Intraspecies Gene Variation within Putative Epitopes of Immunodominant Protein P48 of Mycoplasma agalactiae.

P48 protein of Mycoplasma agalactiae is used to diagnose infection and was identified as potential vaccine candidate. According to the genetic nature of mycoplasma and variable sensitivity in P48-based serological diagnosis tests, intra species variation of P48 nucleotide sequence investigated in 13 field isolates of difference province of Iran along with three vaccine strains. Samples were collected from sheep and goat and were cultured in modified PPLO broth. Two pair of primer employed to confirm genus and species of isolates and a pair of primer has developed to amplify the P48 gene. The sequencing results of PCR products were aligned and analyzed besides published sequences in GenBank. T-Cell and B-Cell epitopes and antigenicity of sequence were computationally predicted. The results have shown P48 nucleotide sequences are 99.9% identical in field isolates and vaccine strain of Iran, but analysis of GenBank published sequences have shown divergence up to 5.3% at the nucleotide level and up to 4.9% divergence in protein level of P48 sequences of Iran isolates and other available sequences in GenBank. Single nucleotide polymorphism exists in 89 positions and variable amino acid was observed at 25 residues. Phylogenetic analyses have shown that Mycoplasma agalactiae isolates fall into three main groups based on P48 nucleotide sequences. Immunoinformatics analysis of all available P48 nucleotide sequences have revealed that gene variation lead to differences in immunological properties, but the gene in Iranian isolates are conservative and stable. The sequence variation in epitopes can be underlying source of antigen heterogeneity as a result, affect serological tests accuracy. Due to the high level of divergence in worldwide isolates and high degree of similarity in P48 protein of Iranian isolates, designing recombinant P48 protein based on local pattern can increase the sensitivity and consistency of serological test.

Molecular characterization of Mycoplasma agalactiae reveals the presence of an endemic clone in Spain.

Mycoplasma agalactiae isolates from Spain were genetically characterized to investigate their genomic diversity and to better understand their relationship to isolates from other countries. Molecular typing revealed a high genomic homogeneity in Spanish M. agalactiae isolates, which clearly shows the circulation of one endemic clonal population.

Phylogeny and molecular typing of Mycoplasma agalactiae and Mycoplasma bovis by multilocus sequencing.

Mycoplasma agalactiae and Mycoplasma bovis are important pathogens producing similar pathologies in small ruminants and cattle, respectively. They share many phenotypic and genotypic traits and comparison of their 16S rDNA sequences lacks sufficient resolution for phylogenetic analysis. The aim of this study was to develop a multilocus sequence typing (MLST) scheme to analyse the phylogenetic relationships between M. agalactiae and M. bovis and to assess its use for unequivocal strain characterisation and molecular typing. An MLST based on fusA, gyrB, lepA and rpoB was applied to a sample of strains from both species, some of which could not be classified by serology or PCR. A robust phylogenetic tree was inferred, where the two species were clearly resolved. The use of this tool for the molecular typing of M. agalactiae strains was further evaluated on 19 presumably unrelated isolates, resulting in the discrimination of 14 sequence types (ST). The discriminatory power was increased (17 ST) by including an alternative target located in a more variable region. The diversity of M. agalactiae in Turkey (9 strains) and Israel (15 strains) was also assessed. Five closely related ST were evidenced in Turkey and 6 in Israel, with one ST common to both countries. Each country showed a predominant type that persisted over years. The MLST scheme developed here constitutes a universal tool for unequivocal strain characterisation and global, long-term screening of dissemination of M. agalactiae and M. bovis, whereas addition of more variable, non-housekeeping gene targets allows precise epidemiological investigations.

Molecular typing of Mycoplasma agalactiae: tracing European-wide genetic diversity and an endemic clonal population.

Mycoplasma agalactiae causes chronic infections in small ruminants and remains endemic in many regions of the world, despite intensive and costly eradication programs. In this study, the innate genomic plasticity of M. agalactiae was exploited to design and assess a combination of molecular epidemiological tools to trace the pathogen in different geographic locations and to understand its emergence or re-emergence after eradication campaigns. For this purpose, two collections of M. agalactiae isolates, representing European outbreaks or localized endemic disease in a single region of France, were subjected to RFLP (Restriction Fragment Length Polymorphism) analyses using two sets of DNA probes (distributed across the genome and specific for the vpma gene locus), and a previously described VNTR (Variable Number Tandem Repeats) analysis. A combination of four genome-specific DNA probes and two VNTRs gave the highest discriminative power. Molecular typing revealed that, while isolates from diverse geographical origins fell into clearly different groups, the endemic disease repeatedly observed in the Western Pyrenees region over the past 30 years has been caused by a unique subtype of M. agalactiae. This indicates that the re-emergence of the pathogen after seemingly successful eradication programs is not due to the importation of exotic strains, but to the persistence of local reservoirs of infection.

Emergence of atypical Mycoplasma agalactiae strains harboring a new prophage and associated with an alpine wild ungulate mortality episode.

The bacterium Mycoplasma agalactiae is responsible for contagious agalactia (CA) in small domestic ruminants, a syndrome listed by the World Organization for Animal Health and responsible for severe damage to the dairy industry. Recently, we frequently isolated this pathogen from lung lesions of ibexes during a mortality episode in the French Alps. This situation was unusual in terms of host specificity and tissue tropism, raising the question of M. agalactiae emergence in wildlife. To address this issue, the ibex isolates were characterized using a combination of approaches that included antigenic profiles, molecular typing, optical mapping, and whole-genome sequencing. Genome analyses showed the presence of a new, large prophage containing 35 coding sequences (CDS) that was detected in most but not all ibex strains and has a homolog in Mycoplasma conjunctivae, a species causing keratoconjunctivitis in wild ungulates. This and the presence in all strains of large integrated conjugative elements suggested highly dynamic genomes. Nevertheless, M. agalactiae strains circulating in the ibex population were shown to be highly related, most likely originating from a single parental clone that has also spread to another wild ungulate species of the same geographical area, the chamois. These strains clearly differ from strains described in Europe so far, including those found nearby, before CA eradication a few years ago. While M. agalactiae pathogenicity in ibexes remains unclear, our data showed the emergence of atypical strains in Alpine wild ungulates, raising the question of a role for the wild fauna as a potential reservoir of pathogenic mycoplasmas.

Multilocus sequence typing of Mycoplasma agalactiae.

Mycoplasma agalactiae is the main cause of contagious agalactia, a serious disease of sheep and goats, which has major clinical and economic impacts. We have developed a multilocus sequence typing (MLST) scheme using the sequenced genomes of the M. agalactiae strains PG2 and 5632. An MLST scheme based on the genes gltX, metS, gyrB, tufA and dnaA was designed and in total 3468 bp of sequence were analysed for each strain. MLST offers a highly discriminatory typing method for M. agalactiae and was capable of subdividing 53 strains into 17 distinct sequence types, largely according to geographical origin. MLST detected unexpected diversity in recent isolates from Spain, identifying two novel outliers, and enabled typing of novel Mongolian isolates for the first time. Genetic diversity in the sequenced regions was largely due to mutation, with recombination playing a much smaller role. A web-accessible database has been set up for this MLST scheme for M. agalactiae: http://pubmlst.org/magalactiae/. MLST offers a robust, objective molecular epidemiological tool for M. agalactiae that that enables interlaboratory comparison of data.

VNTR analysis reveals unexpected genetic diversity within Mycoplasma agalactiae, the main causative agent of contagious agalactia.

BACKGROUND: Mycoplasma agalactiae is the main cause of contagious agalactia, a serious disease of sheep and goats, which has major clinical and economic impacts. Previous studies of M. agalactiae have shown it to be unusually homogeneous and there are currently no available epidemiological techniques which enable a high degree of strain differentiation. RESULTS: We have developed variable number tandem repeat (VNTR) analysis using the sequenced genome of the M. agalactiae type strain PG2. The PG2 genome was found to be replete with tandem repeat sequences and 4 were chosen for further analysis. VNTR 5 was located within the hypothetical protein MAG6170 a predicted lipoprotein. VNTR 14 was intergenic between the hypothetical protein MAG3350 and the hypothetical protein MAG3340. VNTR 17 was intergenic between the hypothetical protein MAG4060 and the hypothetical protein MAG4070 and VNTR 19 spanned the 5' end of the pseudogene for a lipoprotein MAG4310 and the 3' end of the hypothetical lipoprotein MAG4320. We have investigated the genetic diversity of 88 M. agalactiae isolates of wide geographic origin using VNTR analysis and compared it with pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) analysis. Simpson's index of diversity was calculated to be 0.324 for PFGE and 0.574 for VNTR analysis. VNTR analysis revealed unexpected diversity within M. agalactiae with 9 different VNTR types discovered. Some correlation was found between geographical origin and the VNTR type of the isolates. CONCLUSION: VNTR analysis represents a useful, rapid first-line test for use in molecular epidemiological analysis of M. agalactiae for outbreak tracing and control.

Characterisation of protein and antigen variability among Mycoplasma mycoides subsp. mycoides (LC) and Mycoplasma agalactiae field strains by SDS-PAGE and immunoblotting.

Mycoplasma mycoides subsp. mycoides (LC) (Mmm LC) and Mycoplasma agalactiae are the most important mycoplasma species involved in the contagious agalactia syndrome. A total of 25 field strains from Spain and the two type strains were analysed by SDS-PAGE and immunoblotting. Two polyclonal antisera (PAbs) raised against a pool of strains of each mycoplasma species were used. The results revealed a high degree of protein variability among the field strains. The type strain of Mmm LC appeared to be representative of the field strains of this species, whereas this was not the case with the M. agalactiae type strain. Whereas M. agalactiae is known to possess a gene family regulating surface antigen diversity, there is a need to study the mechanisms used byMmm LC to generate antigenic variability in more detail.

Suppression subtractive hybridization as a basis to assess Mycoplasma agalactiae and Mycoplasma bovis genomic diversity and species-specific sequences.

The phylogenically related Mycoplasma agalactiae and Mycoplasma bovis species are two ruminant pathogens difficult to differentiate and for which a limited amount of sequence data are available. To assess the degree of genomic diversity existing between and within these mycoplasma species, sets of DNA fragments specific for M. bovis type-strain PG45 or for M. agalactiae type-strain PG2 were isolated by suppression subtractive hybridization and used as probes on a panel of M. agalactiae and M. bovis field isolates. Results indicated that approximately 70 % of the DNA fragments specific to one or the other type strain are represented in all field isolates of the corresponding species. Only one M. bovis isolate, which was first classified as M. agalactiae, reacted with 15 % of the PG2-specific probes, while several M. agalactiae isolates reacted with 15 % of the PG45-specific probes. Sequence analyses indicated that most of the genomic diversity observed within one species is related to ORFs with (i) no homologies to proteins recorded in the databases or (ii) homologies to proteins encoded by restriction modification systems. Reminiscent of gene transfer as a means for genomic diversity, a PG45-specific DNA fragment with significant homologies to a central protein of an integrative conjugative element of Mycoplasma fermentans (ICEF) was found in most M. bovis field isolates and in a few M. agalactiae isolates. Finally, sequences encoding part of DNA polymerase III were found in both sets of M. agalactiae- and M. bovis-specific DNA fragments and were used to design a species-specific PCR assay for the identification and differentiation of M. agalactiae and M. bovis.

Evaluation of PCR systems for the identification and differentiation of Mycoplasma agalactiae and Mycoplasma bovis: a collaborative trial.

Diagnostic differentiation between the ruminant pathogens Mycoplasma agalactiae and Mycoplasma bovis is known to be problematic when only conventional serological and biochemical tests are used. The main reason for this is that both agents share a considerable number of related proteins and common epitopes. DNA-based detection methods offer advantages in terms of specificity and sensitivity. However, there is an urgent need to compare currently used PCR assays because they target different genomic regions and, therefore, may perform differently. In the present work, five laboratories, which use PCR routinely, evaluated the specificity of four different PCR systems for M. agalactiae and three systems for M. bovis on a total of 41 strains of the two Mycoplasma species including six previously unidentified strains. As the vast majority of PCR examinations (97.1% of all tests) correctly identified the strains the specificity of all seven detection systems appears to be high. In four cases, incorrect identification by conventional diagnostic methods was rectified by PCR. Isolates from non-typical hosts, i.e. three M. bovis strains from small ruminants and two M. agalactiae strains from cattle, were characterised by sequencing the 16S and part of the 23S ribosomal RNA genes.