Mycoplasma hyopneumoniae, Mycoplasma flocculare and Mycoplasma hyorhinis live in swine respiratory tracts. M. flocculare, a commensal bacterium, is genetically closely related to M. hyopneumoniae, the causative agent of enzootic porcine pneumonia. M. hyorhinis is also pathogenic, causing polyserositis and arthritis. In this work, we present the genome sequences of M. flocculare and M. hyopneumoniae strain 7422, and we compare these genomes with the genomes of other M. hyoponeumoniae strain and to the a M. hyorhinis genome. These analyses were performed to identify possible characteristics that may help to explain the different behaviors of these species in swine respiratory tracts.M. flocculare and M. hyopneumoniae display a high degree of similarity with respect to the gene content, only some genomic regions display considerable synteny. Genes encoding proteins that may be involved in host-cell adhesion in M. hyopneumoniae and M. flocculare display differences in genomic structure and organization. Some genes encoding adhesins of the P97 family are absent in M. flocculare and some contain sequence differences or lack of domains that are considered to be important for adhesion to host cells. The phylogenetic relationship of the three species was confirmed by a phylogenomic approach. The set of genes involved in metabolism, especially in the uptake of precursors for nucleic acids synthesis and nucleotide metabolism, display some differences in copy number and the presence/absence in the three species.The overall genome organization of three species was analyzed, revealing that the ORF clusters (OCs) differ considerably and that inversions and rearrangements are common. Although M. hyopneumoniae and M. flocculare display many differences that may help to explain why one species is pathogenic and the other is considered to be commensal. However, it was not possible to identify specific virulence determinant factors that could explain the differences in the pathogenicity of the analyzed species. The M. hyorhinis genome contains differences in some components involved in metabolism and evasion of the host’s immune system that may contribute to its growth aggressiveness. Several horizontal gene transfer events were identified. The phylogenomic analysis places M. hyopneumoniae, M. flocculare and M. hyorhinis in the hyopneumoniae clade.The comparative analyses of three mycoplasma species that inhabit the swine respiratory tract facilitated the identification of some characteristics that may be related to their different behaviors.  Mycoplasmas belong to the class Mollicutes, which is a taxon of bacteria that is characterized by the absence of a cell wall, a relatively small genome size and a strong dependence on nutrients supplied by the host environment . More thMycoplasma hyopneumoniae, Mycoplasma flocculare and Mycoplasma hyorhinis are the most important species that have been identified in porcine respiratory systems [M. hyopneumoniae and M. flocculare are known to be closely related [M. hyopneumoniae is the etiological agent of porcine mycoplasmal pneumonia, while M. hyorhinis, which causes polyserositis and arthritis, is also frequently found in swine respiratory tracts [M. flocculare is also widespread in swine herds, but no disease has been associated with this species [M. hyopneumoniae can adhere to the cilia of tracheal epithelial cells and causes damage. Although M. flocculare can also adhere to cilia, no resulting damage has been observed, suggesting that M. hyopneumoniae and M. flocculare may possess different adhesins, facilitating the recognition of different receptor sites on the cilia [M. flocculare is restricted to the swine respiratory tract, M. hyopneumoniae and M. hyorhinis can also colonize other sites, such as cardiac or joint tissues [M. hyorhinis has been detected in human carcinoma tissues [M. flocculare ATCC 27716 and M. hyopneumoniae strain J have been compared, revealing that at least three chromosomal inversions have occurred since the divergence of both species [ systems -11. Base related . M. hyopy tracts . M. floc species . M. hyophe cilia . Additio tissues ,16. Thes tissues ,18. The  species .M. hyopneumoniae (four strains), M. hyorhinis (four strains) and Mycoplasma suis (two strains) have been sequenced, facilitating the comparison of metabolic pathways and evidencing specific mechanisms that can be utilized to survive in different host environments [In recent years, the genomes of several mycoplasma species have been sequenced. The absence of several metabolic pathways, which was suggested by genetic and biochemical studies , has beeronments -28.M. flocculare and M. hyopneumoniae strain 7422 have now been completed, the current study presents a comprehensive comparison of the M. flocculare, M. hyopneumoniae and M. hyorhinis genomes. These three mycoplasma species can inhabit swine respiratory tracts. We have assessed the overall genome organizations including analyses of the open reading frame (ORF) clusters (OCs), inversions and rearrangements, and coding capacities, including analyses of encoded metabolic pathways and surface protein repertoires. Potential mechanisms of interaction with host cells are evidenced, and their implications on pathogenicity are discussed. Additionally, a phylogenomic approach using 32 mycoplasma genomes (including two that are reported here for the first time) was implemented to reconstruct the evolutionary history of the swine mycoplasma genomes, individual genes and/or portions of their genomes, including horizontal gene transfer analysis.Because the genome sequences of M. flocculare and M. hyopneumoniae 7422 genomes are composed of single, circular chromosomes of 763,948 and 899,887 bp with 28.9% and 28.4% GC contents, respectively. The M. flocculare genome, which by assembly remained with 13 gaps, contains 585 coding sequences (CDSs), of which 356 have known functions and 229 are annotated as hypothetical. The M. hyopneumoniae 7422 genome, completely closed in one contig by assembly, comprises 692 CDSs, of which 414 correspond to proteins with known functions and 278 are annotated as hypothetical. The protein-coding regions occupy approximately 87% of each M. flocculare and M. hyopneumoniae 7422 chromosome, and the average ORF length is 1,145 bp. Each genome contains one gene encoding the ribosomal RNAs (rRNAs) 16S and 23S, one gene encoding rRNA 5S and 30 genes encoding the transfer RNAs (tRNAs) representing all 20 amino acids. The general genome features of the five strains, M. hyopneumoniae , M. flocculare, and M. hyorhinis HUB-1, were compared in this study and are listed in Table The M. flocculare and M. hyopneumoniae 7422 CDSs that encode proteins with known functions, 380 and 403 CDSs, respectively, were classified into COG families comprising 18 functional categories  approach and plotted the data in a Venn diagram  and the inositol metabolism pathway, we identified 76 genes that are unique to M. flocculare, 69 to M. hyopneumoniae and 234 to M. hyorhinis.To identify the genes that constitute the core and pan-genome of m Figure . We idenM. hyopneumoniae, the genome of strain 7422 displays a highly similar gene composition and organization, with the exception of the localization of the integrative conjugative element (ICEH), which is positioned from 139,715 to 162,049 bp in the 7422 genome and from 518,376 to 540,705 bp in the 7448 genome. The similarity between M. hyopneumoniae gene repertories was 88% approximately. The small, but significative difference in the M. hyopneumoniae 7422 genome is the presence of an exclusive region of genes encoding transposases, hypothetical proteins and an ortholog of subtilisin-like serine protease . In comparison to 7422 genome, just one exclusive region was found in the 7448 genome, which is composed of genes encoding hypothetical proteins .When compared to other sequenced strains of M. flocculare, M. hyopneumoniae 7448 and M. hyorhinis HUB-1 was analyzed, and the gene localization patterns were compared to detect ORFs with order conservation. The ORF cluster composition, organization and localization in the genomes were analyzed to determine the conservation level among the OC organization. Two groups of ORF clusters were created for each species, the OC group  and an excinuclease ABC subunit C (uvrC) were found to display monocistronic organization in the three genomes  encode hypothetical proteins, transport-related proteins, myo-inositol utilization proteins, the integrative conjugative element (ICEH) and an additional copy of the P97 protein , which have been described as being involved in a complex system involving bacterial-host interactions [A detailed, genomic-scale analysis of the OC organization in es Table D, and thractions .M. flocculare contigs (MFL contigs) and M. hyopneumoniae genome, considering the ORF string organization and OC distribution  were identified as being involved in inversions or rearrangements  and M. hyorhinis (represented by the HUB-1 strain). The results of this survey are summarized in Additional files M. flocculare CDSs, 277 (47.5%) were predicted to encode surface proteins; this number was similar to that of M. hyopneumoniae 7448  and higher than that of M. hyorhinis HUB-1 A comparative M. flocculare, M. hyopneumoniae and M. hyorhinis, respectively, with respect to the other two species , they also include some proteins with assigned functions. Among these proteins with predicted functions, M. hyopneumoniae includes proteins involved in myo-inositol catabolism, a permease and a protein encoded by the integrative conjugative element (ICEH), and M. hyorhinis includes some variable antigens, secretory system components, transporters and lipoproteins.The repertoire of M. flocculare surface proteins is shared with M. hyopneumoniae and/or M. hyorhinis  [M. hyopneumoniae and M. hyorhinis virulence factors [M. flocculare P97 copy 2 and P97-like orthologs present relatively high overall identities to their M. hyopneumoniae counterparts , but in the case of P97 copy 2, the M. flocculare ortholog lacks a domain (R1) regarded to be important for virulence in M. hyopneumoniae; instead, it contains a second R2 domain  and M. hyorhinis (P95).The presence of surface virulence determinants even in the non-pathogenic  strain) ,45 sugge strain) . Differe factors ,41,47,48M. flocculare, M. hyopneumoniae and M. hyorhinis. However, because all (in the case of M. flocculare) or most (in the cases of M. hyopneumoniae and M. hyorhinis) of these unshared CDS products are hypothetical or conserved hypothetical proteins, their potential contributions to pathogenicity remain elusive. However, considering the nature of the unshared CDS products annotated in M. hyopneumoniae and M. hyorhinis, these ‘exclusive’ and unknown proteins are likely to include players of processes that are important for pathogen-host interactions, such as proteins involved in secretion, the uptake of certain molecules, conjugation and immune evasion/modulation. The variation in M. hyorhinis surface lipoproteins (Vlp) is considered important to protect the organism from the humoral response and may be a primary adaptive strategy for immune evasion during infection and disease [Cell-surface features with implications for virulence may also reside in the 10 to 18% of the repertoires of surface proteins that are not shared between  disease ,50. TherM. flocculare, M. hyopneumoniae and M. hyorhinis was performed based on the COG classification of the predicted surface protein sets for each species , and for the no-COG category, which included 45-53% of the proteins, most of which  were represented by hypothetical proteins or in the additional category of antigen, adhesin or lipoprotein, in which proteins were included based on their prior immunological or functional characterization according to published studies. Overall, the surface protein set for M. flocculare, taken from the correspondent COG profile, was shown to be very similar to those of M. hyopneumoniae and M. hyorhinis. This result suggests that the species have equivalent genetic backgrounds for metabolic and growth processes. Such functional similarities may be the result of common selective pressures associated with the colonization of the same environment .An additional comparison of the repertoires of surface proteins from M. flocculare (and M. hyorhinis) contained approximately half of the number of proteins as M. hyopneumoniae. Protein sets assigned to the L category are heterogeneous, and the relative excess of proteins in M. hyopneumoniae corresponds to transposases that cannot be found in either M. flocculare or M. hyorhinis. Conversely, category V is enriched with ATP-binding cassette (ABC) transporter system proteins related to defense mechanisms, such as the M. hyorhinis ABC-type multidrug-like transport system ATP-binding proteins and their orthologs in M. flocculare and M. hyopneumoniae. The remaining transporters, including ABC and non-ABC transporter system components  appear in other COG categories, such as E, G, R or P. However, the overall number of transporters unrelated to defense mechanisms (non-V) is roughly equivalent in the three species, with 50, 49, and 47, in the M. flocculare, M. hyopneumoniae and M. hyorhinis surface protein sets, respectively. Although M. hyorhinis have 19 genes encoding transposases, and M. hyopneumoniae 9 genes, notably, the presence of transposases among M. hyopneumoniae predicted surface proteins may be an artifact due to the occurrence of helical structures in these enzymes [M. hyopneumoniae capabilities that are unavailable in both M. flocculare and M. hyorhinis. A larger number of transport proteins is usually related to a species’ capacity to persist in different tissue environments [M. flocculare, M. hyopneumoniae or M. hyorhinis because they share a large portion of the transporter repertoire. This situation is similar to that observed for secretory system components. However, according to the COG (see below), M. hyopneumoniae has approximately two-fold more transporters associated with defense mechanisms than the other two species. In this aspect, M. hyopneumoniae is more similar to Mycoplasma bovis[M. flocculare or M. hyorhinis. The implications of the larger M. hyopneumoniae repertoire of defense mechanisms proteins (COG V) for its survival in the swine respiratory tract have not yet been investigated.The L  and V (defense mechanisms) categories displayed differences;  enzymes ; these hronments , but thisma bovis, which iM. flocculare genes, 179 gene sets were retrieved that contained at least one gene representative for each swine mycoplasma analyzed here (BLAST cut off E-10). Overall, 179 ortholog-like files representing different CDSs were concatenated, leading to an aligned file containing 104,097 amino acid residues. The neighbor-joining method (NJ) and maximum parsimony (MP) tree topologies did not differ significantly, especially when major clades were considered , hominis (Mycoplasma hominis and Mycoplasma arthritidis), hyopneumoniae , hemotrophic mycoplasma (Mycoplasma suis and Mycoplasma haemofelis), genitalium-pneumoniae , and mycoides . All of these clades displayed high bootstrap values. The synoviae-pulmonis  group did not form a monophyletic cluster, but they are closely related to the bovis clade. M. penetrans HF-2 is near the genitalium-pneumoniae clade.The M. flocculare, M. hyopneumoniae 7448, and M. hyorhinis HUB-1 were located in the hyopneumoniae clade.Our mycoplasma phylogenomic tree Figure  corroborM. flocculare, M. hyorhinis HUB-1 and M. hyopneumoniae 7448 genomes, several paralog clusters were identified through the bidirectional best hit (BBH) approach, wherein a paralog cluster was defined as a gene set in which every gene is a BBH with at least one other element. Fourteen of these paralogs  were submitted to phylogenetic analyses to understand the evolutionary history of those paralogs.When comparing the M. flocculare and M. hyopneumoniae  events. HGTs are an important source of genome innovation and evolution in prokaryotes , and it M. hyorhinis HUB-1 and M. conjunctivae HRC. The events involving species belonging to the hyopneumoniae group occurred in ribosomal proteins, GTP-binding proteins, heat-shock proteins, DNA primase, signal recognition particle protein, ABC transporter ATP-binding proteins, phosphoesterases, cell division protein, elongation factor, fructose-biphosphate aldolase, DNA polymerase, glutamyl-tRNA synthetase, helicases, and hypothetical proteins. Regions encoding ABC transporters were likely transferred between M. synoviae and M. gallisepticum  in the M. hyorhinis HUB-1 genome and a single copy in M. hyopneumoniae 7448 (MHP7448_0580), M. hyopneumoniae 7422 (MX03145) and M. flocculare (MF00420). Another set represented by two copies was observed in the genomes of the three species. The cell surface-exposed exonuclease (mhp379) from M. hyopneumoniae 232, a representative of the latter set, has been analyzed, and it has been proposed that the exonuclease activity of mhp379 may be important for importing nucleic acid precursors [M. hyorhinis may represent a potential advantage of this species in acquiring nucleic acids precursors. Mycoplasmas cannot de novo synthesize purines and pyrimidines; therefore, they depend on salvage and interconversions to supply the cell with the nucleic acid precursors [M. hyopneumoniae, MHR_0566 - M. hyorhinis and MF01198 - M. flocculare) and pyrimidine  metabolism , allowing the conversion of dUMP to dTMP, and dihydrofolate reductase (DHFR), which catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF). The presence of TS and DHFR in M. hyorhinis may also contribute to its ability to overgrow the other Mycoplasma species in the swine respiratory tract [It has been shown that nuclease activities can be detected in Mollicutes and that these activities are primarily associated with the membrane and may be essential for growth and survival . Genes eecursors . The preecursors . The thr7422 MX035 and M. M. hyopneumoniae strain 7422 genome displays a similar organization as to the other previously described strains, but it contains rearrangements and an altered position of ICEH in the genome. The genomes of M. hyopneumoniae and M. flocculare, two closely related species, contain some blocks of synteny, but they also display many differences that may help to explain why one species is pathogenic and the other is commensal. However, it was not possible to correlate specific virulence determinant factors to the pathogenicity differences of the analyzed species. A large proportion of the repertoire of M. flocculare surface proteins is shared with M. hyopneumoniae and/or M. hyorhinis, which would be expected because the organisms may occupy the same niche. However, certain members of the p97 family are absent in M. flocculare, and some display sequence differences or lack domains that are considered to be important for host-cell adhesion. M. hyorhinis contains some metabolic genes that are absent in the other species, suggesting a possible advantage in the growth of this species. The differences in some components involved in evasion of the host immune system may also contribute to the aggressive growth of M. hyorhinis. The phylogenomic analysis confirmed previous results, placing M. hyopneumoniae, M. flocculare and M. hyorhinis in the hyopneumoniae clade. Several horizontal gene transfer events were identified, and several of them occurred between M. hyorhinis and M. conjunctivae.The comparative analyses of three mycoplasma species that inhabit the swine respiratory tract facilitated the identification of some characteristics that may promote the understanding of their different behaviors. The M. hyopneumoniae strain 7422 was isolated from an infected swine in Lindóia do Sul, Santa Catarina, Brazil. M. flocculare (ATCC 27716) was acquired by Embrapa Suínos e Aves  from the American Type Culture Collection. Both of the strains were cultivated in 5 mL of Friis medium [s medium  at 37°C s medium .M. flocculare was sequenced in one region of a two-region PicoTiterPlate (PTP), and M. hyopneumoniae 7422 was sequenced in two regions of an eight-region PTP. The contigs were assembled using the Newbler software program version 2.6 with the default parameters. The PCR assisted contig extension (PACE) strategy [M. hyopneumoniae 7422 and M. flocculare, the estimated genome coverage for both genomes was 23X. The M hyopneumoniae 7422 genome was completely closed in one contig, and the M. flocculare genome retained 13 gaps.For each species, one 454 shotgun library was prepared with approximately 5 μg of gDNA. The library construction, titration, emulsion PCR and sequencing steps were performed explicitly according to the manufacturer’s protocol. Sequencing was performed using Roche 454 GS FLX Titanium platform. strategy  was used-5, query coverage of 60%, and positive similarity value of 50%. The comparative databank is available at http://www.genesul.lncc.br/comparative/.The annotation and analysis of the sequences of both genomes were performed using the System for Automated Bacterial Integrated Annotation (SABIA) . The comM. flocculare and PRJNA47327 ID: 47327 for M. hyopneumoniae 7422.To store and analyze the data, a databank was developed using the MySQL and Perl programming languages. This databank integrates tools and information from numerous biological databases, such as The Integrated Resource of Protein Domains and Functional Sites (Interpro) , ProteinM. flocculare, M. hyorhinis and M. hyopneumoniae genomes was established based on the occurrence of clusters with two or more tandem genes in the same DNA strand. This procedure was performed by a systematic annotation comparison of the protein sequences encoded in all of the ORFs from the analyzed genomes. According to the complexity of the adjacent ORF rearrangements, two groups were created; the OC group was characterized by the presence of two or more ORFs in the same DNA strand until the occurrence of ORFs in the opposite strand, and the monocistronic (mC) group represented single ORFs. Differences in the annotation were evaluated by comparing the protein sequences from these three genomes using the NCBI/BLASTP program. The OC groups predicted for M. flocculare and M. hyorhinis were compared with the OC organization found in M. hyopneumoniae[M. hyorhinis and M. flocculare to predict OC organization. The M. flocculare analysis was performed with each contig sequence, while the M. hyorhinis analysis was performed with the whole genome sequence.The prediction of OCs was performed by the Artemis Release 10.5.2 software program  accordinneumoniae. MoreoveM. flocculare genome and from the M. hyopneumoniae 7448 and M. hyorhinis HUB-1 genomes  were analyzed using the default parameters by the following software programs: SVMtm Transmembrane Domain Predictor [M. flocculare, M. hyopneumoniae 7448 and M. hyorhinis HUB-1 (see below), the CDSs predicted to encode surface proteins just for one species (by the aforementioned criteria) had their corresponding orthologs (when existent) included in the list of surface-protein-encoding CDSs for the other two species. The clusters of orthologous group (COG) functional classification of M. flocculare predicted surface proteins using NCBI COGnitor [M. hyopneumoniae 7448 and M. hyorhinis HUB-1 predicted surface proteins obtained from NCBI.For the surface protein predictions, all of the translated CDS entries from the redictor , TMHMM Sredictor , SCAMPI redictor , and PSOredictor . The firredictor , and wheCOGnitor , and thoM. flocculare predicted surface protein encoding CDSs and all of the CDSs from the other two species using the BioEdit Sequence Alignment Editor [-20, 10-10 or 10-6; manual inspection of query coverage, identity and similarity scores; and, if required, consideration of peculiarities of specific gene families.For the ortholog and paralog identifications, bidirectional local BLAST searches  were pert Editor . The ortM. flocculare (ATCC 27716) was used as query organism. We began with all 582 genes identified in this strain and performed a BLAST search against the gene set of all other 32 organisms . Multi-FASTA files were created containing ortholog gene sets for each query gene in the M. flocculare strain. Only gene sets containing at least one representative in each genome were selected for further phylogenomic analysis .M. flocculare deduced protein sequence for all of the analyzed swine mycoplasmas were used as the input for multiple alignments using the CLUSTALw algorithm with default parameters.Multi-FASTA putative ortholog files containing the best representative of each The SCaFos software program  was used-6 e-value cutoff; all of the sequences were subsequently aligned using COBALT [Selected gene clusters of paralogs were subjected to a phylogenetic analysis. BLAST searches were first conducted for each gene using a 10g COBALT . DistancHorizontal gene transfer (HGT) was analyzed with Tree and Reticulogram reconstruction (T-Rex)  using thCDS: Coding DNA sequence; COG: Clusters of orthologous groups; HGT: Horizontal gene transfer; ORF: Open read frames; OC: ORF cluster.The authors declare they have no competing interests.in silico analyses. ISS, HBF, ATV and AZ conceived, designed and coordinated the study, participated in the interpretation of the results and in the writing of the manuscript. All authors read and approved the final manuscript.FMS performed the genomes organization analyses and comparative analyses of genomes, participated in the interpretation of the results and in the writing of the manuscript. CET, FP and MMBF carried out the phylogenomics and phylogenetics analyses and in the interpretation of the results. LGPA and RS carried out the assemblies genomes and participated in the comparative analyses of genomes. VGV, LR and TG participated in the surface proteins OCs organization in M. flocculare genome.Click here for fileOCs in M. hyorhinis genome.Click here for fileMonocistronic group (mC).Click here for fileOC organization similarities in the M. hyorhinis and M. flocculare genomes in relationship of M. hyopneumoniae OC organization.Click here for fileMonocistronic ORFs presents in the MHP, MHR and MFL genomes.Click here for fileA. Exclusive OCs of M. hyopneumoniae genome. 6B. Exclusive OCs of M. flocculare genome. 6C. Conserved Ocs among the three mycoplasmas species. 6D. Exclusive OCs of M. hyorhinis genome.Click here for fileA. M. flocculare contigs with conservation regions. B. M. flocculare contigs with inversion regions.Click here for fileVenn diagram of the predicted surface protein sets from M. flocculare, M. hyopneumoniae 7448 and M. hyorhinis HUB-1.Click here for fileCOG functional classifications of the predicted surface protein sets from M. flocculare (A), M. hyopneumoniae 7448 (B) and M. hyorhinis HUB-1 (C).Click here for file List of predicted surface proteins ofM. flocculare (A), M. hyopneumoniae 7448 (B), and M. hyorhinis HUB-1 (C). B: List of predicted surface proteins of M. flocculare (A), M. hyopneumoniae 7448 (B), and M. hyorhinis HUB-1 (C). List of predicted surface proteins of M. flocculare (A), M. hyopneumoniae 7448 (B), and M. hyorhinis HUB-1 (C).Click here for fileA. List of predicted surface proteins of M. flocculare not shared with M. hyopneumoniae 7448 and M. hyorhinis HUB_1. B. List of predicted surface proteins of M. hyopneumoniae 7448 not shared with M. flocculare and M. hyorhinis HUB-1. C. List of predicted surface proteins of M. hyorhinis HUB-1 not shared with M. flocculare and M. hyopneumoniae 7448.Click here for fileList of ortholog surface protein coding CDSs between M. flocculare, M. hyopneumoniae 7448 and M. hyorhinis HUB-1.Click here for fileSummary of adhesins associated to pathogenicity and Genome organization comparison.Click here for fileM. flocculare, M. hyopneumoniae and M. hyorhynis P97 and P97-like adhesin orthologs and paralogs.Click here for fileEvolutionary history of mycoplasmas obtained through a phylogenomic approach. The Neighbor-Joining method, using p-distance to compute the evolutionary distances and complete deletion of gaps was implemented by MEGA 5 software. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test  are shown next to the branches. S. pyogenes was used as outgroup.Click here for fileEvolutionary history of mycoplasmas obtained through a phylogenomic approach. The Neighbor-Joining method, using Poisson correction to compute the evolutionary distances and complete deletion of gaps was implemented by MEGA 5 software. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test  are shown next to the branches. S. pyogenes was used as outgroup.Click here for fileEvolutionary history of mycoplasmas obtained through a phylogenomic approach. The Neighbor-Joining method was the same description of the Additional file 16.Click here for fileEvolutionary history of mycoplasmas obtained through a phylogenomic approach. The Maximum Parsimony method using the close-neighbor-interchange algorithm, and the complete deletion of gaps was implemented by MEGA 5 software. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches. S. pyogenes was used as outgroup.Click here for fileEvolutionary history of DNA methylases from mycoplasmas obtained through a phylogenetic analysis. The Neighbor-Joining method, using p-distance to compute the evolutionary distances and complete deletion of gaps was implemented by MEGA 5 software. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test  is shown next to the branches.Click here for fileEvolutionary history of ATP synthases from mycoplasmas obtained through a phylogenetic analysis. The Neighbor-Joining method was the same description of the Additional file 19.Click here for fileEvolutionary history of ribulose-phosphate-3epimerases from mycoplasmas obtained through a phylogenetic analysis. The Neighbor-Joining method was the same description of the Additional file 19.Click here for fileEvolutionary history of oligoendopeptidases from mycoplasmas obtained through a phylogenetic analysis. The Neighbor-Joining method was the same description of the Additional file 16. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test  is shown next to the branches. (PDF 9 kb)Click here for fileEvolutionary history of single-strand binding proteins from mycoplasmas obtained through a phylogenetic analysis. The Neighbor-Joining method was the same description of the Additional file 19.Click here for fileEvolutionary history of fructose-bisphosphate aldolase proteins from mycoplasmas obtained through a phylogenetic analysis. The Neighbor-Joining method was the same description of the Additional file 19.Click here for fileEvolutionary history of dihydrolipoamide dehydrogenase proteins from mycoplasmas obtained through a phylogenetic analysis. The Neighbor-Joining method was the same description of the Additional file 19.Click here for fileEvolutionary history of glucose-6-phosphate-isomerase proteins from mycoplasmas obtained through a phylogenetic analysis. The Neighbor-Joining method was the same description of the Additional file 19.Click here for fileEvolutionary history of lypoate protein ligases from mycoplasmas obtained through a phylogenetic analysis. The Neighbor-Joining method was the same description of the Additional file 19.Click here for fileEvolutionary history of acyl carrier phosphodiesterases from mycoplasmas obtained through a phylogenetic analysis. The Neighbor-Joining method was the same description of the Additional file 19.Click here for fileEvolutionary history of lactate dehydrogenases from mycoplasmas obtained through a phylogenetic analysis. The Neighbor-Joining method was the same description of the Additional file 19.Click here for fileEvolutionary history of membrane nucleases from mycoplasmas obtained through a phylogenetic analysis. The Neighbor-Joining method was the same description of the Additional file 19.Click here for fileEvolutionary history of TRSE-like proteins from mycoplasmas obtained through a phylogenetic analysis. The Neighbor-Joining method was the same description of the Additional file 19.Click here for fileEvolutionary history of P97 proteins from mycoplasmas obtained through a phylogenetic analysis. The Neighbor-Joining method was the same description of the Additional file 19.Click here for fileHGT events in mycoplasmas species.Click here for fileBacterial strains used in the phylogenomic analyses.Click here for file


Lawsonia intracellularis is the causative agent of proliferative enteropathy, an endemic disease in pigs and an emerging concern in horses. Enterocyte hyperplasia is a common lesion in every case but there are differences regarding clinical and pathological presentations among affected species. We hypothesize that host susceptibility to L. intracellularis infection depends on the species of origin of the bacterial isolate. The objective of this study was to evaluate the susceptibilities of pigs and horses to L. intracellularis infection using either a porcine or an equine isolate.9L. Intracellularis/challenged animal), and a saline solution (negative control group). The animals were monitored regarding clinical signs, average of daily weight gain, fecal shedding of the bacteria by PCR and humoral serological response.Twelve foals and eighteen pigs were equally divided into three groups and infected with either a porcine or an equine isolate (10Foals infected with the equine isolate developed moderate to severe clinical signs and maintained a lower average of weight gain compared to control foals. Fecal quantitative PCR in equine isolate-infected foals revealed higher amounts of bacterial DNA associated with longer duration of shedding compared with porcine isolate-infected foals. All four foals infected with the equine isolate demonstrated higher IgG titers in the serum compared with porcine isolate-infected foals. In the pig trial, diarrhea and seroconversion were only observed in animals infected with the porcine isolate. Pathological changes typical of proliferative enteropathy were observed in the necropsied foal infected with equine isolate and in the two necropsied pigs infected with the porcine isolate.L. intracellularis strain.Evident clinical signs, longer periods of bacterial shedding and stronger serologic immune responses were observed in animals infected with species-specific isolates. These results show that host susceptibility is driven by the origin of the isolated  Lawsonia intracellularis is an obligate intracellular bacterium and the etiologic agent of proliferative enteropathy (PE), an intestinal hyperplasic disease characterized by thickening of the mucosa of the intestine due to enterocyte proliferation [feration . The disferation ,3. Sinceferation . In the feration -8.Although hyperplasic lesions are present in every case of PE, there are some differences regarding clinical and pathological presentations among affected species. In pigs, there are two major clinical forms: a sporadic, acute, haemorrhagic diarrhea and a chronic, mild diarrhea . A haemoL. intracellularis has only been achieved by using dividing cells in culture under strict microaerophilic conditions. These fastidious properties restrict opportunities to study the dynamics of inter-species transmission, potential reservoirs for the bacterium and host susceptibilities to different bacterial isolates. The disease has been experimentally reproduced in hamsters, pigs and horses using species-specific isolates or intestinal homogenate derived from infected animals [L. intracellularis isolates have consistently reproduced subclinical disease and mild lesions in infected animals [L. intracellularis infections.Isolation and cultivation of  animals -12. Resu animals -15. TherL. intracellularis infection is capable of driving the susceptibilities of pigs and horses depending on the species origin of the isolate. The objective of this study was to evaluate the susceptibilities of horses and pigs to L. intracellularis infection using porcine and equine isolates. The present study reports clinical signs, longer periods of fecal shedding of bacteria and stronger serologic immune responses in pigs and foals infected with their species-specific isolates.We hypothesize that host adaptation to L. intracellularis strain isolated from a gilt and a foal, respectively, both affected with the acute form of PE. The pathogenicity of each of these isolates was previously established in a porcine and an equine experimental model [175 cell culture flasks containing Dulbecco’s Modified Eagles Medium with 1% L-glutamine, 0.5% amphotericin B and 7% fetal bovine serum (FBS) were infected with 2 mL of L. intracellularis (with approximately 106 organisms). Infected flasks were placed in an anaerobic jar, from which the atmospheric air was evacuated by a vacuum pump to 500 mmHg and replaced with hydrogen gas. The infected flasks were then placed in a Tri-gas incubator with 83.2% nitrogen gas, 8.8% carbon dioxide and 8% oxygen gas and incubated with a temperature of 37°C for seven days [2PO4, 0.0072 M K2HPO4 and 0.0049 M potassium glutamate) solution with 10% FBS and stored at −80°C until the day of infection [L. intracellularis. For the horse trial, porcine and equine isolates were separately preserved in dry ice and shipped to the University of California-Davis Center for Equine Health. For both equine and porcine isolates the molecular identities were determined by multi-locus variable number tandem repeat (VNTR) analysis [The present study used a porcine (PHE/MN1-00) and an equine (E40504) al model ,12. Bothal model ,17. Brieven days . After anfection . The inoanalysis .L. intracellularis organisms was assessed by direct counting after immunoperoxidase staining of serial 10-fold dilutions prepared in sterile phosphate-buffered saline (PBS) using polyclonal L. intracellularis-specific antibody [The number of antibody . Additioantibody .n = 4); Horse/Equine isolate (n = 4) and Horse/Negative control (n = 4). Similarly, 18 Duroc-Landrace cross pigs at 3-weeks-of-age were allocated into three groups: Pig/Porcine isolate (n = 6); Pig/Equine isolate (n = 6) and Pig/Negative control (n = 6). The animals were obtained from a herd with no history of PE and each treatment group was housed in a different pen. Prior to study commencement, all animals were evaluated for any signs of illness by a full physical examination. In addition, blood and fecal samples from all animals were collected and tested for L. intracellularis-specific antibodies by immunoperoxidase monolayer assay (IPMA) and qPCR  in order to document the negative status for each animal. Throughout the study, the foals were housed at the University of California-Davis Center for Equine Health and fed with free choice of grass and alfalfa hay and water and were supplemented daily with a commercial foal supplement. The pigs were housed in the isolation barns at the College of Veterinary Medicine of the University of Minnesota and fed with non-medicated nursery feed and water ad libitum. Horse and pig pens were cleaned once daily. All procedures were approved by the Institutional Animal Care and Use Committees of the University of California (horse study) and University of Minnesota (pig study). The horse and pig studies were conducted in the summer of 2010 and 2011, respectively.Twelve Quarter Horse foals between 4 and 5 months of age were randomly divided into three groups: Horse/Porcine isolate (a (0.01 mg/kg BWT) and inoculated with 50 mL of the inoculum via nasogastric intubation [On the challenge day (day 0), the frozen inoculum was thawed at 37°C and administered within 1 h of thawing. The foals were sedated with detomidine hydrochloridetubation . The pigtubation . ControlMonitoring and sample collection were conducted similarly in the horse and pig studies. All animals were observed daily for general attitude and appetite for 56 days post-inoculation (pi). Once weekly, the weight of each animal was recorded in order to determine average daily weight gains throughout the study period. Feces were collected directly from the rectum every other day and submitted for qPCR . Blood sb  for 10 days. Additional supportive treatment including intravenous flunixine megluminec  and replacement fluidsd  were given based on the patient’s need.Due to the non-terminal design of the horse study, foals developing either moderate to severe acute  or chronic  signs and/or hypoproteinemia (<5.0 g/dL) were treated with doxycycline hyclateL. intracellularis DNA at the School of Veterinary Medicine of the University of California-Davis, as described elsewhere [e, according to the manufacturer’s recommendations. Absolute quantification was calculated using a standard curve for L. intracellularis and expressed as copy numbers of the aspA gene of L. intracellularis per gram of feces. The standard curve was determined by using 10-fold dilutions of L. intracellularis derived from cell culture in McCoy cells added to L. intracellularis-free equine feces. Furthermore, a qPCR assay targeting a universal sequence of the bacterial 16S rRNA gene was used as quality control (i.e. efficiency of DNA purification and amplification) and as an indicator of fecal inhibition [All fecal samples were analyzed by qPCR for lsewhere . Brieflyhibition .L. intracellularis specific IgG by IPMA at the College of Veterinary Medicine of the University of Minnesota, as previously reported [Blood samples for the collection of serum were drawn from all animals in order to determine the concentration of total solids using a refractometer and to measure anti-reported . PositivL. intracellularis[Two pigs from each group were euthanized on day 21 pi and evaluated for typical PE lesions. Intestinal samples from jejunum, ileum, cecum and colon were collected, fixed in 10% buffered formalin, processed routinely for histology, embedded in paraffin, and sectioned 5 μm thick. Two sections were prepared: one section was stained by haematoxylin and eosin  and the ellularis. The levellularis. HistoloL. intracellularis, were calculated using trapezoid rule in the SAS software (version 9.2). The Wilcoxon–Mann–Whitney test was used to compare the experimental groups based on AUC. Statistical significance was defined at values of p < 0.05.Descriptive analyses were used to describe clinical findings among the different groups due to the limited number of animals. Statistical analysis was performed by use of Wilcoxon–Mann–Whitney tests to assess differences in daily weight gain among the different groups. The area under curve (AUC) for the amount and duration of fecal shedding, as well as for the magnitude and duration of measureable IgG titers against L. intracellularis organisms , which was confirmed by qPCR, each animal received 109 bacteria intragastrically. In addition, the molecular identities of the porcine and equine isolates were confirmed after the experimental infection by VNTR typing using PCR positive samples.The challenge doses were standardized for the horse and pig trials in order to avoid any influence of dose effect on the clinical, pathological or immune response. Based on the quantification of p < 0.05) were observed in pigs infected with the porcine isolate and in foals infected with the equine isolate, as shown in Table Lawsonia-specific antigen identified by IHC  between foals infected with equine and porcine isolates. As a result, equine isolate-infected foals revealed higher amounts of bacterial DNA in the feces associated with longer duration of shedding compared with porcine isolate-infected foals.Results of fecal shedding of p < 0.05)  on day 2 pi. All foals and pigs in the negative control group remained negative for the entire study period.In the pig trial, animals infected with the porcine isolate showed higher and longer shedding of bacteria in the feces throughout the study period  Figure b. In theL. intracellularis in the serum compared with the porcine isolate-infected foals (≤1920)  between foals infected with equine and porcine isolates. However, because there was no serum from day 28 pi in the foal euthanized on day 21, the area under the curve for this animal was underestimated in the analysis. Simulating IgG titers of at least 960 on days 28 and 42 for this foal, significant difference (p < 0.03) would be observed in the time—titer curve of the IPMA between foals infected with porcine and equine isolates.All four equine isolate-infected foals demonstrated higher IgG titers ≥3840) against 840 againL. intracellularis from day 21 pi. The serological responses were persistent throughout the study in foals and pigs infected with species-specific isolates. However, foals infected with the equine isolate had much stronger immune responses (p = 0.057) than those pigs infected with the porcine isolate.The serologic responses in the pig experiment are summarized in Figure L. intracellularis used in the horse and pig experimental infections and support the hypothesis that host susceptibilities are driven by the origin of the isolated strain. Pigs and foals demonstrated a similar pattern of fecal shedding, which lasted until 39 days pi, but the bacterial DNA was identified earlier in pigs (day 2 pi) compared to foals (day 8 pi). In those animals infected with species-specific isolates, foals exhibited a more robust serological IgG response compared with pigs which may reflect a characteristic related to the host response, regardless of the L. intracellularis isolate. Additionally, hypoproteinemia was an important sign of PE in the affected horses. Despite the phenotypic differences regarding the susceptibilities of these two species, gross and histological lesions characterized by thickening of intestinal mucosa due to proliferation of enterocytes were identical in those animals infected with species-specific isolates.Marked clinical signs and pathological changes typical of PE associated with longer periods of bacterial shedding and stronger serologic immune responses were observed in pigs and foals infected with species-specific isolates. These observations were made despite the identical doses of L. intracellularis is a fastidious organism and extremely difficult to isolate and propagate, mucosal homogenate challenge models have been reported in pigs using intestinal mucosa from PE-affected pigs [L. intracellularis isolates have revealed inconsistent results. Smith et al. [-) mice infected with a porcine isolate. Go et al. [- mice but not in three wild-type mice strains . A more recent study showed mild histological lesions in four mice strains, including BALB/c and C57BL/6 [L. intracellularis isolates from different host species has limited the experimental infection studies to the use of porcine isolates. Recently, our research group isolated a new equine strain and its virulence was confirmed in a foal experimental model [L. intracellularis and to evaluate different host susceptibilities and adaptation to species-specific bacterial isolates.PE has been previously reproduced using pure cultures or intestinal mucosa homogenates derived from infected animals ,12,25. Sted pigs -28. Hamsted pigs ,29. Howeted pigs ,25. Studh et al.  describeo et al.  reported C57BL/6 . As descL. intracellularis infection, especially regarding inter-species transmissions, source or reservoir hosts of L. intracellularis and potential biological or mechanical vectors. Based on the pattern of bacterial shedding (Figure L. intracellularis DNA in the feces during a short period of time at the earlier stages of the experimental infection. Based on these findings and the uncommon opportunity of pigs and foals to share the same environment in the modern pig production system or horse breeding farms, any direct cross-species transmission in the field between these two species is unlikely.The challenge models previously described have contributed to advances in the prevention and control of PE by determining the efficacy of different antimicrobial drugs and vaccination protocols ,27,32. NL. intracellularis in the wild population with successive introductions into domestic pig or horse farms. Pusterla et al. [L. intracellularis in the pig production system has also been investigated. Using quantitative PCR, Collins et al. [5L. intracellularis/g of feces. However, these authors also reported that a small proportion of rats shed more than 108 bacteria per gram of feces. Associating these observations with the results from our study leads us to speculate on the presence of different sources or reservoir species for L. intracellularis infections in horses and pigs. In addition, the lack of evidence supporting inter-species transmission between horses and pigs helps confirm the host adaptation to species-specific isolates demonstrated in the present study.Free-living animals have been speculated to be a reservoir of PE by allowing the maintenance of a et al.  detecteds et al.  recentlyL. intracellularis isolates. Comparison of the 16S ribosomal DNA sequences showed a high degree of similarity among L. intracellularis from pigs, hamsters, deer and ostriches [L. intracellularis genome were used in the development of the VNTR technique [L. intracellularis isolates is crucial to identify any genomic variations associated with different host adaptations and susceptibilities.In order to support these hypotheses, the phenotypic observations described in the present study may be linked with genotypic differences between striches . Four seechnique . This teL. intracellularis isolate. Comparative genomic analysis is a promising route to associate phenotypic characteristics with potential genomic variations between porcine and equine isolates and may help to characterize species-specific L. intracellularis strains and potentially novel bacterial subspecies or genotypes.In conclusion, the present study demonstrated marked clinical signs, longer periods of bacterial shedding and stronger serologic immune responses in foals and pigs experimentally infected with their species-specific isolates. This evidence of host adaptation in these species suggests that host susceptibilities to PE are driven by the origin of the a Dormosedan, Pfizer Animal Health, Exton, Pa.b Doxycycline, Reva Pharmaceuticals, Sellersville, Pa. c Banamine, Schering-Plough Animal Health, Union, Nj.d Plasma-Lyte A, Baxter Healthcare Corporation, Deerfield, Il. e CAS-1820 X-tractor Gene, Corbett Life Science, Sydney, Australia.PE, Proliferative enteropathy; FBS, Fetal bovine serum; VNTR, Variable number tandem repeat; PBS, Phosphate-buffered saline; IPMA, Immunoperoxidase monolayer assay; pi, Post-inoculation; qPCR, Quantitative PCR; IHC, Immunohistochemistry; AUC, Area under curve; IFN-γ R-, IFN-γ receptor knockout.The authors declare they have no competing interests.Conceived and designed the experiments: FAV, NP, CG. Performed the experiments and analyzed the data: FAV, NP, SMM, CG. Wrote the paper: FAV, NP. Coordinated and helped to draft the manuscript: CG. All authors read and approved the final manuscript.


Some forms of tinnitus are likely to be perceptual consequences of altered neural activity in the central auditory system triggered by damage to the auditory periphery. Animal studies report changes in the evoked responses after noise exposure or ototoxic drugs in inferior colliculus and auditory cortex. However, human electrophysiological evidence is rather equivocal: increased, reduced or no difference in N1/N1m evoked amplitudes and latencies in tinnitus participants have been reported.The present study used magnetoencephalography to seek evidence for altered evoked responses in people with tinnitus compared to controls  in four different stimulus categories . Results revealed that amplitudes of the evoked responses differed depending on the tone category. N1m amplitude to the dominant tinnitus pitch and the frequency within the area of hearing loss were reduced compared to the other two categories. Given that tinnitus pitch is typically within the area of hearing loss, the differences in the evoked responses pattern in tinnitus participants seem to be related more to the hearing loss than to the presence of tinnitus. •We use MEG to compare evoked responses in participants with tinnitus and controls.•We compare N1m responses to control, edge, tinnitus and hearing loss tone.•N1m amplitudes to the tinnitus and hearing loss tones are reduced.•Differences in N1m amplitudes are related to hearing loss rather than tinnitus.•There are no tinnitus-specific changes in the pattern of N1m responses. While lSeveral mechanisms have been proposed to explain changes in the evoked responses associated with tinnitus. One view proposes an unmasking of the excitatory activity due to loss of lateral inhibition as a result of hearing loss . A seconWhatever the precise neural mechanism for tinnitus, the implication for human neuroimaging studies using electroencephalography (EEG) or magnetoencephalography (MEG) is that an increase in neural excitability probably elevates the amplitude of the sound-evoked response, and possibly also affects its latency. Moreover, we might expect differences in the amplitude and/or latency of the evoked responses to predominantly affect either the edge frequency of the hearing loss and/or a frequency corresponding to the dominant tinnitus pitch relative to a control tone that falls within the region of normal hearing. The N1 (EEG) or N1m (MEG) component of the auditory evoked response would seem relevant for studying tinnitus-related activity because it is a reliable cortical response that reflects stimulus properties such as frequency . The chaPrevious studies have investigated the N1/N1m response as a correlate of tinnitus, but these have yielded rather inconsistent results. A number of studies have predicted generalised hyperexcitability, testing this question by measuring activity evoked by a low-frequency tone (often 1 kHz) corresponding to the region of normal hearing thresholds. A prediction from the viewpoint of tonotopic reorganisation is that there should be an enhanced response to a frequency corresponding to the audiometric edge of a sloping high-frequency hearing loss. One MEG study found a significant increase of cortical strength values (dipole moments) for the audiometric edge frequency compared to lower frequencies in people with hearing loss, with seven out of eight of these also experiencing tinnitus . These aOther studies have sought evidence for enhanced responses corresponding to the tinnitus frequency, which is often within the region of hearing loss, by investigating intensity dependence of a tone corresponding to the dominant tinnitus pitch . They poIn addition to the N1m, some of the above MEG studies have also analysed the P2m response . Some ofper se, separate from hearing loss, we and others have previously argued that it is important to closely match tinnitus individuals and controls for hearing loss  is a somewhat inappropriate stimulation paradigm for directly addressing questions about neuroplasticity in the deafferented frequency region. To the best of our knowledge, no study to-date has assessed responses across frequency categories and yet such an experimental design could be highly informative. Second, some authors chose to compare their tinnitus group with normally hearing controls, while others used controls matched for hearing loss. Many studies do not fully report data on audiometric profile e.g.  or simploss e.g. . Indeed,oss e.g. . On the oss e.g.  that hasoss e.g. . Again, oss e.g. .In this study, we used MEG to investigate the amplitude and latency of evoked magnetic fields in response to a range of audible frequencies in participants with and without tinnitus. We tested the hypothesis that tinnitus-related activity enhancement is evident in the amplitude of the N1m response, even after accounting for explanations in terms of hearing loss and hyperacusis. We also investigated whether any such abnormal response patterns are frequency dependent. To separate the effect of reorganisation due to prolonged tinnitus from that due to sensorineural hearing loss, our study recruited two control groups; one with normal hearing and no tinnitus and the other with matched hearing loss, but without tinnitus.22.1For the MEG study we recruited 22 participants experiencing tinnitus  and 20 controls . Five had normal hearing defined as pure tone average (PTA) for the frequencies between 0.25 and 8 kHz≤20. However, four of these had some degree of high-frequency hearing loss above 8 kHz. Three had an asymmetric hearing loss defined as a pure-tone average  difference of ≥15 dB (The Food and Drug Administration guidelines). One of the participants had low-frequency hearing loss with normal thresholds from 4 kHz.Of the 22 people with tinnitus, most had a high-frequency hearing loss  > 20; mean age 64 years 7 months), while the other comprised 13 people with normal hearing ; where normal hearing was defined as PTA(0.25–8 kHz)≤20. For the individual hearing profiles see 2.2Auditory stimuli were 300-ms tone bursts presented at 40 dB SL to adjust for the degree of individual hearing loss at each particular frequency. Frequency conditions were defined according to the individual audiogram for each ear and tinnitus spectrum for the prevailing tinnitus sound. Conditions were: (i) a ‘control’ tone in the region of normal hearing, (ii) a tone corresponding to the individual audiometric edge frequency, (iii) a tone corresponding to the dominant tinnitus pitch, and (iv) a tone within the area of hearing loss. The audiometric edge was determined objectively using the ‘broken stick’ procedure performed for log frequency, as previously described in N = 3) or dominant tinnitus pitch (N = 2). In those cases, the chosen control tone was instead a 0.5 kHz tone. For the one participant with low-frequency hearing loss, an 8-kHz tone, which was within normal hearing range, was selected as a control tone. For ten tinnitus participants, their hearing loss was sufficiently severe that the tinnitus tone could not be presented at 40 dB SL within the output capabilities of the sound delivery system. In those cases, an alternative tone was selected that was near to the tinnitus tone and within the region of hearing loss, but could be presented at 40 dB SL. That frequency was categorised separately as (iv) a hearing loss tone (mean = 4.6 kHz). For two individuals with asymmetric hearing, tones in condition (iv) were presented to the worse ear at maximum output. However, the corresponding evoked responses were not included in the analysis because these tones did not reach 40 dB SL. For the tinnitus group, the geometric mean of frequency of the selected tones was as follows: (i) control = 1.2 kHz, (ii) edge = 3.1 kHz, (iii) tinnitus pitch = 3.1 kHz. It was not possible in all cases to easily determine tones in those conditions. For some tinnitus participants, 1 kHz corresponded to the audiometric edge (Condition (iii) corresponding to the tinnitus pitch was not applicable for control groups. For the hearing-loss controls, the geometric mean of the frequencies of the selected tones was as follows: (i) control = 1.1 kHz, (ii) edge = 2.4 kHz, and (iv) hearing loss = 4.8 kHz. Normal-hearing controls were presented with tones corresponding to 1.0, 4.0 and 8.0 kHz and for the purpose of analysis, these were all considered to be control tones. Geometric means of the frequencies presented in the MEG across the three groups of participants are shown in Tone stimuli were generated in advance and presented using Presentation software  and attenuated using TDT PA5 attenuators . The maximum sound level output for any tone frequency was limited to 100 dB SPL. Tones were delivered to left and right ears using Etymotic transducers (Etymotic Research – ER2) inside the magnetically shielded room. To protect the MEG sensors from potential magnetic interference due to the proximity of the transducers to the MEG helmet, each transducer was placed in specially designed mu-metal casing for shielding.2.3MEG data were recorded with participants in a supine position and in a magnetically shielded room using a whole cortex 275-channel CTF MEG system  equipped with 3rd order gradiometer sensor configuration. A sampling rate of 600 Hz was used, applying a 150-Hz low-pass anti-aliasing hardware filter. The MEG data was collected in trials of 1 s duration which included a 100 ms prestimulus baseline. Each tone condition was repeated 120 times delivered in a random sequence to each ear with a varying inter-stimulus interval between 1.5 and 3.0 s. Participants were instructed to listen attentively without performing any task. To minimise contamination of the evoked potentials with alpha rhythm, subjects were asked to keep their eyes opened and focus on a set point. The experiment lasted about 25 min.During data acquisition, the position of the participants' head inside the scanner was monitored by placing three coils at the nasion and the left and right pre-auricular fiducial points. A head motion tolerance of 5 mm was applied; none of the datasets violated this limitation. Following the data collection, the position of the coils and the head shape on the scalp surface were recorded using a 3D digitiser .The dataset was mean corrected and a 3rd order synthetic gradiometer was applied. Trials with artefacts were excluded from the analysis following visual inspection. The data was then filtered in the 1–20 Hz band and separately averaged for each tone condition to determine contra- and ipsi-lateral N1m responses in each participant. The N1m response was identified by visual inspection as a dipole pattern distributed over the temporal region with a peak in the time window between about 80 and 190 m after sound onset. Ten channels with the largest amplitude responses (5 sinks and 5 sources) in each hemisphere were identified and chosen for further analysis. For each hemisphere, the root mean square (RMS) of the field strength was calculated for each sample timepoint, averaged across the chosen channels see . The pea3A linear mixed effects analysis of variance (ANOVA) was performed using PASW Statistics 17 which enabled within- and between-subject comparisons. This was performed separately for both peak N1m amplitude and latency. We tested the main effect of group , tone category , laterality  and ear of presentation . In order to adjust for frequency differences between participants within each tone category log frequency was included in the analysis as a linear covariate. The logarithmic transformation was applied to allow for the non-linear relationship between frequency and the response. We also tested all interactions of main variables and covariate with group  as well as tone category x laterality.p = 0.011 and F = 7.69, p = 0.006, respectively). However, the data plotted in p = 0.12), indicating that both tinnitus participants and controls showed an equivalent pattern of frequency dependence. Although latency for hearing-loss controls decreased as a function of frequency more rapidly than the other two groups , absolute changes in latency were very small.For the covariate, N1m amplitude and latency significantly decreased as a function of frequency . Overall, the responses to the frequency corresponding to the dominant tinnitus pitch were smaller than for control and edge frequencies (p = 0.013) , nor group on N1m latency . We infer that the overall pattern of N1m responses for the tinnitus group did not differ from the controls as there was no interaction between group and tone category for amplitude  and latency . In general, we therefore found no evidence of tinnitus-specific changes in the pattern of N1m responses.Having accounted for the effect of frequency in the model, our results indicate a significant residual effect of tone category on N1m amplitude  . Howeverp = 0.001), with shorter latency . This pattern was found regardless of the group, tone category and ear of presentation. Contralateral dominance reflects the cross-over organisation of the ascending auditory pathway and our result concurs with previous human neuroimaging studies in this regard , but N1m latency was significantly shorter when stimuli were presented to the left than to the right ear . As in the case for frequency, the magnitude of the latency difference across ears was so small as to be not functionally meaningful (3.4 ms).As for the ear of presentation, there was no effect on amplitude . However, similarly to the results of the main analysis we found significant effects of tone category on amplitudes of N1m, where amplitudes for the tinnitus pitch were lower than for control (p = 0.031) and edge frequency (p = 0.015) and similar to the hearing loss frequency (p = 0.28).To further discriminate the effects of hearing loss from the effects of tinnitus on N1m amplitudes we compared tinnitus participants with hearing loss with controls with hearing loss. Mixed effects ANOVA testing all the effects assessed in the main analysis was performed. There was no difference between amplitudes of N1m between tinnitus with hearing loss participants and controls with hearing loss  nor between tinnitus and non-tinnitus ear . Similar to the main analysis, we found a significant effect of tone category  and frequency  on N1m amplitudes.To further explore the effects of tinnitus laterality  and ear (tinnitus vs non-tinnitus ear) on amplitudes of N1m responses additional linear mixed effects ANOVA was performed. We tested the main effect of group , tone category , laterality  and ear of presentation  with log frequency included in the analysis as a linear covariate. We did not find differences in amplitudes of N1m responses between unilateral and bilateral tinnitus patients  in tinnitus participants and to compare them with two non-tinnitus control groups; normal hearing subjects and with subjects with hearing loss. In general, we predicted tinnitus-related differences most likely in the N1m amplitude for tone frequencies corresponding to the dominant tinnitus pitch and/or the audiometric edge frequencies. Our results have shown that amplitudes of N1m response indeed depended on the tone category, even after controlling for frequency, but crucially there was no difference in this pattern across tinnitus and control groups. In general, the N1m for the dominant tinnitus pitch was significantly smaller than for the control and audiometric edge frequencies and was similar to the tone within the hearing-loss region. Therefore we postulate that differences in evoked responses pattern across tone category seem more related to the hearing loss than to the presence of tinnitus.4.1In agreement with the previous literature we found more pronounced frequency dependent changes for N1m amplitude than for latency . The amp4.2As reviewed in the introduction, many of the current models of tinnitus generation attribute the tinnitus sensation to altered neuronal activity within the central auditory system triggered by damage to the auditory periphery. Depending on the precise details of the proposed neural mechanism, we might expect differences in the amplitude and/or latency of the evoked responses for tones corresponding to the edge frequency of the hearing loss and/or to the dominant tinnitus pitch compared to a control tone, and for this to be different in the tinnitus group compared to controls.Enhancement of the N1m amplitude for a tone corresponding to the audiometric edge frequency might be directly predicted by those models which consider tinnitus to be the consequence of a shift in the frequency tuning properties of neurons above the audiometric edge towards those of lower frequencies . SimilarEnhancement of the N1m amplitude for a tone corresponding to the area of hearing loss would be consistent with the view that tinnitus is generated by synchronous spontaneous activity which develops among hyperactive neurons in the deafferented region . Again, No change in the N1m amplitude would be consistent with homeostatic plasticity models that postulate tinnitus to be a side effect of plasticity mechanisms that normally ensure proper function of the auditory brain through an elevation of central spontaneous activity in the frequency range that is affected by hearing loss , 2009. AAmong our tinnitus group, we had five patients with normal hearing thresholds and it may be argued that these should form a separate experimental group. However, four of these had various degrees of hearing loss for frequencies above 8 kHz. In the context of tinnitus, these cannot be considered as normally hearing. This is because at least in theory, hearing loss at any frequency can be related to tinnitus. Moreover, recently The fact that the frequency corresponding to the dominant tinnitus pitch typically lies within the area of hearing loss  is a maj5In line with recommendations in the literature, the current study controlled for comorbid symptoms such as hyperacusis and compared tinnitus participants to a hearing loss matched control group. We did not find any tinnitus specific changes in the pattern of N1m responses. The reduction of amplitudes of N1m for the tinnitus frequency and lack of difference between amplitudes for tinnitus frequency in comparison to the frequency within the area of hearing loss suggests that the differences in the evoked responses pattern in tinnitus participants point to the hearing loss rather than tinnitus itself as a causal factor.


Cochlear implantation (CI) has become the method of choice for the treatment of patients with bilateral deafness. With the development of technology and matured experience, candidacy is changing and becoming broader. Decision makers in charge of public health are faced with the decision for whom to include cochlear implants in the basic medical package. These decisions are based not only on the effectiveness of certain healthcare interventions but also on the costs that are involved. The first study systematically reviews the relevant studies on costs relating to cochlear implantation, analyzing the different methods used.The second paper is an extensive literature review on the changing candidacy. Topics like cochlear implantation in very young children, cochlear implantation and hearing preservation, and cochlear implantation for unilateral deafness and tinnitus are discussed.Since the introduction of universal newborn hearing screening, the vast majority of infants are screened for hearing loss within the first few days of life. Consequently, more patients are being detected with hearing loss early in life, and more paediatric patients are getting CIs in the course of their hearing rehabilitation. However, even with universal newborn hearing screening, still a number of deaf newborns are missed. As a result, some deaf infants receive late intervention and experience devastating language delays in which they spend a lifetime trying to catch up. The purpose of the third paper is to cross disciplines and to extend the understanding of the medical and audiological profession in seeing the language and literacy challenges deaf implanted children face whether they are early or late implanted.Besides late implantation, other challenges, limitations, and potential risks remain in the treatment of deaf children with CI. Meningitis is still leading major causes of acquired severe sensorineural hearing loss. Labyrinthitis ossificans can lead to complete obliteration of the cochlea with limited possibility of cochlear implantation, consequently inducing poor auditory results. The 4th paper describes the audiological, anesthesiological and surgical key points of cochlear implantation after bacterial meningitis in very young infants.With the increasing number of implant users worldwide, there is a growing need for assessing the overall survival of the various devices used and the safety and efficacy of reimplantation procedures. In paper 5 the authors investigate the effects of a perimodiolarly placed CI electrode on the cochlear microstructure. They also look at the possible damage to the modiolar wall at a microstructural level when pulling out an electrode. Based on their observations, they formulate some suggestions for a safe pull-out procedure.The last paper highlights several areas where challenges remain for implant clinics, parents, and educators. While making clear the benefits of CI and its important role in extending opportunities for profoundly deaf children, challenges remain most notably in the areas of children's academic achievement and social development and participation with hearing peers. Ongoing attempts to address these challenges are essential if children with CI are to be fully supported to reach their potential personally, educationally, and socially.Ingeborg DhoogeIngeborg DhoogeCraig BuchmanCraig BuchmanLevent SennarogluLevent SennarogluEmmanuel MylanusEmmanuel MylanusFrançoise DenoyelleFrançoise Denoyelle


Hearing capability plays a principal role on human's communication. Noise-induced hearing loss (NIHL) caused by exposure to high noise levels is a serious socio-economic problem in modern societies. NIHL can either be reversible, resulting in a temporary threshold shifts (TTS) or irreversible, resulting in a permanent threshold shifts (PTS). PTS is often confirmed in the time span of between 2 - 6 weeks. NIHL may be prevented by avoidance of excessive amounts of noise or reducing the sound energy entering the inner ear using hearing protective devices. However, there are some conditions that such prevention is not possible such as noise exceeding the protective capabilities of the hearing protection device, working in military or the person does not tolerate the protection device. Thus the protective agent for preventing NIHL would be useful.Free radical molecules and consequence oxidative stress have been shown to play a significant role in noise-induced hearing loss. Silymarin is an antioxidant flavonoid complex derived from the herb milk thistle has ability to mitigating the oxidative stress, scavenge free radicals. In the current study, we aimed to evaluate the protective effect of silymarin on noise induced hearing loss in guinea pig by auditory brain stem response.Twenty guinea pigs randomly divided into 2 groups. The animals in the experimental group were intraperitoneally injected with 100 mg/kg/day silymarin dissolved in propylene glycol for 6 consecutive days. The control subjects were intraperitoneally injected with propylene glycol for 6 consecutive days. All animals were exposed to 4 kHz octave band noise at 120 dB SPL for 6 hours. Auditory brainstem responses (ABRs) at frequencies of 2, 4, 6, 8, 12, 16 and 20 kHz were precisely recorded before intervention and then on intervals of 0, 3, 10 and 15 days after noise exposure. Data were analyzed using repeated measures ANOVA.Threshold shifts for the experimental group at all frequencies immediately, 3, 10 and 15 days after noise exposure were significantly reduced compared to the control group (P < 0.01).The findings indicate a protective effect of silymarin on temporary and permanent noise-induced hearing loss. Hearing capability plays a principal role on human's communication. Noise-induced hearing loss (NIHL) caused by exposure to high noise levels is a serious socio-economic problem in modern societies. NIHL can either be reversible, resulting in a temporary threshold shifts (TTS) or irreversible, resulting in a permanent threshold shifts (PTS). PTS is often confirmed in the time span of between 2 - 6 weeks , 2. NIHLSince oxidative stress is involved in the mechanism of NIHL, and in accordance with latter reports that silymarin has antioxidant properties, the aim of this study was to evaluate the protective capacity of silymarin on noise induced hearing loss in guinea pig by auditory brain stem response.Free radical molecules and consequence oxidative stress have been shown to play a significant role in noise-induced hearing loss. Silymarin is an antioxidant flavonoid complex derived from the herb milk thistle has ability to mitigating the oxidative stress, scavenge free radicals. In the current study, we aimed to evaluate the protective effect of silymarin on noise induced hearing loss in guinea pig by auditory brain stem response.Twenty male six weeks old albino guinea pigs (290 ± 10g) were prepared from Pasteur Institute . Since sex differences have been associated with a deferring ability to detoxify ROS, only male guinea pigs were used . The subAfter baseline ABR threshold measurements animals were randomly divided into two groups . In the experimental group, subjects were i.p. injected for 6 consecutive days with silymarin dissolved in propylene glycol at a dose of 100 mg/kg/day. In the control group, the animals were i.p. injected for six consecutive days with propylene glycol. One hour after the final intervention each animal was separately exposed for six hours to 4 kHz octave band noise at 120 dB SPL. Noise exposure was carried out in a lighted and ventilated anechoic chamber while animals having free access to food and water. The chamber was ﬁtted with speakers driven by a noise generator and a power ampliﬁer. Sound levels were measured and calibrated at the location of animal cage by a B&K 2231 sound level meter. Immediately, 3, 10 and 15 days after noise exposure, ABR thresholds were repeated. Sample of ABR thresholds in guinea pig were shown in ABR thresholds were analyzed by repeated measures ANOVA in both groups at all test frequency in 5 days . Statistical comparisons were made using the SPSS 17 software package and P < 0.01 was statistically considered significant.Baseline ABR thresholds at 2, 4, 6, 8, 12, 16 and 20 kHz were observed as displayed on Hearing loss was observed immediately after noise exposure in all subjects. As shown in In two groups recovery of threshold shifts were occurred over time; however, the degree of recovery differed among the groups. Fifteen days after noise exposure, ABR thresholds shifts were ranging from 16.5 dB at 2 kHz to 41 dB at 8 kHz in control group. In experimental group, the minimum and maximum threshold shifts were 5 dB at 20 kHz and 30 dB at 8 kHz, respectively. Data indicated that silymarin had reduced the PTS up to 19 dB compared to control group. Hearing loss in experimental group was less than control group 15 days after noise exposure. These differences were statistically significant (P = 0.00).In this study, guinea pigs were exposed to 4 kHz octave band noise at 120 dB SPL for six hours to evaluate the protective capacity of silymarin on TTS and PTS. The results indicated that this antioxidant agent could effectively attenuate the temporary and permanent noise-induced ABR threshold shift in guinea pigs. The ABR thresholds showed that silymarin reduced the TTS up to 32 dB and PTS up to 19 dB compared to control group. When we observed the ABR threshold shifts at different time scales, we concluded that the recovery was greater when silymarin pretreatment implemented. Therefore, data from current study indicated that silymarin is capable to protect the cochlea from noise-induced hearing loss. The cochlear hair cell damage due to ROS and/or RNS production following loud sound exposure has already been demonstrated . It is a


Military personnel are usually exposed to high levels of impulse noise (IN) which can lead to hearing loss.The purpose of this study was to assess the effects of relatively low level exposure of impulse noise (IN) during shooting practice on hearing using pure tone audiometry (PTA) and transiently evoked otoacoustic emission (TEOAE) in military personnel.Forty male soldiers (mean age 20.08 years) were recruited for the study. Prior to their first shooting practice, PTA and TEOAE were recorded. After 15 minutes and one week post- practice PTA and TEOAE were compared.Immediately after shooting practice significant differences in PTA at 500, 1000, and 4000 Hz were observed for the right ear and no significant difference at any frequency for the left ear. There was a significant difference in the amplitude of TEOAE 15 minutes after shooting practice at 500, 1000, 2000, 3000, and 4000 Hz in the right ear, while for the left ear the difference was significant at 1000 and 2000 Hz. One week after exposure a significant difference at 500 and 4000 Hz was found only in the right ear and a significant difference in the amplitude of TEOAE was observed at 500, 1000, 2000, 3000, and 4000 Hz.Even exposure lower than permissible levels may lead to acoustic trauma. TEOAE is more sensitive than PTA in detecting early hearing loss after military shooting exercises. Hearing protection equipment and appropriate surveillance programs are recommended. Impulse noise (IN) is a transient noise which consists of one or more bursts of sound energy. Military personnel are usually exposed to high levels of IN and can suffer from a wide range of IN effects on hearing including acoustic trauma -5. It haSince military environments expose army personnel to very high levels of IN -13, it iThe aim of this study was to evaluate the effect of low level IN produced by gunfire in 40 military personnel after their first firing practice and to compare the sensitivity of TEOAE and pure tone audiometry in detection of TTS and PTS following firing practice.Forty male soldiers (mean age 20.08 years) were recruited for this study to be evaluated following their first firing practice. Those with any current or past history of hearing problems, history of systemic diseases, history of using ototoxic drugs, previous excessive exposure to noise, ototoxic chemicals or abnormal hearing results (hearing threshold more than 20 dB) were not included in the study. The Institutional Review Board of Tehran University of Medical Sciences approved the protocol of the study. The training exercise consisted of one round of 10 single shots and one round of 10 continuous shots using a Kalashnikov rifle in recumbent position. The soldiers did not use hearing protectors during training. All were right-handed. The C-weighted peak sound pressure level (SPL) (LCs) and an equivalent continuous A-weighted SPL (LAs ) during shooting were determined using a real-time frequency analyzer (type 2131 B&K with expansion unit type 5765 B&K). Frequency analysis was also performed for 500, 1000, 2000, 4000, and 8000 Hz frequencies.Before firing, 15 minutes post-firing, and one week after the practice all subjects completed a questionnaire and underwent otoscopic assessment and PTA as well as TEOAE were recorded. PTA was recorded in an acoustic cabin using a Danflex DA65 audiometer at the range of frequency from 0.5 to 8 kHz. Otoacoustic emissions were recorded using an ILO 292 Echoport version 5.0 (Otodynamics Ltd). TEOAE recordings were collected for every subject at stimuli levels of 80 ± 2 dB SPL using 80 ms duration clicks in a nonlinear pattern. An artifact rejection level of 4.6 mPa (47.3 dB SPL) was used throughout the recording session. Each response was windowed from 2.5 to 20 ms post-stimulus and band pass filtered from 0.5 to 6 kHz. Values of TEOAE and signal to noise ratio (SNR) were examined in 1 kHz bands from 1 to 5 kHz. Only emissions with an amplitude of at least two standard deviations above the noise were accented as a true OAE. A threshold shift which was detected 15 minutes after exposure and reversed after one week was considered as TTS; a threshold shift which did not recover at one-week postexposure measurements was considered PTS. The effect of exposure to the impulse noise was evaluated using t-Student test comparing pre-exposure measurements to 15-minute and one-week post-exposure measurements for PTA, TEOAE, and SNR.Im dB SPL was between 105 and 113 (mean 108.3) for the right ear and between 100 and 110 (mean 105.7) for the left ear. The impulse C-weighted sound level LCIm dB SPL was between 104 and 112 (mean 107.6) for the right ear and between 102 and 109 (mean 105.9) for the left one after the single-shot round. The broadband noise analysis for the single-shot round showed that the highest noise levels were found in the frequencies 2 KHz and 4 KHz near the left and the right ear  with a mean ± SD age of 20.08 ± 2.61 were studied. All 40 were right-handed and held the weapon with their dominant arm. The mean duration of exposure to gunshot noise was 190 seconds. The noise level for single-shot round for the impulse A-weighted sound level LAight ear . For conight ear .P < 0.001), 1000 Hz (P = 0.04), and 4000 Hz (P = 0.029)in the right ear. In the left ear, however, there was no significant difference at any frequency. Whole wave reproducibility (WWR) of the right ear and WWR of the left ear 15 minutes after exposure were significantly different from before exposure. There was a significant difference in the amplitude of TEOAE 15 minutes after shooting at 500, 1000, 2000, 3000, and 4000 Hz in the right ear with a mean change of 2.5 dB SPL at 500 Hz (P <0.02), 2.4 dB SPL at 1000 Hz (P <0.02), 2.6 dB SPL at 2000 Hz (P <0.03), 3.1 dB SPL at 3000 Hz (P <0.03) and 5.1 dB SPL at 4000 Hz (P <0.01). Decrease in TEOAE amplitude 15 minutes after exposure was significant at 1000 Hz  and 2000 Hz  for the left ear  had abnormal PTAs. Comparison between PTA results in different frequencies before and 15 minutes after exposure  showed tleft ear .P < 0.05) in the right ear, however significant difference at any frequency was not observed in the left ear. WWR of the right ear one week after exposure was significantly different from before exposure (P =0.023); however, the difference was not significant for the left ear. There was a significant difference in the amplitude of TEOAE one week after shooting at 500, 1000, 2000, 3000, and 4000 Hz in the right ear with a mean change of 2.1 dB SPL at 500 Hz (P < 0.03), 2.2 dB SPL at 1000 Hz (P < 0.02), 2.4 dB SPL at 2000 Hz (P < 0.03), 2.9 dB SPL at 3000 Hz (P < 0.03) and 4.9 dB SPL at 4000 Hz (P < 0.01) both for the whole response as well as for 1/2-octave band responses. Decrease in TEOAE amplitude for the left ear at one week was present at 1000 Hz (1.3 dB SPL) and 2000 Hz (0.4 dB SPL) but as mentioned above these differences were not statistically significant (P > 0.05)  had abnormal PTAs. Comparison between PTA results in different frequencies before and one week after exposure  showed t > 0.05) .Immediately after exposure, four acquired clinical deafness of the right ear and two acquired clinical deafness of both ears. After one week, 4 had clinical hearing loss of the right ear and 1 had clinical hearing loss of the both ears. After shooting, extra-auditory symptoms included tinnitus (25 subjects), dizziness (16 subjects), discomfort when exposed to loud sounds (29 subjects) and problems in speech discrimination (30 subjects). In one subject, rupture of the tympanic membrane was evident with a 30-dB decrease in all frequencies at audiometry. Three participants had earache, inflammation and limitation of tympanic membrane movement and were treated. However, examination of the pharynx and tonsils was normal. Of the aforementioned three subjects, one had clinical hearing loss. These problems completely resolved after one week.Our study noted that exposure to lower levels of IN can disturb cochlear function in which TEOAE is more sensitive than PTA. Also indices of TEOAE demonstrated other than 3000-6000 Hz, low frequency hearing spectrum (500-2000 Hz) may be involved. Peak level of impulse noise varies according to different standards. For example NATO has notified a value limit of 160 dB for military noises . The Occet al. (et al.(et al.(The small-caliber weapons have been considered to produce a peak level of 132-165 dB with a frequency spectrum between 150 to 2500 Hz -24. Even. (et al.. They stl.(et al. noted thl.(et al.. Neverthl.(et al.. In our et al. (et al. (Typically, in audiogram the first sign due to occupational noise exposure is change at 3,000, 4,000, or 6,000 Hz, with recovery at 8,000 Hz . In the et al. , althouget al. . Involveet al. , but int (et al. . Who sta25. The most common clinical finding after exposure to IN is tinnitus. Current consensus is that the functional integrity of outer hair cells and nerve fibers is disrupted (In this study, temporary threshold shift was evident among the subjects and permanent threshold shift was documented in the right ear. However, no significant difference existed in the number of subjects with clinical hearing loss before, immediately after and one week after practice which represents the necessity of occupational hearing surveillance by means of audiologic tests. Furthermore all of our subjects were right-handed and their right ears were positioned closer to the source of noise during shooting. This may explain the greater changes of hearing results in the right ears. In other studies involvement of right ear is more commonisrupted , 9. Howeisrupted . In our isrupted  stated tisrupted , 37.According to our findings exposure to IN produced by small caliber types of guns is clinically important and personal protection and occupational health surveillance should be considered for all military personnel who fire guns Also assessment of hearing should be performed at the end of the training course for better evaluation of permanent changes. We also recommend that hearing assessment be performed for the frequencies that we did not assess in this study and particularly for higher frequencies, as reduced TEOAE levels in these frequencies may be the earliest sign of hearing loss , 39. The