bp and 1,017,183 bp for strains F38 and ILRI181, respectively.