Multi-locus sequence analysis reveals great genetic diversity among Mycoplasma capricolum subsp. capripneumoniae strains in Asia.

Multi-Locus Sequence Analysis (MLSA) of Mycoplasma capricolum subsp. capripneumoniae (Mccp) strains from Asia revealed unforeseen diversity and a central position for genotyping groups representing strains from Central/East Asia, suggesting a possible origin of contagious caprine pleuropneumonia in this continent. A better assessment of the emergence, diversity and distribution of Mccp in Asia and Africa calls for renewed efforts to dramatically enlarge the sample of strains. Availability and affordability in the field, added to superior typeability (directly from poor samples) and high stability, discriminatory power and concordance with epidemiological and phylogenetic analyses, make MLSA an excellent tool for such investigations.

A library preparation optimized for metagenomics of RNA viruses.

Our understanding of the viral communities associated to animals has not yet reached the level attained on the bacteriome. This situation is due to, among others, technical challenges in adapting metagenomics using high-throughput sequencing to the study of RNA viromes in animals. Although important developments have been achieved in most steps of viral metagenomics, there is yet a key step that has received little attention: the library preparation. This situation differs from bacteriome studies in which developments in library preparation have largely contributed to the democratisation of metagenomics. Here, we present a library preparation optimized for metagenomics of RNA viruses from insect vectors of viral diseases. The library design allows a simple PCR-based preparation, such as those routinely used in bacterial metabarcoding, that is adapted to shotgun sequencing as required in viral metagenomics. We first optimized our library preparation using mock viral communities and then validated a full metagenomic approach incorporating our preparation in two pilot studies with field-caught insect vectors; one including a comparison with a published metagenomic protocol. Our approach provided a fold increase in virus-like sequences compared to other studies, and nearly-full genomes from new virus species. Moreover, our results suggested conserved trends in virome composition within a population of a mosquito species. Finally, the sensitivity of our approach was compared to a commercial diagnostic PCR for the detection of an arbovirus in field-caught insect vectors. Our approach could facilitate studies on viral communities from animals and the democratization of metagenomics in community ecology of viruses.

Sepsis modeling in mice: ligation length is a major severity factor in cecal ligation and puncture.

BACKGROUND: The cecal ligation and puncture (CLP) model, a gold standard in sepsis research, is associated with an important variability in mortality. While the number of punctures and needle size is well described in CLP animal studies, the length of cecal ligation is often not. The relationship between cecal ligation and survival in mice is briefly reported in the literature; therefore, we devised an investigation in mice of the consequences of three standardized cecal ligation lengths on mortality and the severity of the ensued sepsis. METHODS: Male C57BL/6J mice underwent standardized CLP. The cecum was ligated at 5, 20, or 100 % of its total length and further perforated by a single 20-G puncture. Mortality was analyzed. We assessed blood lactate, serum creatinine levels, and serum cytokines (TNF-α, IL-1ß, IL-6, and IL-10) after procedure in a control group and in ligated mice. RESULTS: Mortality was directly related to ligation length: median survival was 24 h for the "100 %" group and 44 h for the "20 %" group. Blood lactate increased proportionally with the ligation length. At 6 h post-procedure, pro-inflammatory cytokines significantly increased in the ligated group with significantly higher serum levels of IL-6 in the 100 % group compared to the other ligated groups. The 20 % group exhibited the characteristics of septic shock with hypotension below 65 mmHg, pro-inflammatory balance, organ dysfunction, and hyperlactatemia. CONCLUSIONS: Cecal ligation length appears to be a major limiting factor in the mouse CLP model. Thus, this experimental model should be performed with high consistency in future protocol designs.

A large-scale genomic approach affords unprecedented resolution for the molecular epidemiology and evolutionary history of contagious caprine pleuropneumonia.

Contagious caprine pleuropneumonia (CCPP), caused by Mycoplasma capricolum subsp. capripneumoniae (Mccp), is a devastating disease of domestic goats and of some wild ungulate species. The disease is currently spreading in Africa and Asia and poses a serious threat to disease-free areas. A comprehensive view of the evolutionary history and dynamics of Mccp is essential to understand the epidemiology of CCPP. Yet, analysing the diversity of genetically monomorphic pathogens, such as Mccp, is complicated due to their low variability. In this study, the molecular epidemiology and evolution of CCPP was investigated using a large-scale genomic approach based on next-generation sequencing technologies, applied to a sample of strains representing the global distribution of this disease. A highly discriminatory multigene typing system was developed, allowing the differentiation of 24 haplotypes among 25 Mccp strains distributed in six genotyping groups, which showed some correlation with geographic origin. A Bayesian approach was used to infer the first robust phylogeny of the species and to date the principal events of its evolutionary history. The emergence of Mccp was estimated only at about 270 years ago, which explains the low genetic diversity of this species despite its high mutation rate, evaluated at 1.3 × 10(-6) substitutions per site per year. Finally, plausible scenarios were proposed to elucidate the evolution and dynamics of CCPP in Asia and Africa, though limited by the paucity of Mccp strains, particularly in Asia. This study shows how combining large-scale genomic data with spatial and temporal data makes it possible to obtain a comprehensive view of the epidemiology of CCPP, a precondition for the development of improved disease surveillance and control measures.

SNaPshot and CE-SSCP: Two Simple and Cost-Effective Methods to Reveal Genetic Variability Within a Virus Species.

The multiplex SNaPshot and the capillary electrophoresis-single-strand conformation polymorphism (CE-SSCP) procedures are here used for rapid and high-throughput description of the molecular variability of viral populations. Both approaches are based on (1) standard amplification of genomic sequence(s), (2) labeled primers or labeled single-stranded DNA, and (3) migration of fluorescent-labeled molecules in capillary electrophoresis system. The SNaPshot technology was used to describe the diversity of 20 targeted single nucleotide polymorphisms (SNPs) selected from alignment of viral genomic sequences retrieved from public database. The CE-SSCP procedure was applied to identify the polymorphisms of two small (<500 bases in length) genomic regions of viral genomes. The different steps of SNaPshot and CE-SSCP setup procedures are presented using Potato virus Y (PVY, Potyvirus) and Plum pox virus (PPV, Potyvirus) RNA viruses as molecular targets, respectively.

Complete Genome Sequence of Mycoplasma capricolum subsp. capripneumoniae Strain 9231-Abomsa.

Mycoplasma capricolum subsp. capripneumoniae is the etiological agent of contagious caprine pleuropneumonia. We report here the complete and annotated genome sequence of M. capricolum subsp. capripneumoniae strain 9231-Abomsa.

Draft Genome Sequences of Mycoplasma auris and Mycoplasma yeatsii, Two Species of the Ear Canal of Caprinae.

We report here the draft genome sequences of Mycoplasma auris and Mycoplasma yeatsii, two species commonly isolated from the external ear canal of Caprinae.

Draft Genome Sequences of Mycoplasma alkalescens, Mycoplasma arginini, and Mycoplasma bovigenitalium, Three Species with Equivocal Pathogenic Status for Cattle.

We report here the draft genome sequences of Mycoplasma alkalescens, Mycoplasma arginini, and Mycoplasma bovigenitalium. These three species are regularly isolated from bovine clinical specimens, although their role in disease is unclear.

Complete Genome Sequence of Mycoplasma putrefaciens Strain 9231, One of the Agents of Contagious Agalactia in Goats.

Mycoplasma putrefaciens is one of the etiologic agents of contagious agalactia in goats. We report herein the complete genome sequence of Mycoplasma putrefaciens strain 9231.

Finishing bacterial genome assemblies with Mix.

MOTIVATION: Among challenges that hamper reaping the benefits of genome assembly are both unfinished assemblies and the ensuing experimental costs. First, numerous software solutions for genome de novo assembly are available, each having its advantages and drawbacks, without clear guidelines as to how to choose among them. Second, these solutions produce draft assemblies that often require a resource intensive finishing phase. METHODS: In this paper we address these two aspects by developing Mix , a tool that mixes two or more draft assemblies, without relying on a reference genome and having the goal to reduce contig fragmentation and thus speed-up genome finishing. The proposed algorithm builds an extension graph where vertices represent extremities of contigs and edges represent existing alignments between these extremities. These alignment edges are used for contig extension. The resulting output assembly corresponds to a set of paths in the extension graph that maximizes the cumulative contig length. RESULTS: We evaluate the performance of Mix on bacterial NGS data from the GAGE-B study and apply it to newly sequenced Mycoplasma genomes. Resulting final assemblies demonstrate a significant improvement in the overall assembly quality. In particular, Mix is consistent by providing better overall quality results even when the choice is guided solely by standard assembly statistics, as is the case for de novo projects. AVAILABILITY: Mix is implemented in Python and is available at https://github.com/cbib/MIX, novel data for our Mycoplasma study is available at http://services.cbib.u-bordeaux2.fr/mix/.

Evolutionary history of contagious bovine pleuropneumonia using next generation sequencing of Mycoplasma mycoides Subsp. mycoides "Small Colony".

Mycoplasma mycoides subsp. mycoides "Small Colony" (MmmSC) is responsible for contagious bovine pleuropneumonia (CBPP) in bovidae, a notifiable disease to the World Organization for Animal Health (OIE). Although its origin is not documented, the disease was known in Europe in 1773. It reached nearly world-wide distribution in the 19(th) century through the cattle trade and was eradicated from most continents by stamping-out policies. During the 20(th) century it persisted in Africa, and it reappeared sporadically in Southern Europe. Yet, classical epidemiology studies failed to explain the re-occurrence of the disease in Europe in the 1990s. The objectives of this study were to obtain a precise phylogeny of this pathogen, reconstruct its evolutionary history, estimate the date of its emergence, and determine the origin of the most recent European outbreaks. A large-scale genomic approach based on next-generation sequencing technologies was applied to construct a robust phylogeny of this extremely monomorphic pathogen by using 20 representative strains of various geographical origins. Sixty two polymorphic genes of the MmmSC core genome were selected, representing 83601 bp in total and resulting in 139 SNPs within the 20 strains. A robust phylogeny was obtained that identified a lineage specific to European strains; African strains were scattered in various branches. Bayesian analysis allowed dating the most recent common ancestor for MmmSC around 1700. The strains circulating in Sub-Saharan Africa today, however, were shown to descend from a strain that existed around 1810. MmmSC emerged recently, about 300 years ago, and was most probably exported from Europe to other continents, including Africa, during the 19(th) century. Its diversity is now greater in Africa, where CBPP is enzootic, than in Europe, where outbreaks occurred sporadically until 1999 and where CBPP may now be considered eradicated unless MmmSC remains undetected.

Genetic architecture of sexual selection: QTL mapping of male song and female receiver traits in an acoustic moth.

Models of indirect (genetic) benefits sexual selection predict linkage disequilibria between genes that influence male traits and female preferences, owing to non-random mate choice or physical linkage. Such linkage disequilibria can accelerate the evolution of traits and preferences to exaggerated levels. Both theory and recent empirical findings on species recognition suggest that such linkage disequilibria may result from physical linkage or pleiotropy, but very little work has addressed this possibility within the context of sexual selection. We studied the genetic architecture of sexually selected traits by analyzing signals and preferences in an acoustic moth, Achroia grisella, in which males attract females with a train of ultrasound pulses and females prefer loud songs and a fast pulse rhythm. Both male signal characters and female preferences are repeatable and heritable traits. Moreover, female choice is based largely on male song, while males do not appear to provide direct benefits at mating. Thus, some genetic correlation between song and preference traits is expected. We employed a standard crossing design between inbred lines and used AFLP markers to build a linkage map for this species and locate quantitative trait loci (QTL) that influence male song and female preference. Our analyses mostly revealed QTLs of moderate strength that influence various male signal and female receiver traits, but one QTL was found that exerts a major influence on the pulse-pair rate of male song, a critical trait in female attraction. However, we found no evidence of specific co-localization of QTLs influencing male signal and female receiver traits on the same linkage groups. This finding suggests that the sexual selection process would proceed at a modest rate in A. grisella and that evolution toward exaggerated character states may be tempered. We suggest that this equilibrium state may be more the norm than the exception among animal species.

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.

Type 2 diabetes mellitus in mice aggravates the renal impact of hemorrhagic shock.

The objectives of this study were to determine whether type 2 diabetic mice would exhibit a more severe renal impact of hemorrhagic shock (HS) based on a recently described model of acute kidney injury and to determine the impact of HS on renal responses to hypoxia. We induced HS or sham procedure in type 2 diabetic and obese db/db mice. Creatininemia, glomerular filtration rate, urine output, histologic injury score, and kidney inductible molecule 1 mRNA were used to investigate the renal impact of HS. Tissular hypoxia and its impact were quantified using pimonidazole immunostaining and mRNA of hypoxic inducible factor, vascular endothelial growth factor receptors 1 and 2, Tie-2, endothelial nitric oxide synthase, and inducible nitric oxide synthase. Diabetic mice exhibiting mild diabetic nephropathy express hypoxic signals at baseline. The renal impact of HS was more severe in diabetic mice, with a worsening of tissular hypoxia and an altered response to hypoxia. Furthermore, endothelial nitric oxide synthase was highly overexpressed in diabetic shocked mice when compared with nondiabetic shocked mice. Renal impact of HS in type 2 diabetic mice is more intense than in nondiabetic ones. Preexisting hypoxia during diabetes could result in a renal preconditioning that modifies endothelial and tissular responses to acute kidney injury.

Fatal outbreak of Mycoplasma capricolum pneumonia in endangered markhors.

A pneumonia outbreak reduced the numbers of a wild population of endangered markhors (Capra falconeri) in Tajikistan in 2010. The infection was diagnosed by histologic examination and bacteriologic testing. Mycoplasma capricolum subsp. capricolum was the sole infectious agent detected. Cross-species transmission from domestic goats may have occurred.

High molecular variability of sugarcane bacilliform viruses in Guadeloupe implying the existence of at least three new species.

Thirty-five unique partial sugarcane bacilliform virus (SCBV) sequences extending over 529 bp were identified in sugarcane samples from Guadeloupe diagnosed by Immunocapture-PCR (IC-PCR) using specific badnavirus primers. Phylogenetic analysis of these sequences along with the two known genome sequences of Sugarcane bacilliform Mor virus (SCBMV) and Sugarcane bacilliform IM virus (SCBIMV) revealed high molecular variability in the SCBV genome. Seven phylogenetic groups, named A to G, were characterized: virus isolates from groups A-B, C and D are proposed to be members of three additional SCBV species. The two (7446 and 7444 bp) and one (7317 bp) complete sequences of SCBV isolates from groups A and D, respectively, likely represented the genome of two new species. Phylogenetic analysis of the complete genome and RT/RNase H sequences confirmed the polyphyletic structure of SCBV isolates and the absence of a clear separation between SCBV and Banana streak virus (BSV) isolates within badnavirus group 1. These results showed that reconsideration of taxonomy and classification of SCBV and BSV are necessary.

Multi-locus sequence analysis of Mycoplasma capricolum subsp. capripneumoniae for the molecular epidemiology of contagious caprine pleuropneumonia.

Mycoplasma capricolum subsp. capripneumoniae (Mccp) is the causative agent of contagious caprine pleuropneumonia (CCPP), a devastating disease of domestic goats. The exact distribution of CCPP is not known but it is present in Africa and the Middle East and represents a significant threat to many disease-free areas including Europe. Furthermore, CCPP has been recently identified in Tajikistan and China. A typing method with an improved resolution based on Multi-Locus Sequence Analysis (MLSA) has been developed to trace new epidemics and to elucidate whether the recently identified cases in continental Asia were due to recent importation of Mccp. The H2 locus, a polymorphic region already in use as a molecular marker for Mccp evolution, was complemented with seven new loci selected according to the analysis of polymorphisms observed among the genome sequences of three Mccp strains. A total of 25 strains, including the two new strains from Asia, were analysed by MLSA resulting in the discrimination of 15 sequence types based on 53 polymorphic positions. A distance tree inferred from the concatenated sequences of the eight selected loci revealed two evolutionary lineages comprising five groups, which showed good correlation with geographic origins. The presence of a distinct Asian cluster strongly indicates that CCPP was not recently imported to continental Asia. It is more likely that the disease has been endemic in the area for a long time, as supported by historical clinical descriptions. In conclusion, this MLSA strategy constitutes a highly discriminative tool for the molecular epidemiology of CCPP.

Mycoplasma mycoides, from "mycoides Small Colony" to "capri". A microevolutionary perspective.

BACKGROUND: The Mycoplasma mycoides cluster consists of five species or subspecies that are ruminant pathogens. One subspecies, Mycoplasma mycoides subspecies mycoides Small Colony (MmmSC), is the causative agent of contagious bovine pleuropneumonia. Its very close relative, Mycoplasma mycoides subsp. capri (Mmc), is a more ubiquitous pathogen in small ruminants causing mastitis, arthritis, keratitis, pneumonia and septicaemia and is also found as saprophyte in the ear canal. To understand the genetics underlying these phenotypic differences, we compared the MmmSC PG1 type strain genome, which was already available, with the genome of an Mmc field strain (95010) that was sequenced in this study. We also compared the 95010 genome with the recently published genome of another Mmc strain (GM12) to evaluate Mmc strain diversity. RESULTS: The MmmSC PG1 genome is 1,212 kbp and that of Mmc 95010 is ca. 58 kbp shorter. Most of the sequences present in PG1 but not 95010 are highly repeated Insertion Sequences (three types of IS) and large duplicated DNA fragments. The 95010 genome contains five types of IS, present in fewer copies than in PG1, and two copies of an integrative conjugative element. These mobile genetic elements have played a key role in genome plasticity, leading to inversions of large DNA fragments. Comparison of the two genomes suggested a marked decay of the PG1 genome that seems to be correlated with a greater number of IS. The repertoire of gene families encoding surface proteins is smaller in PG1. Several genes involved in polysaccharide metabolism and protein degradation are also absent from, or degraded in, PG1. CONCLUSIONS: The genome of MmmSC PG1 is larger than that of Mmc 95010, its very close relative, but has less coding capacity. This is the result of large genetic rearrangements due to mobile elements that have also led to marked gene decay. This is consistent with a non-adaptative genomic complexity theory, allowing duplications or pseudogenes to be maintained in the absence of adaptive selection that would lead to purifying selection and genome streamlining over longer evolutionary times. These findings also suggest that MmmSC only recently adapted to its bovine host.

Development of 10 microsatellite loci in the pulmonate snail Biomphalaria kuhniana (Mollusca, Gastropoda).

We characterized 10 variable microsatellite loci in the freshwater snail Biomphalaria kuhniana, as well as conditions for multiplexing and co-loading sets of loci. Two to five alleles were detected per locus over the two studied populations in Venezuela. High inbreeding coefficients suggest high selfing rates. Cross-species amplification provided some variability at eight and three loci in the other species belonging to the Biomphalaria straminea complex (B. straminea and B. intermedia), but was unsuccessful in more divergent species.

Development of eight microsatellite markers in the parsley frog (Pelodytes punctatus).

We characterized eight microsatellite loci to study spatial and temporal population structure of Pelodytes punctatus, a European anuran that has a peculiar breeding pattern among amphibians. The eight loci proved to be highly polymorphic with the number of alleles per locus ranging from two to 15 within two populations. Cross-amplification indicates that those markers may be also useful for closely related species from the same family.