Response to somatic cell count-based selection for mastitis resistance in a divergent selection experiment in sheep

A divergent selection experiment in sheep was implemented to study the consequences of log-transformed somatic cell score (SCS)-based selection on resistance to natural intramammary infections. Using dams and progeny-tested rams selected for extreme breeding values for SCS, we created 2 groups of ewes with a strong divergence in SCS of approximately 3 genetic standard deviations. A survey of 84 first-lactation ewes of both the High and Low SCS lines indicated favorable responses to SCS-based selection on resistance to both clinical and subclinical mastitis. All clinical cases (n = 5) occurred in the High SCS line. Additionally, the frequency of chronic clinical mastitis,as detected by the presence of parenchymal abscesses, was much greater in the High SCS line (n = 21) than in the Low SCS line (n = 1). According to monthly milk bacterio-logical examinations of udder halves, the prevalence of infection was significantly greater (odds ratio = 3.1) in the High SCS line than in the Low SCS line, with predicted probabilities of 37 and 16%, respectively. The most frequently isolated bacteria responsible for mastitis were staphylococci: Staphylococcus auricularis(42.6% of positive samples), Staphylococcus simulans, Staphylococcus haemoliticus, Staphylococcus xylosus, Staphylococcus chromogenes, Staphylococcus lentus, Staphylococcus warneri, and Staphylococcus aureus. The incidence of positive bacteriology was greater in the High SCS line (39%) than in the Low SCS line (12%)at lambing, indicating that High SCS line ewes were especially susceptible to postpartum subclinical mastitis. Negativation of bacteriological results from one sampling time point to the next was markedly different between lines after weaning (e.g., 41 and 84% in the High and Low SCS lines, respectively). This result was consistent with differences in the duration of infection, which was much greater in the High SCS line compared with the Low SCS line. Finally, ewes from the High SCS line consistently had greater SCS in positive milk samples than did ewes from the Low SCS line (+2.04 SCS, on average), with an especially large difference between lines during the suckling period (+3.42 SCS). Altogether, the preliminary results suggest that the better resistance of Low SCS line ewes, compared with High SCS line ewes, was principally characterized by a better ability to limit infections during the peripartum period, to eliminate infections during lactation, and quantitatively to limit the inflammation process and its clinical consequences

Genome-Scale Analysis of Mycoplasma agalactiae Loci Involved in Interaction with Host Cells

Mycoplasma agalactiae is an important pathogen of small ruminants, in which it causes contagious agalactia. It belongs to a large group of “minimal bacteria” with a small genome and reduced metabolic capacities that are dependent on their host for nutrients. Mycoplasma survival thus relies on intimate contact with host cells, but little is known about the factors involved in these interactions or in the more general infectious process. To address this issue, an assay based on goat epithelial and fibroblastic cells was used to screen a M. agalactiae knockout mutant library. Mutants with reduced growth capacities in cell culture were selected and 62 genomic loci were identified as contributing to this phenotype. As expected for minimal bacteria, “transport and metabolism” was the functional category most commonly implicated in this phenotype, but 50% of the selected mutants were disrupted in coding sequences (CDSs) with unknown functions, with surface lipoproteins being most commonly represented in this category. Since mycoplasmas lack a cell wall, lipoproteins are likely to be important in interactions with the host. A few intergenic regions were also identified that may act as regulatory sequences under co-culture conditions. Interestingly, some mutants mapped to gene clusters that are highly conserved across mycoplasma species but located in different positions. One of these clusters was found in a transcriptionally active region of the M. agalactiae chromosome, downstream of a cryptic promoter. A possible scenario for the evolution of these loci is discussed. Finally, several CDSs identified here are conserved in other important pathogenic mycoplasmas, and some were involved in horizontal gene transfer with phylogenetically distant species. These results provide a basis for further deciphering functions mediating mycoplasma-host interactions

The Integrative Conjugative Element (ICE) of Mycoplasma agalactiae: Key Elements Involved in Horizontal Dissemination and Influence of Coresident ICEs

ABSTRACT The discovery of integrative conjugative elements (ICEs) in wall-less mycoplasmas and the demonstration of their role in massive gene flows within and across species have shed new light on the evolution of these minimal bacteria. Of these, the ICE of the ruminant pathogen Mycoplasma agalactiae (ICEA) represents a prototype and belongs to a new clade of the Mutator-like superfamily that has no preferential insertion site and often occurs as multiple chromosomal copies. Here, functional genomics and mating experiments were combined to address ICEA functions and define the minimal ICEA chassis conferring conjugative properties to M. agalactiae. Data further indicated a complex interaction among coresident ICEAs, since the minimal ICEA structure was influenced by the occurrence of additional ICEA copies that can trans-complement conjugation-deficient ICEAs. However, this cooperative behavior was limited to the CDS14 surface lipoprotein, which is constitutively expressed by coresident ICEAs, and did not extend to other ICEA proteins, including the cis-acting DDE recombinase and components of the mating channel whose expression was detected only sporadically. Remarkably, conjugation-deficient mutants containing a single ICEA copy knocked out in cds14 can be complemented by neighboring cells expressing CDS14. This result, together with those revealing the conservation of CDS14 functions in closely related species, may suggest a way for mycoplasma ICEs to extend their interaction outside their chromosomal environment. Overall, this report provides a first model of conjugative transfer in mycoplasmas and offers valuable insights into understanding horizontal gene transfer in this highly adaptive and diverse group of minimal bacteria. IMPORTANCE Integrative conjugative elements (ICEs) are self-transmissible mobile genetic elements that are key mediators of horizontal gene flow in bacteria. Recently, a new category of ICEs was identified that confer conjugative properties to mycoplasmas, a highly adaptive and diverse group of wall-less bacteria with reduced genomes. Unlike classical ICEs, these mobile elements have no preferential insertion specificity, and multiple mycoplasma ICE copies can be found randomly integrated into the host chromosome. Here, the prototype ICE of Mycoplasma agalactiae was used to define the minimal conjugative machinery and to propose the first model of ICE transfer in mycoplasmas. This model unveils the complex interactions taking place among coresident ICEs and suggests a way for these elements to extend their influence outside their chromosomal environment. These data pave the way for future studies aiming at deciphering chromosomal transfer, an unconventional mechanism of DNA swapping that has been recently associated with mycoplasma ICEs

Mycoplasmas under experimental antimicrobial selection: The unpredicted contribution of horizontal chromosomal transfer.

Horizontal Gene Transfer was long thought to be marginal in Mycoplasma a large group of wall-less bacteria often portrayed as minimal cells because of their reduced genomes (ca. 0.5 to 2.0 Mb) and their limited metabolic pathways. This view was recently challenged by the discovery of conjugative exchanges of large chromosomal fragments that equally affected all parts of the chromosome via an unconventional mechanism, so that the whole mycoplasma genome is potentially mobile. By combining next generation sequencing to classical mating and evolutionary experiments, the current study further explored the contribution and impact of this phenomenon on mycoplasma evolution and adaptation using the fluoroquinolone enrofloxacin (Enro), for selective pressure and the ruminant pathogen Mycoplasma agalactiae, as a model organism. For this purpose, we generated isogenic lineages that displayed different combination of spontaneous mutations in Enro target genes (gyrA, gyrB, parC and parE) in association to gradual level of resistance to Enro. We then tested whether these mutations can be acquired by a susceptible population via conjugative chromosomal transfer knowing that, in our model organism, the 4 target genes are scattered in three distinct and distant loci. Our data show that under antibiotic selective pressure, the time scale of the mutational pathway leading to high-level of Enro resistance can be readily compressed into a single conjugative step, in which several EnroR alleles were transferred from resistant to susceptible mycoplasma cells. In addition to acting as an accelerator for antimicrobial dissemination, mycoplasma chromosomal transfer reshuffled genomes beyond expectations and created a mosaic of resistant sub-populations with unpredicted and unrelated features. Our findings provide insights into the process that may drive evolution and adaptability of several pathogenic Mycoplasma spp. via an unconventional conjugative mechanism

Genome Mosaicism in Field Strains of Mycoplasma bovis as Footprints of In-Host Horizontal Chromosomal Transfer

Rights Retained by the Author ASM grants the Author and the employer for whom the Work was created (if applicable) a royalty-free, non-exclusive limited license to: a) post the Work (after publication by ASM) on the Author's personal or university-hosted website.Horizontal gene transfer was long thought to be marginal in Mollicutes, but the capacity of some of these wall-less bacteria to exchange large chromosomal regions has been recently documented. Mycoplasma chromosomal transfer (MCT) is an unconventional mechanism that relies on the presence of a functional integrative conjugative element (ICE) in at least one partner and involves the horizontal acquisition of small and large chromosomal fragments from any part of the donor genome, which results in progenies composed of an infinite variety of mosaic genomes. The present study focuses on Mycoplasma bovis, an important pathogen of cattle responsible for major economic losses worldwide. By combining phylogenetic tree reconstructions and detailed comparative genome analyses of 36 isolates collected in Spain (2016 to 2018), we confirmed the mosaic nature of 16 field isolates and mapped chromosomal transfers exchanged between their hypothetical ancestors. This study provides evidence that MCT can take place in the field, most likely during coinfections by multiple strains. Because mobile genetic elements (MGEs) are classical contributors of genome plasticity, the presence of phages, insertion sequences (ISs), and ICEs was also investigated. Data revealed that these elements are widespread within the M. bovis species and evidenced classical horizontal transfer of phages and ICEs in addition to MCT. These events contribute to wide-genome diversity and reorganization within this species and may have a tremendous impact on diagnostic and disease control

Experimental Infections with <i>Mycoplasma agalactiae</i> Identify Key Factors Involved in Host-Colonization

<div><p>Mechanisms underlying pathogenic processes in mycoplasma infections are poorly understood, mainly because of limited sequence similarities with classical, bacterial virulence factors. Recently, large-scale transposon mutagenesis in the ruminant pathogen <i>Mycoplasma agalactiae</i> identified the NIF locus, including <i>nifS</i> and <i>nifU</i>, as essential for mycoplasma growth in cell culture, while dispensable in axenic media. To evaluate the importance of this locus <i>in vivo</i>, the infectivity of two knock-out mutants was tested upon experimental infection in the natural host. In this model, the parental PG2 strain was able to establish a systemic infection in lactating ewes, colonizing various body sites such as lymph nodes and the mammary gland, even when inoculated at low doses. In these PG2-infected ewes, we observed over the course of infection (i) the development of a specific antibody response and (ii) dynamic changes in expression of <i>M. agalactiae</i> surface variable proteins (Vpma), with multiple Vpma profiles co-existing in the same animal. In contrast and despite a sensitive model, none of the knock-out mutants were able to survive and colonize the host. The extreme avirulent phenotype of the two mutants was further supported by the absence of an IgG response in inoculated animals. The exact role of the NIF locus remains to be elucidated but these data demonstrate that it plays a key role in the infectious process of <i>M. agalactiae</i> and most likely of other pathogenic mycoplasma species as many carry closely related homologs.</p></div

Stochastic changes in <i>M. agalactiae</i> antigenic structure upon colonization of the animal host.

<p>(<b>A</b>) SDS-PAGE analysis of Triton X-114 soluble proteins extracted from cultures of <i>M. agalactiae</i> recovered from milk samples collected from animals A5 and A8 at different days post-inoculation (0 to 28). (<b>B</b>) Characterization of the Vpma expression profile using specific polyclonal antibodies (see Materials and Methods). A lipoprotein P80 antiserum was used as control. Molecular weight standards (MW) are in kDa.</p

Overview of the experimental infections with <i>M. agalactiae</i> strain PG2 and NifS mutants in lactating ewes using the subcutaneous route.

a<p>Mean mycoplasma CFUs inoculated to each experimental group;</p>b<p>number of animals with reduced milk production;</p>c<p>number of animals with at least one milk sample found positive for <i>M. agalactiae</i>;</p>d<p>number of animals that developed a serological response to <i>M. agalactiae</i>;</p>e<p>number of animals with at least one lymph node found positive for <i>M. agalactiae</i> (the lymph nodes analyzed are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093970#pone-0093970-g002" target="_blank">Fig. 2</a>);</p>f<p>number of animals with subclinical mammary infections with <i>Escherichia coli</i> or coagulase-negative Staphylococci.</p

Mycoplasma excretion and serological response in animals inoculated with <i>M. agalactiae</i> strain PG2 and NifS mutants.

a<p>The excretion of <i>M. agalactiae</i> in the mammary gland is indicated with a letter indicating udder halves found positive: right (R), left (L) or both (LR);</p>b<p>the detection of specific IgG antibodies to Triton X-114 soluble antigens of <i>M. agalactiae</i> in animal sera are indicated (+).</p