Beauveria bassiana is an entomopathogenic fungus that grows both in vivo and in vitro. In vivo it can colonize live insect hosts, and tissue digestion occurs by secreted hydrolytic exoenzymes. It can also colonize dead insect tissue provided this is free from competing microorganisms. Depending on whether the host is alive or dead the expression  of the exoenzymes may vary. We have grown several isolates of B. bassiana in shaking flasks for 120 h at 25 °C in order to evaluate the maximal exoenzyme production using two diet regimes. As sole carbon, nitrogen, and phosphate sources we used 1% shrimp chitin and either 0.5% w/v of dead intact American cockroach (Periplaneta americana) or their isolated cuticles. This is the first report of a differential proteomics of B. bassiana exoenzymes performed by label-free nano-LC MS/MS. Total proteolytic enzyme activity was mainly due to Pr1A or Pr1B depending on the isolate and the diet regime. The most differentially secreted enzymes were: the cuticle-degrading subtilisin Pr1A, GH13 alpha-glycosidase, glucan endo-1,3-beta-glucosidase, subtilisin-like proteinase Spm1, lipase 1, beta-1,3 exoglucanase, and endo-1,3-beta-glucosidase. Among the B. bassiana isolates analyzed, Bb 678 and Bb BG were the most active in Pr1A secretion. Beauveria bassiana  is a facultative saprophytic fungus with capacity to infect and kill insects and other arthropods and also to colonize a range of plant species as endophytes [Periplaneta americana) [N-acetyl-β-glucosaminidases) from entomopathogens showed no detectable activity against insect cuticle either alone or in combination with protease or other chitin-degrading enzymes  [B. bassiana exoenzymes is sequential as previously established by macroscopic observation of the cuticle [B. bassiana isolates in order to establish qualitative and quantitative aspects of secreted hydrolases, including proteases, chitinases, lipases, and phosphates a.o. during aerobic liquid growth using intact cockroaches versus isolated cuticles as sole N/C/P sources. By increasing the information on the secretome of B. bassiana, the use of this fungus as a control agent against insect pests such as P. americana can potentially be supplemented by B. bassiana culture supernatant coming from aerobic fermentation or by using some of the listed exoenzymes discovered here as potential recombinant candidates for a fungus-free control approach, where appropriate. In addition, other uses of B. bassiana exoenzymes can be envisioned, for example, in various biotechnological applications such as recombinant lipases to be used in biodiesel production [dophytes . Insectsdophytes ,3,4,5. Edophytes . Proteinericana) . Most enericana) . Fungal s, etc.) . The firs, etc.) ,10, mean cuticle . To date cuticle ,13,14. Hoduction .Metarhizium sp., Cordyceps militaris, Ophiocordyceps sinensis, and Beauveria sp. for OMICS studies [About 200 species of entomopathogenic fungi from a range of genera have been described and many species are effective biocontrol agents . At pres studies ,18.B. bassiana isolates. This has potential as a tool for identifying important exoenzymes which maybe at a later stage can be introduced as recombinant into other B. bassiana isolates in order to improve their performance or produced in vitro as recombinant  in order to be used as a fungus-free bio-insecticide mixture.Here we aim at using a novel and up-to-date approach to study the proteomics of exoenzymes from B. bassiana isolates were used: Bb 715, isolated from Stomoxys calcitrans (Diptera: Muscidae); Bb 678, isolated from Musca domestica (Diptera: Muscidae); Bb BG, re-isolated from the commercial product BotaniGard 22 WP® , Butte, MT, USA), originally isolated from Diabrotica undecimpunctata (Coleoptera: Chrysomelidae); Bb 813, isolated from Varroa destructor (Acari: Varroidae); Bb 949, isolated from V. destructor; and Bb 893, isolated from V. destructor. All isolates except Bb BG originated in Denmark and were kept stored in glycerol at −80 °C .Six B. bassiana was based both on microscopic features and molecular identification using PCR on fungal genomic DNA extracted by the CTAB method [Assignment to B method , targetiP. americana, from an insect rearing) or isolated cuticle diet from crushed cockroaches. Isolated cuticles were prepared by crushing dead cockroaches with mortar and pestle in MilliQ water and filtering the homogenate through three layers of Miracloth . The filtrate was centrifuged at 4000× g for 10 min and the pellet was washed twice with distilled water and centrifuged again as described above. Subsequently, the liquid media containing either 1% shrimp chitin with 0.5% w/v intact cockroaches or 0.5% w/v of isolated cuticle were sterilized by autoclaving. Fungal isolates were subcultured on 1% shrimp chitin agar  before growth in liquid media see . contain8 spores/mL was added to 250 mL Erlenmeyer baffled flasks containing 50 mL of sterilized shrimp chitin/cockroach medium: 1% shrimp chitin, 0.5% w/v cockroach-isolated cuticle or 1% shrimp chitin, and 0.5% w/v intact cockroach as described above. Shaking was performed at 25 °C on a rotary shaker at 200 rpm for six days. One milliliter of media per sample was collected and the supernatant obtained by centrifugation every 24 h during six days, and the final mycelia wet weight recorded. The supernatant was filter sterilized through a 0.2 µm syringe filter (Sartorius) and used for enzyme assay or concentrated 10 times by Vivaspin 15 (Sartorius) centrifugal concentrators . The fungus did not grow much more rapidly in the media containing isolated cuticles as compared with the intact insect diet. In both cases the wet weight at the fourth day was comparable (1.2 ± 0.15 g/50 mL media). Considering that the initial pellet containing chitin and cuticle for 50 mL media used was 0.9 ± 0.12 g/50 mL media, the net wet weight recorded at day four was around 0.3 ± 0.14 g/50 mL media. All experiments were performed in triplicate.Fungal isolates were sub-cultured on 1% shrimp chitin agar at 25 °C for six days. Conidia were harvested by adding an aqueous solution of 0.01% Tween 80 and filtering through sterile absorbent cotton. One mL of spore suspension containing a concentration of 3 × 10N-Phenylanaline-Arginine-p-nitro-anilide) and Suc-Ala-Ala-Pro-Phe-pNA . The assay consisted of incubating 10 µL of culture supernatant for 5 min at 37 °C in the presence of 0.1 mM of the chromogenic synthetic peptides as substrate in 0.4 mL of 0.1 M Tris-HCl pH 8.0. The reaction was terminated by adding 0.2 mL of 1.5 M HCl followed by diazotization by 0.1 mL of 0.1% NaNO2, 0.1 mL of 1% ammonium-sulfamate, and 0.1 mL of 0.1% of NNED (N-(1-Naphthyl)ethylenediamine dihydrochloride), respectively [−1·min−1) were calculated by dividing enzymes units (U·mL−1·min−1) to their protein concentrations (mg/mL). Assays were performed in triplicate and standard deviation calculated. We focused on protease activity that was assayed using two chromogenic substrates (from Bachem) for trypsin and chymotrypsin-like activities: Z-Phe-Arg-pNA  at 56 °C for 30 min. Cysteine alkylation was performed by adding 30 μL of 0.5 M iodoacetamide in 0.1 M ABC buffer and vortexed briefly before incubating for 45 min at room temperature (RT) in the dark. Desalting of alkylated proteins was performed using Vivaspin 500 (MWCO 3000) and the buffer was exchanged as well into 0.1 M ABC buffer.Supernatants from day four of liquid cultures of iana see  were conHundred μL of concentrated and alkylated proteins were digested with 1 μg of proteomics grade trypsin . The digestion was performed at 37 °C overnight and then stopped by adding 2 μL of 50% formic acid (FA). To the undesalted peptides 100 fmol/mL final concentration of tryptic digest yeast Enolase 1 was spiked in. Sample preparation was performed in triplicate.Per sample, 4 µL of desalted peptides were analyzed also in triplicate onto an Xbridge BEH130 C18 5 μm desalting/trap column on-line with a BEH300 C18 1.7 μm nanoUPLC analytical capillary column (100 μm × 100 mm) on an Acquity nanoUPLC-LC system interfaced with a nano source to a Q-TOF Premiere MS . The entire length of the LC run was 66 min starting with a gradient of ACN/0.1 FA (0 to 40%) from 0 to 50 min followed by 90% ACN wash and re-equilibration. Data acquisition was performed in V positive mode with Glu-Fib as calibrant (m/z 785.8426) and lock mass. MS and MS/MS data were recorded in MSe mode . MSe runs were also analyzed by Protein Lynx Global Server 2.5 software for validation of protein hits and peptide coverage map. Saccharomyces cerevisiae) standard tryptic digest . Peptides were identified using the Progenesis QI for proteomics internal MSe search engines based on PLGS 3.0 and using B. bassiana proteins FASTA databases created according to the respective Uniprot sequences. The quantification in Progenesis was performed by the built-in Hi-3 algorithm from Waters which allowed, after Apex 3D peptide identification of the reference protein digest, the quantification of any protein based on their three most abundant peptides. The fixed modification was the carbamidomethyl cysteine (ΔMass +57.02), and other variable modifications were also accounted for in the search, such as: deamination NQ (+0.98), oxidation MHW (15.99), carbamilation N-term (+43.01), dehydration DSTY, C-term (−18.01), hydroxylation DKNPRY (+15.99), etc. The protein normalization method chosen in Progenesis, as mentioned previously, was performed according to ENO1 as well as the absolute quantification. ANOVA statistics were made by the built-in Progenesis statistics module. Protein identification having p-value(s) > 0.05 was not included in the analysis.Progenesis QI for Proteomics ver. 2.0  was used to analyze the different MSe runs in triplicate. Samples were compared according to their exact mass versus retention time ratio after normalization with 100 fmol/µL of yeast Enolase 1 , Pr1B , and a putative fungistatic producing metabolite oxidase (A0A0A2VRL8) judging from their number of peptides found and quantitative spectral counting and ENO1 normalized . Other aroteases . Other groteases . Exoenzyroteases . In addiB. bassiana exoenzymes and focuses on the effect of intact insects versus isolated cuticles (of the American cockroach) as nutrition and sole N/C/P source for B. bassiana. The main objective was to identify and quantify exoenzymes of B. bassiana from supernatants when cultivated under these two diet regimes. A range of B. bassiana isolates from different hosts were studied to evaluate the qualitative/quantitative nature of their secretome in liquid cultures. The highest total chymotrypsin-like proteolytic activity was observed for the time point at day four for all isolates  and/or a chitinase (Bbchit1) resulted in an increased virulence [Lecanicillium lecanii increased its virulence against the aphid Aphis gossypii when Pr1A was over-expressed as compared to the wild type [M. anisopliae showed significantly lower Pr1 and elastase activities and were less virulent towards the coleopteran Tenebrio molitor, but no change was observed for the lepidopteran Galleria mellonella [The entomopathogenic fungi bstrates ,23. Receublished ,24,25. Tectively ,25. Compecretome , our stut aphids . In addiobserved . Furtherve hosts . Even inirulence . Likewisild type . These ollonella .B. bassiana with Z-FR-pNA as substrate. This is in contrast with previous studies with M. anisopliae documenting that Metarhizium secretes trypsin-like proteases . The troteomics  and by coteomics  could coB. bassiana isolates seems to depend foremost on the capability to hyper produce both proteases and chitinases [B. bassiana isolates has a variable baseline of expression with the highest level only observed when the intact cockroach diet regime was used  [Serratia sp. or Bacillus sp.Chitin represents the second most abundant polymer in insect cuticle. enzymes) . Fungal Since many chitinases in entomopathogenic fungi are often under catabolic repression, their secretion prior to host invasion has to be continuously induced ,39 by meB. bassiana to aphids [We were able to detect an endo-chitinase D (A0A0A2VG14) as main chitinase differentially expressed more on intact insect diet in Bb 813. Actually, when compared to isolated cuticle diet in Bb813, it increased almost twice as much , but chio aphids ,40. EndoB. bassiana virulence, the recovery of which from the culture media may have been adversely affected by being strongly bound to the chitin and hence having poor solubility. Two other endo-chitinases (A0A0A2VB74 and A0A0A2V532) were only detected using the isolated cuticle diet in Bb 813 . TogetheB. bassiana, one of which  activity against the bacterial cell wall [Cladosporium fulvum (uniprot B3VBK9). Microbial pathogens secrete effector proteins to suppress PAMP (Pathogen-Associated Molecular Pattern)-triggered host immunity in order to establish infection. The LysM domain (CBM50) of Ecp6 mediates virulence through perturbation of chitin-triggered host immunity [B. bassiana are also plant endophytes. In B. bassiana the LysM-containing proteins (A0A0A2V6S0) might be involved in the recognition of the endophytic phase for target plants [B. bassiana the LysM protein (A0A0A2V6S0), homologue of some Piriformospora indica LysM proteins, could also inhibit chitin oligosaccharide-triggered and PRR-mediated activation of host immunity and establish root symbiont mutualistic relationships [We also detected two secreted LysM pfam PF01476) proteins in ell wall . The othimmunity . The perimmunity . Differet plants ,45. In pionships ,47. 6 proteinM. anisopliae Pr1A) [Manduca sexta hemolymph phenoloxidase, whose activation is achieved by a serine proteinase cascade. This pathway is a component of the defensive response of insects to microbial/fungal infection. Phenoloxidase catalyzes reactions that produce quinolic substances that either cross-link proteins and chitins in cuticular sclerotization or polymerize to form melanin [From the host immune response point of view , in partae Pr1A) . One of  melanin . Entomop melanin .B. bassiana exoenzymes from several isolates elucidating their quantitative and qualitative aspects. Previously, B. bassiana exoenzymes were only reported as enzyme activities or partially purified activities from extracellular artificial media containing intact insect or crushed/purified cuticles. Here we describe a precise quantitative/qualitative secretome of several fungal isolates according to the nutrition based on either intact dead cockroach diet or isolated cuticle diet.In conclusion, our study represents the first report of a detailed up-to-date proteomics study on These exoenzymes, produced as recombinant enzymes (recombinant fungus-free bio-insecticides), could potentially overcome the endogenous inhibitors in insects, when used in high amounts. Spray formulation of such complex mixtures  could maybe increase their bioinsecticidal efficacy by reducing the killing time as compared to the entomopathogenic fungi per se.


RMR) is tightly associated with life history strategies, thermoregulation, diet use, and inhabited latitude and could therefore moderate differences in fitness responses to fluctuations in local environments, particularly when species have adapted to different climates in allopatry. We work in a long‐term study of collared  and pied flycatchers (Ficedula hypoleuca) in a recent hybrid zone located on the Swedish island of Öland in the Baltic Sea. Here, we explore whether differences in RMR match changes in relative performance of growing flycatcher nestlings across environmental conditions using an experimental approach. The fitness of pied flycatchers has previously been shown to be less sensitive to the mismatch between the peak in food abundance and nestling growth among late breeders. Here, we find that pied flycatcher nestlings have lower RMR in response to higher ambient temperatures (associated with low food availability). We also find that experimentally relaxed nestling competition is associated with an increased RMR in this species. In contrast, collared flycatcher nestlings did not vary their RMR in response to these environmental factors. Our results suggest that a more flexible nestling RMR in pied flycatchers is responsible for the better adaptation of pied flycatchers to the typical seasonal changes in food availability experienced in this hybrid zone. Generally, subtle physiological differences that have evolved when species were in allopatry may play an important role to patterns of competition, coexistence, or displacements between closely related species in secondary contact.Variation in relative fitness of competing recently formed species across heterogeneous environments promotes coexistence. However, the physiological traits mediating such variation in relative fitness have rarely been identified. Resting metabolic rate ( We predict that pied flycatchers either have a generally lower RMR, demanding less food, or, that they have the ability to adjust their RMR to fluctuations in food availability. The main aim of this study was to experimentally test these predictions using a cross‐fostering design to control for genetic effects while varying the environment. Specifically, we investigated how nestling RMR responded to (1) natural fluctuations in food availability estimated as temperature, which indicates the parents’ onset of breeding in relation to the progress of the spring through the degree of match with the temporal peak in food abundance, and (2) experimentally changed brood size, which influenced individual nestlings access to food through affects on nestling competition.2To isolate variation in intrinsic differences between nestlings of the two species and to remove effects of parental territory, quality, and behavior, we performed a reciprocal, cross‐fostering experiment on a monitored mixed population of nestbox–breeding collared and pied flycatchers on Öland , Sweden in 2013, 2014, and 2015. In these years, in the general population, there was no difference in clutch size between collared and pied flycatchers (collared flycatchers mean clutch size = 6.49 ± 0.7 (2013), 6.70 ± 0.8 (2014), 6.52 ± 0.7 (2015), pied flycatchers mean clutch size = 6.51 ± 0.8 (2013), 6.48 ± 0.8 (2014), 6.81 ± 0.7 (2015)). We matched nests based on species, laying date, brood size, and average nestling mass, to ensure that nestling growth would not be affected by initial biases between the cross‐fostered broods . SMHI has two weather stations close to our study areas on both Öland and in Kalmar. All nestboxes were within 25 km of a weather station, and 95% of boxes were less than 20 km away. We used mean temperature estimates from the nearest weather stations from when the nestlings were 3 days old until when they were 12 days old , computed separately for each experimental nest. We refer to this mean environmental temperature as “ambient temperature.” We used ambient temperature as a proxy for food availability because it has been used as a proxy in other studies . Collection of frass in this system is described in detail in , which retained the original number of nestlings. In total, we had 148 experimental nests but 33 were predated or died for other reasons  prior to 8 days posthatching, resulting in 115 remaining experimental nests across 3 years. Experimental sample sizes are outlined in Table 2.22 monitor was span calibrated using a known concentration of 0.5% CO2 in nitrogen. All calibrations were performed according to the manufacture's recommendation. This respirometry set up allowed us to measure seven birds at a time, with a reference chamber. Nestlings were placed in an individual glass chamber (8 cm by 30 cm) for 4 hr; each chamber was measured six times for five minutes, and since we were able to run the respirometer twice per night, we could measure 14 birds each night, between either 8 p.m. until 12 a.m. or 12 a.m. until 4 a.m. Chamber and cycle assignment was performed haphazardly. We used a flow rate of approximately 400 ml/min, consistent with previous studies of passerines of a similar size  to ensure that they were in their thermoneutral zone , carbon dioxide (CO2), and water vapor pressure and used equations 9.3 and 9.4 in Lighton , as RMR. As we measured metabolic rate six times for each bird, we needed to decide on the most representative measurement. We always discarded the first measurement to allow the bird to settle down. From the remaining measurements, we chose the one with the lowest standard deviation, as this lack of variation was assumed to be indicative that the animal was very still or sleeping . We judged this lag by eye and applied it equally to all files. We measured oxygen levels , although we recognize that these environmental variables tested in different models may explain some of the same variance. We also included foster nest and nest of origin as random effects to account for nonindependence in all models. For all models except for the model assessing an effect of brood manipulation, we used only control nestlings , so as to not need to account for the experimental effects while testing other factors. To test for an effect of food availability, we included control nestlings in addition to nestlings that were part of the brood size experiment.t‐values, and p‐values and false discovery rate adjusted p‐values to account for multiple testing  comparisons below, and all estimates, standard errors, 3F1,423.5 = 0.571, p = .450, Table F1,97.4 = 3.912, p = .0232, Table F2,194.6 = 0.553, p = .576, Table We weighed 831 nestlings at 3 days old in 2013, 2014, and 2015, and cross‐fostered 321 nestlings between nests. We found that collared flycatcher nestlings that survived to 12 days old gained an average of 0.971 ± 0.2 g/day and pied flycatcher nestlings gained an average of 0.948 ± 0.2 g/day. We found no effect of foster status on growth rate . Mass, species, and the interaction between mass and species explained 14.2% of the variation in RMR.We measured the RMR of 64, 84, and 51 nestlings in 2013, 2014, and 2015 , and a marginal interaction between treatment, species, and mass as we might have expected . Examination of specific contrasts showed that this relationship was driven by a difference between pied flycatcher nestlings in reduced nests and those in control nests, with an interaction with mass  and nestling RMR and the effects of a brood size manipulation on nestling RMR differed between the two species. Pied flycatcher nestling RMR was lower in higher ambient temperatures, when their parents had bred relatively late in relation to the peak in food availability , such as when we measured it here. The estimates of RMR that we report here are approximately two times as high as those that have been reported for other bird species that are between 10 and 20 g (0.84 ml/min ± 0.2; Stager et al., 4.2−1 min−1, which can be translated to 2.18e‐5 kcal for an average sized pied flycatcher nestling (Schmidt‐Nielsen, To emphasize the importance of the pied flycatcher nestlings response to the environment, we can determine the actual energetic savings of a nestling with a lower RMR compared to one with a higher RMR. The predicted difference in RMR between the first ambient temperature quartile experienced by nestling pied flycatcher nestlings in our study and the third ambient temperature quartile in pied flycatchers is 4.6e‐3 ml gThe robustness to a mismatch between breeding and peak of food abundance (Qvarnström et al., 4.3In contrast with the patterns found in pied flycatchers, collared flycatcher nestling RMR did not vary with ambient temperature experienced during growth and did not appear to be affected by the brood size manipulation experiment. This difference in plasticity could stem from the narrower climatic niche that collared flycatchers have inhabited before colonizing the Swedish Baltic islands (Qvarnström et al., An important question then becomes whether nestling RMR will remain less flexible in collared flycatchers as compared to pied flycatchers in the Swedish hybrid zone? Collared flycatchers are under stronger selection to adjust to the changing food peak (Sirkiä et al., 5Studies in community ecology have shown that competitive interactions and relative fitness responses of species to environmental variation can drive further niche differentiation (McGill, Enquist, Weiher, & Westoby, None declared.SEM and AQ conceptualized the study; SEM, MÅ, and PS performed the fieldwork, including cross‐fostering, and respirometry; SEM analyzed the data; SEM and AQ wrote the manuscript; and all authors revised and approved the final version.


Globally, one in three adults has some level of measurable hearing loss, and 1.1 billion young persons are at risk for hearing loss attributable to noise exposure. Although noisy occupations such as construction, mining, and manufacturing are primary causes of hearing loss in adults, nonoccupational noise also can damage hearing. Loud noises can cause permanent hearing loss through metabolic exhaustion or mechanical destruction of the sensory cells within the cochlea. Some of the sounds of daily life, including those made by lawn mowers, recreational vehicles, power tools, and music, might play a role in the decline in hearing health. Hearing loss as a disability largely depends on a person’s communication needs and how hearing loss affects the ability to function in a job. The loss of critical middle and high frequencies can significantly impair communication in hearing-critical jobs .A recent analysis of 2011–2012 National Health and Nutrition Examination Survey (NHANES) data estimates that approximately 14% of U.S. adults aged 20–69 years (27.7 million persons) have hearing loss. After adjustments for age and sex, hearing impairment was nearly twice as prevalent in men as in women; age, sex, ethnicity, and firearm use were all important risk factors for hearing loss  estimates that 22 million workers are exposed to hazardous levels of noise in their workplaces (For high exposure levels such as firearm or aircraft noise above 140 decibels sound pressure level (dB SPL), engineering and administrative controls might not reduce noise exposures adequately. Such situations require hearing protection devices (HPDs) providing upwards of 30–40 dB of noise reduction when worn properly. Despite the existence of occupational regulations for hearing protection, many workers fail to achieve adequate protection because their earplugs or earmuffs do not fit properly. Hearing protector fit testing provides an opportunity to train workers to properly fit hearing protectors and to encourage effective use. The NIOSH HPD Well-Fit hearing protector fit-test system is a simple, portable solution for testing in quiet office spaces. Other fit-testing systems are commercially available . In 2016, CDC began initiatives to raise awareness about the risk for permanent hearing damage attributable to nonoccupational noise exposures, including the development of new communication tools about noise-induced hearing loss. An analysis of 2011–2012 NHANES audiometric data from 3,583 adults aged 20–69 years identified persons with high-frequency audiometric notches suggestive of noise-induced hearing loss .The weighted prevalence of an audiometric notch was 24%, extrapolated to represent nearly 40 million U.S. adults. Unilateral audiometric notches were three times more prevalent than were bilateral audiometric notches and were more prevalent in men than in women. Participants who reported having exposure to loud noise at work were twice as likely to have evidence of hearing damage as were those who did not. However, 20% of persons with no occupational exposure to loud noise had an audiometric notch, suggesting that 21 million U.S. adults likely have hearing damage from noise at home or in their communities .Regardless of whether participants’ exposure was to work or recreational noise, 24% of those with such damage reported that their hearing was excellent or good, suggesting that many persons might be either unaware of or ignoring noise-induced hearing damage. Although most noise-induced hearing loss is preventable, the NHANES analysis found that 70% of persons exposed to loud noise in the past 12 months had seldom or never worn hearing protection  is an evidence-based intervention program that has changed knowledge, attitudes, beliefs, and behaviors of both youths and adults for the prevention of noise-induced hearing loss and tinnitus. The messaging incorporates three strategies for hearing loss prevention: 1) turn it down, 2) walk away, and 3) protect your ears. Originally developed for youths, Dangerous Decibels has been successfully adapted for civilian adults and the military, and its effectiveness was demonstrated in randomized trials among children in the United States and in studies in New Zealand and Brazil (Dangerous Decibels (http://www.safeinsound.us/) developed by NIOSH and the National Hearing Conservation Association  and the Make Listening Safe initiative (http://www.who.int/pbd/deafness/activities/MLS/en/) (Two principal advocacy initiatives promoted by WHO include World Hearing Day (Noise reduction and avoidance can prevent hearing loss or slow its progression. Persons can protect themselves by moving away or taking breaks from loud sounds, using quieter consumer products, lowering volumes on personal listening devices, reducing time listening to loud levels of music, and using hearing protectors. Hearing protectors need to fit well to reduce noise exposures effectively. Health care providers can inform patients about hearing loss symptoms, early diagnosis of hearing loss, and prevention strategies.Policymakers, governments, and manufacturers of equipment can develop policies to reduce noise levels and limit noise exposures of the public. In parts of Europe, community noise and the effect of urban soundscapes on public health have received considerable attention. In the United States, national, state, and local community noise-control efforts are largely uncoordinated, potentially resulting in higher levels of community noise. Increasing awareness and reducing needless exposures to loud noise might help the public take appropriate steps to protect their hearing.


Mycoplasma hyopneumoniae (M. hyopneumoniae). However, in case of positive serology, further decisions on farm health status and the related consequences should be based on additional confirmation tests.A dedicated program to monitor for freedom of several economically important diseases is present within most of the breeding companies that currently deliver high health breeding animals to their customers. Serology is therefore the preferential approach in order to screen for most of these diseases, including M. hyopneumoniae-seropositive situation. A Central-European SPF herd was shown positive  for M. hyopneumoniae using the conventional ELISA serology (Idexx HerdChek Mhyo ELISA) and a second ELISA test (IDEIA™ Mycoplasma hyopneumoniae EIA kit) did not exclude potential M. hyopneumoniae infection . Further follow-up remained inconclusive on both tests. Throughout the entire monitoring period of 6 months, no coughing, necropsy lesions or lesions at slaughter could be detected which could confirm the M. hyopneumoniae health status. TBS sampling was used to confirm the health status for M. hyopneumoniae. In total, 162 samples were collected at different ages (n = 18 per age category): piglets at 3–6–9-12 and 15 wks of age, rearing gilts at 18–21-24 and 27 weeks of age. Collected TBS samples were negative for M. hyopneumoniae until 15 wks of age, but rearing gilts were highly M. hyopneumoniae-positive from 18 wks onwards with 87–100% M. hyopneumoniae-positive animals and PCR Ct-values between 25 and 33.The current case report demonstrates that tracheo-bronchial swab (TBS) sampling is a suitable alternative to confirm a suspect M. hyopneumoniae infection status of a breeding herd was able to provide additional information to serology in order to make crucial decisions concerning health management and eradication strategies within the breeding herd.This case report shows that collection of TBS samples to confirm the  Mycoplasma hyopneumoniae (M. hyopneumoniae), the primary pathogen of enzootic pneumonia, occurs worldwide and causes major economic losses to the pig industry. The pathogen adheres to and damages the ciliated epithelium of the respiratory tract. Affected pigs usually show chronic coughing, are more susceptible to other respiratory infections and have a reduced performance [M. hyopneumoniae plays a key role in the Porcine Respiratory Disease Complex (PRDC) through interactions with several other respiratory pathogens.formance ). MoreovM. hyopneumoniae during the suckling period and many studies have shown M. hyopneumoniae-positive animals from weaning onwards [M. hyopneumoniae, animals can excrete the pathogen over a long period of time, with total clearance lasting till 254 days post-infection [M. hyopneumoniae well across their first pregnancy into their first lactation cycle, infecting their offspring with M. hyopneumoniae in early life.Piglets can become infected with  onwards –7. Moreonfection . This imM. hyopneumoniae have been developed within breeding companies that currently deliver high health breeding animals to their customers. Serology using ELISA is the preferential approach in order to screen for M. hyopneumoniae [M. hyopneumoniae infections is often low and the positive herd predictive value of a serological result decreases progressively with the decreasing herd prevalence [M. hyopneumoniae infection was 13 days, whereas the mean time between onset of coughing and seroconversion as measured by ELISA was 9 days [M. hyopneumoniae infection using one of the commercially available M. hyopneumoniae ELISAs remains relatively low (16–22% at 21 days and 35–45% at 28 days post-infection) [M. hyopneumoniae within the monitored herd. In the Danish SPF program, the final verification of herd infection with M. hyopneumoniae is consequently performed by demonstration of the agent [M. hyopneumoniae detection showed that laryngeal swabs were a reliable option to establish early detection of M. hyopneumoniae, followed by brocho-alveolar lavage fluids and nasal swabs [M. hyopneumoniae to reliably detect the pathogen of infected animals. The objective of the current case report is to show that TBS sampling is a suitable method to confirm a suspect M. hyopneumoniae-seropositive situation.Therefore, dedicated programs to monitor for freedom of eumoniae –14. In ceumoniae . Howeverevalence . Moreoveevalence , 17, ands 9 days . Recent s 9 days . The perfection) . This imhe agent . A recenal swabs . Other ial swabs  have beeM. hyopneumoniae for more than 20 years using a standard serological monitoring schedule  with a commercially available M. hyopneumoniae ELISA test . Besides freedom for M. hyopneumoniae, the farm was also negative for Pasteurella multocida DNT+, Sarcoptes scabiei var. suis, Brachyspira hyodysenteriae, Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Actinobacillus pleuropneumoniae.A high health breeding farm in Central Eastern Europe (220-sow herd) had been negative for External biosecurity is at the highest level, with no entrance to visitors and strict shower protocols for all farm personnel upon entrance of the farm. Internal biosecurity is also well established with boot hygiene (washing and disinfection) between production groups , clean disinfection baths at entrance of each individual compartment and no movement of other materials  between production groups.The sow farm is run on a 3-week batch management system with 7 groups of 32 sows each. Productive sows, weaned piglets and rearing gilts are housed in separate buildings on the premises. Successive batches of weaned piglets from 4 weeks until 16 weeks of age are housed in separate nursery compartments with strict all-in all-out (AI/AO) strategies. From 16 weeks of age onwards, rearing gilts are housed in a larger barn that is not managed according to AI/AO strategies.M. hyopneumoniae status was performed throughout the last decade, repeatedly confirming the M. hyopneumoniae-negative status. The farm first tested positive for M. hyopneumoniae using the first ELISA serology  in March 2017. Additional monitoring one month later (Idexx) confirmed the M. hyopneumoniae positivity and therefore, a second ELISA test  was performed, demonstrating a clear evolution towards lower M. hyopneumoniae-positivity in both ELISA tests used. This decreasing trend did however not persist in the fourth sampling showing again a gradual increase in M. hyopneumoniae-titers. The number of positive samples obtained with both ELISA tests on each sampling date are given in Table At gilt delivery (27 weeks of age), regular serological sampling to assess Moreover, throughout the entire monitoring period, no coughing, lung lesions at necropsy or at slaughter could be detected.M. hyopneumoniae, is actively exchanged among these different farms. Under the local conditions, wind direction is most often from a western direction with high wind speed (source: http://oze.tzb-info.cz/vetrna-energie/9800-vetrne-podminky-v-ceske-republice-ve-vysce-10-m-nad-povrchem-ii). During the last quarter of 2017, both farms  were detected M. hyopneumoniae-positive on serological monitoring using the conventional ELISA test .Several other swine farms, some belonging to the same production group, are located within a range of 2–3 km from the described SPF farm. Farm VS, a fattening unit directly related to the SPF source farm, is located at 1.35 km air distance in western direction and Farm VH, an unrelated 35-sow herd, is located at 2.25 km air distance in south-south-eastern direction. Epidemiological information on disease state, including Tracheo-bronchial sampling was performed as previously described , 7. BrieM. hyopneumoniae p183 real-time-PCR [g, the pellet suspended in 400 μL lysis buffer, and 400 μL of the suspension was used as the sample. If no pellet was observable, 300 μL of the TBS was used as the sample. Bead mix and lysis/binding solution were added and the mix transferred onto a 96-well plate in the processor. Nucleic acid isolation was performed according to the manufacturer’s instructions. The PCR results were reported as negative (Ct ≥ 37) or positive (Ct < 37) for the presence of M. hyopneumoniae based on a Ct-threshold value. The detection limit range for M. hyopneumoniae reported [M. hyopneumoniae strain J (ATCC 25934) of at least 5 fg/μL.The material collected by TBS was processed in a time-PCR . Nucleicreported  was fromhttp://epitools.ausvet.com.au), using FreeCalc – sample size calculation for freedom testing with imperfect tests using the modified hypergeometric distribution for exact hypothesis testing, with the assumption that maximum 2.5% of the animals (n = 6000) present on the farm were positive for M. hyopneumoniae, assuming test sensitivity and specificity of the TBS/qPCR combination of 75 and 100%, respectively [n = 18 per age category): piglets at 3–6–9-12 and 15 weeks of age and rearing gilts at 18–21-24 and 27 weeks of age.The minimal number of samples needed to show ‘freedom of disease’ in the farm was calculated  on the average Ct values was assessed by means of an F-test in a one-way ANOVA. Post-hoc multiple comparisons of means were performed with Tukey’s method. The overall effect of age (n = 9 age groups) on the probability of a positive qPCR result was assessed by means of the Pearson chi-squared test. Multiple pairwise comparisons were also done with Pearson chi-squared tests, with p-values adjusted with Holm’s procedure.For the assessment of the overall effect of sampling date on the probability of a positive qPCR result, a Pearson chi-squared test was used. Pairwise comparisons between sampling dates was also performed with Pearson chi-squared tests, with All overall tests were performed at the 5% level of significance. Multiple comparison tests were performed simultaneously at the 5% familywise error rate (FWER) level. All data analyses were performed with the R statistical software version 3.4.3. .M. hyopneumoniae could be detected using PCR testing. However, once the animals entered the rearing barn located at the same site, M. hyopneumoniae was clearly present. Among the gilts from 18 until 27 weeks of age, the percentage of positive animals significantly increased (P < 0.05) to 87–100% in the oldest age group  from 37, the cut-off value for PCR test positivity.During lactation and nursery phase (from 3 until 15 weeks of age), no M. hyopneumoniae infection can be difficult using the standard monitoring tools, such as clinical observation, serology [M. hyopneumoniae health status of a breeding herd. This allowed the farmer to make decisions concerning health management within the breeding company.The current case report demonstrated that diagnosis of an serology  and slauserology . It was M. hyopneumoniae-positive results from 18 weeks onwards, which is the age category that enters the gilt rearing barn. Until now, it is unclear how the M. hyopneumoniae infection has entered the farm. Generally, the major source of M. hyopneumoniae introduction into a farm is considered newly arrived subclinically M. hyopneumoniae-infected gilts, especially since M. hyopneumoniae-infected animals can excrete the pathogen for up to 254 days following initial infection [Interestingly, the collected TBS samples only showed M. hyopneumoniae entered the farm by airborne transmission from infected neighboring farms. Under favorable climatic conditions, aerogenous spread of M. hyopneumoniae between neighboring herds over varying distances has been demonstrated [M. hyopneumoniae health status had also happened during this year based on the results of their regular serological monitoring. Unfortunately, we do not have details on which of these swine farms first broke with its M. hyopneumoniae health status, although based on prevailing wind direction, farm VS which is located west from the SPF nucleus herd could be a good candidate.A second hypothesis is that the nstrated –24. IndeM. hyopneumoniae-infected transport vehicle. Especially under winter conditions, cleaning and disinfection of transport vehicles is not always easy to perform. Recently, M. hyopneumoniae survival on stainless steel at 4 °C was demonstrated for at least 2 days (max. 8 days for some strains) [M. hyopneumoniae-free vehicle. Analysis of the M. hyopneumoniae infection kinetics on the farm revealed that the M. hyopneumoniae infection was first detected within the gilt rearing barn. This is also the location from which gilts are selected and loaded for external transport. Therefore, the probability of a biosecurity breach at the gilt loading point seems possible, since external biosecurity at all other levels has always been at the very high level. Moreover, once the M. hyopneumoniae infection entered this section of the farm, M. hyopneumoniae could easily further spread within the age groups from 18 until 27 weeks, which is not managed under strict AI/AO conditions. The reason why no coughing nor typical lung lesions were present is not clear. It may be due to the rather good housing conditions, e.g. air quality and level of dust [Another source of potential introduction might be through an strains) . This wo of dust , 27 and/ of dust .M. hyopneumoniae introduction into the farm. A 4-year research demonstrated that a 1-night downtime period is sufficient to prevent mechanical spread of both PRRSV and M. hyopneumoniae by personnel and fomites [M. hyopneumoniae infection status.Although a break in biosecurity seems unlikely, it should be considered as another option for  fomites . Moreove fomites . In the M. hyopneumoniae infection status of the sow population, since TBS could not detect M. hyopneumoniae-positive piglets till 15 weeks of age. Based on the results of this monitoring, a specified eradication plan will be designed to result in a renewed M. hyopneumoniae-negative gilt outflow to the end customers.Further diagnostics is ongoing to confirm the M. hyopneumoniae infection in an SPF-herd may occur without clinical symptoms and typical lung lesions. In addition, serological testing may be difficult to interpret, and PCR testing on TBS may be needed to establish a conclusive diagnosis.The present case report showed that for several months,


L. intracellularis – Porcilis® Ileitis was evaluated under field conditions.Porcine proliferative ileitis is a major economic burden for the swine industry, affecting growing pigs and young adult pigs. In this study, the protective efficacy of an inactivated, injectable whole-cell bacteria vaccine against L. intracellularis-specific immunohistochemistry, and qPCR of ileal scrapings. Integrity of the intestinal barrier was evaluated to quantify a range of intestinal markers including secreted mucin and intestinal alkaline phosphatase, and innate immune markers including Caspase-3 and Calprotectin. A second subset of pigs was monitored for fecal shedding of L. intracellularis, until resolution of shedding.Eighty-five, three-week-old pigs on a commercial farrow-to-finish farm were vaccinated by the intramuscular route, either with a dose of injectable vaccine, or with saline. A subset of vaccinates and control pigs were necropsied at 21 days post-challenge. Incidence and severity of ileitis were evaluated by gross and microscopic observation of ileal tissues. Colonization of the gut after challenge was examined by p < .05) and preserved normal gut barrier function in the face of an experimental challenge with virulent L. intracellularis.Our investigation indicated that Porcilis Ileitis provided robust protection against ileitis, reduced bacterial shedding 15-fold ( L. intracellularis is a Gram-negative, intracellular bacterium that can infect a number of animal species, but it is of particular economic concern in the swine industry. In pigs, the bacteria cause porcine proliferative enteropathy (ileitis). Clinically affected animals exhibit diarrhea and reduced growth performance, resulting in increased time to market and greater variation in size between pigs. In young adults, the infection can lead to an acute hemorrhagic form of the disease, characterized by dark, tarry diarrhea and which may result in death. L. intracellularis also infects pigs sub-clinically, without clear clinical signs but still resulting in reduced growth performance. Its worldwide distribution and high prevalence have been recognized since the initial characterization of this pathogen in the early 1990s and L. intracellularis is reported to affect 57–100% of herds, globally Ileitis caused by L. intracellularis and host cells is crucial in establishing infection. The bacterium infects the gastrointestinal tract, with a specific tropism for the terminal ileum. The hallmark lesion of L. intracellularis infection is the proliferation of intestinal crypt lining cells (enterocytes) which results in hyperplasia of the mucosal wall. The peak of bacterial burden is associated not only with crypt epithelial cell proliferation but also with down-regulation of specific host mechanisms involved in cell transport and maintenance of mucosal integrity, and with inflammation As an obligate intracellular pathogen, interaction between L. intracellularis infections and limiting the associated economic losses. Infection by the bacterium can be treated with various antibiotics, notably those from the macrolide, pleuromutilin, and quinoxaline groups L. intracellularis is not possible. The use of the oral vaccine requires strict management practices to avoid the simultaneous use of antibiotic treatments. However, prophylactic use of an inactivated vaccine would not be limited in this way.There are some tools available for controlling L. intracellularis was examined. This vaccine was administered to three-week-old pigs under typical field conditions, without restricting the use of antibiotics. Our investigation indicates that Porcilis Ileitis vaccine can provide robust protection against ileitis, help reduce bacterial shedding 15-fold (p < .01), and help maintain gut barrier function integrity.In this study, the effectiveness of a novel inactivated injectable vaccine, Porcilis Ileitis, as an aid in the control of ileitis caused by 22.1The animal trial was conducted by Swine Services Unlimited  as a randomized, blinded study, approved by the SSUI Institutional Animal Care and Use Committee.2.2L. intracellularis bacteria in XSOLVE adjuvant . The vaccine is an oil-in-water emulsion. The adjuvant is based on both mineral oil and alpha tocopherol (Vitamin E).The vaccine used in this study contained inactivated 2.3L. intracellularis infection, and all study pigs were negative for anti-L. intracellularis antibodies as measured by a commercial inhibition ELISA .Eighty-five mixed-breed and mixed-sex pigs were enrolled on a commercial, farrow-to-finish farm, which farrowed 30 litters per week, weaning 300 pigs per week. The herd was health-stable prior to and during the study, with historic pre-weaning mortality rates between 8 and 10% during the previous 2 years, and nursery mortality of approximately 3%. The herd did not show clinical signs of The study design is summarized in The pigs were weaned one day after their vaccination. Upon weaning, the pigs were transported to isolation facilities for the pre-challenge and challenge phases of the study. Pigs from each group were comingled with approximately equal numbers of vaccinates and controls in each pen.L. intracellularis shedding during the post-challenge period  (necropsy groups), and a subset of 15 pigs to be fecal sampled to determine Prior to the start of the study, as well as prior to the challenge, all pigs were bled and sera were prepared. Pigs were bled again at the end of the test, at 21 dpc for the necropsy groups and at 51 dpc for the shedding groups.2.4ad libitum to all animals. Feed met or exceeded the minimum nutritional requirements for animals of this age and complied with standard procedures for the site. During the pre-weaning phase, the piglets had access to the dam’s feed and water dispensers. Sow feed during this phase was a standard, non-medicated lactation diet. Feed was medicated with Mecadox (carbadox) at 50 g/ton, from the day after the vaccination when the pigs were weaned, until two weeks later (14 dpv). Carbadox is a quinoxaline antibiotic indicated for the control of swine dysentery and control of bacterial swine enteritis caused by Salmonella choleraesuis, but also effective against L. intracellularis, with a reported intracellular minimum inhibitory concentration of <0.5 µg/mL Water was provided 2.52.5.1L. intracellularis exposure as detailed in Section L. intracellularis-infected gut homogenate, containing approximately 9.3 Log10 of L. intracellularis, as determined by qPCR. The gut homogenate was prepared from intestines of pigs experimentally challenged with a virulent isolate obtained in 2008 from pigs in the United States displaying clinical signs of ileitis. Briefly, mucosa from guts of pigs with clinical signs of ileitis were collected by scraping, diluted in sucrose-phosphate-glutamate buffer, homogenized in a warren blender, trypsinized, aliquoted and frozen at −70 °C essentially as described previously At the time of challenge, the five sentinel pigs were necropsied and evaluated for evidence of 2.5.2Following the challenge, all pigs were observed daily for clinical signs of ileitis, including body condition, alertness, appetite, and consistency of the feces. Fecal consistency was scored as normal, soft or watery (mild diarrhea), bloody or tarry (severe diarrhea).2.6g. Sera were collected and stored frozen at −20 °C or lower until testing by a commercial competitive ELISA as documented in Section L. intracellularis antibodies, between 20 and 30% are considered suspect, and 30% and higher indicate positive results.Five to 10 mL blood was collected in serum separator tubes, and centrifuged as recommended by the manufacturer, usually for 20 min at 8602.7L. intracellularis (GenBank accession # L157390) was monitored with the forward primer, 5′-GCGCGCGTAGGTGGTTATAT-3′, the reverse primer, 5′-GCCACCCTCTCCGATACTCA-3′ and the fluorescent probe 5′-FAM-CACCGCTTAACGGTGGAACAGCCTT-TAMRA-3′. The qPCR conditions were as described previously L. intracellularis present in the samples and to evaluate the treatment effect, Ct values were transformed to copies per reaction (c/rx) by assuming that 40 Ct represents absence of L. intracellularis DNA (0 c/rx), and assuming 100% efficiency of replication, resulting in a 10-fold increase of DNA for every 3.3 cycles reduction in Ct values. Calculations were therefore carried out as follows: c/rx = 2 ^ (40-Ct).Quantitative real-time PCR (qPCR) of ileal scrapings and fecal samples was conducted at the Iowa State University Veterinary Diagnostic Laboratory, according to previous standard methods 2.8L. intracellularis in intestinal epithelial cells as measured by qPCR. Furthermore a tissue sample was fixed in buffered neutral formalin for microscopic evaluations as described below.The sentinel pigs were necropsied at the time of the challenge, while the necropsy groups were sacrificed at 21 dpc. In each case, the ileum was opened longitudinally, and gross lesions of ileitis were scored. Mucosa were scraped using a glass slide into a 50 mL conical tube, and the collected material was snap-frozen on dry ice and stored at −70 °C until further processing such as the determination of the level of 2.8.1Gross lesions were scored on the basis of the severity of mucosal thickening . Scores 2–4 were considered indicative of clinical ileitis.2.8.2L. intracellularis .Formalin-fixed tissues were submitted to the Iowa State University Veterinary Diagnostic Laboratory for standard histopathological examination following Haematoxylin-Eosin (H&E) staining to evaluate microscopic lesions, mainly proliferation, consistent with 2.8.3For PAS staining, fixed tissues were deparaffinized and rehydrated, immersed in periodic acid and stained with Schiff’s reagent as described previously 2.8.4L. intracellularis antibody required heat-mediated antigen retrieval in citrate buffer at pH6, using a Histo5 Rapid Microwave Histoprocessor (Milestone) with the following protocol: high pressure treatment at 110 °C for 5 min (20 slides per sample). For L. intracellularis immunostaining, sections were incubated in proteinase K  for 10 min at room temperature, followed by two washes in phosphate buffered saline (PBS). Solutions of 0.10% Triton-X in PBS and 0.010% Triton-X in PBS were used to permeabilize the tissue for 10 min at room temperature.For immunofluorescence (IF) all the antibodies tested  except fAll IF slides were incubated in 5% bovine serum albumin (BSA) in PBS at room temperature for 30 min. Excess blocking solution was removed and slides were incubated overnight at 4 °C with primary antibodies in 1% BSA/PBS at the dilutions stated in Intestinal alkaline phosphatase (IAP) was monitored with Vector® Red Alkaline Phosphatase Substrate  according to manufacturer instructions. Slides were stained with DAPI and mounted as described above. The presence of intestinal alkaline phosphatase was viewed with a Texas Red® filter.All sections were observed using LSM700 confocal laser scanning microscope (Carl Zeiss) and 2–4 images per sections were captured and processed blind by a single operator using Zen Blue software (Carl Zeiss).2.8.5For quantitative analysis of PAS staining for L. intracellularis and Mucin 2 (MUC2), two to four random fields from each staining were analyzed using Image J 1.49S. Briefly, the spectrally opposite color channel was selected and intensity threshold was adjusted to highlight the marker. Next, the appropriate color channel (green or purple) was selected, and signal intensity was normalized to the field area and recorded. For cleaved caspase-3 (CASP3) and Calprotectin (CP), and also for IAP, signal intensity was normalized to the section area and then recorded.For quantitative assessment of IF signals of 2.9http://www.graphpad.com/quickcalcs/). qPCR results were evaluated by the Mann-Whitney test (Minitab v 17). Scatterplots were generated using GraphPad Prism v 7.02.Statistical analysis for the bacterial shedding data using qPCR, serological results and quantitative image analysis were carried out using 1-way ANOVA used after assessing normality of the data using SAS package with α = 0.05 33.1All pigs were individually observed every other day for three weeks following vaccination for any evidence of adverse systemic or injection site reactions. No systemic adverse reactions to the vaccination were observed. Local injection site reactions were observed in 10 of 40 (25%) vaccinated animals, ranging in size between 1 × 1 cm and 4 × 4 cm (length × width). All local reactions resolved by 19 days post-vaccination (dpv) without intervention.Signs of clinical disease were monitored during the challenge phase of the experiment. Severe diarrhea was observed in 16 of 40 animals in the control group (40%), but not in any of the 40 vaccinated animals. In the vaccinated group, 11 of 40 animals (28%) presented with mild diarrhea at any time during the post-challenge period. Loss of appetite, lethargy or ill thrift was recorded for four control pigs.p < .0001).Ileum gross lesion scoring is summarized in (p = .0018).The occurrence of clinical ileitis was also evaluated by histopathological examination, on an ordinal scale 0–2 B. Of the3.2L. intracellularis-specific IF. Results are shown in L. intracellularis DNA load of vaccinated pigs was significantly lower than that of the control pigs . Using IF of L. intracellularis specific antigen VPM53, intestinal crypt cells of controls harbored significantly greater bacterial load associated with enhanced cell proliferation than vaccinated pigs (p < .001)  B and C.F3.3L. intracellularis serological profiles using an inhibition assay . Following challenge, all control pigs developed anti-L. intracellularis antibodies similar to vaccinated pigs as monitored at 21 and 51 dpc (p > .05).Vaccinated and control pigs were monitored for anti-on assay . Vaccina3.4p < .01, p < .01, p < .001, respectively). IAP expression in vaccinated pigs was similar to the level found in the sentinels . Expression of apoptotic marker CASP3 was significantly up-regulated in controls (p < .05) pigs compared to the sentinels. CP, a marker associated with neutrophil infiltration, was expressed at a higher level compared to the sentinels, in both controls (p > .05) and vaccinates (p < .05).To assess the integrity of the ileum tissues collected at 21 dpc, a range of intestinal cell lineage markers was evaluated and quantified. In particular, molecular markers that identify secretion , absorption and homeostasis , apoptosis  and immune cell distribution  were monitored  and 6 [63.5L. intracellularis shed during the 51 dpc was 6.2 × 105 copies/reaction/day for the control group, and 4.1 × 104 per day for the test vaccine animals, representing a 15-fold reduction . The median time point where maximum shedding occurred was also earlier in vaccinates (14 dpc versus 23 dpc), although this difference was not statistically significant (p > .05).Fecal shedding was monitored in a subset of the vaccinated (n = 15) and control (n = 15) animals using qPCR over 51 dpc A. The mep < .05, B. Overal4L. intracellularis – Porcilis Ileitis. We found that this adjuvanted L. intracellularis vaccine was safe in pigs when administered under typical field conditions at three weeks of age prior to weaning. Following challenge, signs of severe clinical ileitis were observed in 40% of the controls, but not in the vaccinated pigs. Protection of vaccinates was further confirmed by gross observation of the ileum, as well as histopathology. For each of these parameters, significantly more control animals were affected than vaccinated animals. Taken together these results indicate that the vaccine reduced both the incidence and severity of clinical signs.In this study, we have assessed the efficacy of a novel inactivated, injectable whole-cell bacteria vaccine against L. intracellularis antibodies by ELISA, although three of 85 pigs had test results which were classified as ‘suspect’ by the manufacturer’s criteria, most likely as a result of low levels of maternally-derived antibodies against L. intracellularis. The ‘suspect’ animals were included in the analyses. Vaccinated pigs displayed an antibody response in the majority of pigs, in spite of the vaccine regimen consisting of only a single dose. At three weeks post-vaccination, 75% of vaccinates were classified as suspect or positive. No apparent correlations were found between sero-response following vaccination and outcome parameters of the challenge, such as clinical signs, gross lesions, microscopic scores or PCR results (data not shown). This suggests that at least part of the protective mechanism of this inactivated, parenteral vaccine may be through induction of cell-mediated immunity (CMI) as previously described for other bacterial pathogens, such as Mycoplasma hyopneumoniaeAt the time of vaccination nearly all of the animals tested negative for anti-L. intracellularis-specific IF. Bacterial loads were also significantly lower in the feces of vaccinated animals, both in terms of reduced severity (bacteria shed per day) and in reduction of the duration of fecal shedding. Reduced proliferation of enterocytes in vaccinated pigs compared to controls, as demonstrated by H&E staining of ileum tissues, coincided with significantly greater numbers of MUC2-expressing goblet cells and IAP-expressing brush border cells than controls indicating a restoration of these essential intestinal cell lineages L. intracellularis of the crypt cells, but also helped preserve near normal mucosal architecture and homeostasis.Our study clearly showed that reduced clinical signs in vaccinates coincided with a reduced bacterial load and increased ileum integrity at 21 dpc, compared to nonvaccinated controls. The bacterial load was significantly (200-fold) reduced in the terminal ileum as determined by qPCR, and was significantly lower when examined by L. intracellularis increases the odds ratio for low growth rate by 2. In this study, fecal shed in the vaccinates was reduced 15-fold compared to the controls. Although this study did not record growth parameters, the vaccination can be expected to have prevented the negative impacts of L. intracellularis infection on the economic parameters of this cohort of pigs.According to Johansen et al. The use of a killed Lawsonia vaccine in this study allowed the administration of antibiotics to the pigs around the time of the vaccination. The vaccine was compatible with the use of carbadox administered in the feed as early as one day after vaccination, as demonstrated by the protection that was shown against virulent challenge compared with non-vaccinated control pigs.L. intracellularis vaccine was safe in pigs when administered under typical field conditions as a single, 2 mL dose at three weeks of age prior to weaning. The study demonstrated that the vaccine reduced both the incidence and severity of clinical signs of ileitis due to L. intracellularis, reduced colonization by L. intracellularis, and reduced the severity and duration of fecal shedding of L. intracellularis. The study also found that gut function in the face of L. intracellularis infection was improved following vaccination.In conclusion, the data demonstrated that this inactivated, adjuvanted Merck Animal Health, Madison, NJ USA. The animal trial was designed by FR and ES. End-of-study lesion scoring was conducted by FR while blinded to the grouping. M-HP, AA and TA-A declare no conflict of interest.This study was funded by


CVTree and CVTree with word length of 3, F1-score and their robustness under the influence of different factors.Horizontal gene transfer (HGT) plays an important role in the evolution of microbial organisms including bacteria. Alignment-free methods based on single genome compositional information have been used to detect HGT. Currently, Manhattan and Euclidean distances based on tetranucleotide frequencies are the most commonly used alignment-free dissimilarity measures to detect HGT. By testing on simulated bacterial sequences and real data sets with known horizontal transferred genomic regions, we found that more advanced alignment-free dissimilarity measures such as  As opposed to vertical transmission in which DNA is transferred from parent to offspring, horizontal gene transfer (HGT) or lateral gene transfer (LGT) is defined as the movement of genetic material between organisms that are not in a parent-offspring relationship. HGT plays an important role in bacterial evolution as it is the primary reason underlying the adaptation of bacteria such as metabolic adaptation  composition. These methods are based on the observation that different microbial species have their own genomic word pattern signatures  as the host genome and Bacillus subtilis 168 (B. subtilis), Haemophilus influenzae Rd KW20 (H. influenzae), Helicobacter pylori 26695 (H. pylori), Mycobacterium tuberculosis H37RV (M. tuberculosis), and Streptococcus pneumoniae R6 (S. pneumoniae) as donor genomes. Each time, we picked a fragment randomly from the donor genome with length uniformly chosen from 8kbp to 40kbp and inserted it into a random position uniformly along the E. coli K12 genome until the simulated HGT consists of up to 10% of the artificial genome, since the HGT proportions in most bacteria genomes range from 2 to 15% -th nucleotide w[n + m]:Some dissimilarity measures such as Li) is defined as:The Euclidean distance (Eu) is defined as:d2 distance is defined as:The CVTree dissimilarity is defined as:The CVTree calculates k − 2)-th order Markov chain for genomic sequences.where The where The where E. coli_artificial genome, we divided the genome into overlapped windows of size b with sliding step of 500 bps. As suggested by Dufraigne et al. , we tested it with k = 3, 4, 5. For all methods, a double-strand signature was used to remove strand compositional asymmetry  and precision. Recall is calculated as the length of the overlapped sequence between detected HGT and simulated HGT divided by the total length of simulated HGT fragments. Precision is calculated as the length of the overlapped sequence between detected HGT and simulated HGT divided by the total length of detected HGT. A commonly used measure that combines precision and recall is the harmonic mean of precision and recall, the traditional F1-measure or balanced F1-score, defined asBy comparing the detected HGT and the real transferred fragments in E. coli_artificial genome, for each threshold, we calculated the precision, recall and the F1-score for each method. We then calculated the average precision, recall and the average F1-score for each threshold over 10 simulated genomes and plotted the precision-recall curve. We report the optimal F1-score for each dissimilarity measure.Given an F1-score as our criterion for comparing the different dissimilarity measures.Since most parts of the host genome are not transferred from other genomes, the receiver operating curve (ROC) showing the relationship between the false positive rate  and true positive rate  is not optimal for comparing the different dissimilarity measures since the area under the ROC curve (AUC) and the specificity are generally very high. Therefore, we used the precision recall curve (PRC) and In the simulated genome above, we assumed that all the donor genomes can contribute to the host genome through HGT. Since closely related genomes have similar kmer frequencies, it will be difficult to detect HGT from closely related genomes. On the other hand, if the donor genome has high evolutionary distance from the host genome, it will be relatively easy to identify HGT with any reasonable methods. Therefore, we next investigated how the evolutionary relationship between the the donor genome and the host genome affects the relative performance of the different HGT detection methods.E. coli K12 that is of the Proteobacteria phylum, Gammaproteobacteria class, Enterobacteriales order, Enterobacteriaceae family and Escherichia genus as host genome and chose 20 donor genomes having different evolutionary relationships with E. coli. Four of them are different species of the Escherichia genus , four of them are in different genus of the Enterobacteriaceae family , four of them are in different families of the Enterobacteriales order , four genomes are in different orders of the Gammaproteobacteria class , and four genomes are in different classes of the Proteobacteria phylum . By transferring fragments between 8 and 40 kbp uniformly picked from these genomes into E. coli K12, we constructed 20 artificial genomes, each of them consists of 10% HGT from a certain single donor genome. We then detected the HGT using the different alignment-free methods and compared them using the same criteria as above.In our simulations, we still used F1-score to evaluate the different methods.In order to study the effect of window length, we continued to use the 20 artificial genomes generated above. Instead of using 5 kbp as the length of sliding window, we changed the window size to 3 and 8 kbp, respectively. Finally, we used the E. coli to B. abortus and K. pneumoniae, respectively. Then we did the same analyses as for E. coli.To see if our results are consistent for different host genomes, we changed the host genome from F1-score and the precision-recall curve to compare the different methods.To evaluate the performances of alignment-free methods on HGT detection over real data, we used a data set constructed in Langille et al. . In thisEnterococcus faecalis V583  that contains seven known genes transferred from other genomes. Since we do not know the whole set of HGT genes, we just investigated if these seven genes are ranked higher than other genes. The higher these genes are ranked by a particular method, the better performance the method is in predicting HGT.We also used the different methods to identify HGT regions of F1-score of the different alignment-free methods for different word size k and Markov order m when needed. The highest F1-score of 0.88 is obtained for CVT(4), followed by CVT(3), F1-score at least 0.87. In comparison with background adusted dissimilarity measures, the widely-used Manhattan and Euclidean distances both have F1-score at most 0.80.Table F1 level, we also plotted the precision-recall curves for the different methods shown in Figure Ma, Eu and d2 showed similar performace. Figures k = 3 or k = 4. Among all methods, CVT(3), CVT(4), F1-score or the precision-recall curves.In addition to comparing the different methods at the optimal CVTree, d2) can probably be explained by the following observations. By removing the background counts of the word patterns, the signals from the most relevant kmers representative of the host genome are amplified while the contributions of irrelevant kmers are mitigated. Therefore, the background adjusted dissimilarity measures perform well in HGT detection.The better performance of the background adjusted methods (CVT(3), CVT(4), d2(5) to represent non-background adjusted methods as candidates for the following studies.Based on the performances of the different methods shown in Table CVT(3), CVT(4), d2(5) based on the 20 artificial genomes described in the “Materials and Methods” section and the results are given in Table E. coli K12.We next investigated the influence of evolutionary distance between the donor genome and the host genome on the performance of the different methods F1 value greater than 0.30 indicating that none of them can successfully detect HGT when the donor genome and host genome are very close. For the second group of donor genomes with Manhattan distance between 0.12 to 0.31, for eight out of ten donor genomes except for V. parahaemolyticus and B. abortus, the optimal F1 scores are moderate between 0.32 to 0.71. Except for E. cloacae, E. vulneris and K. pneumoniae, the background adjusted dissimilarity measures outperform the non-background adjusted measures, some times by a significant margin. For example, when the donor genome is V. parahaemolyticus, the F1-scores for CVT(3), CVT(4), F1-scores for Ma(5), Eu(5), and d2(5) are at most 0.58. Within this group of donor genomes, CVT(4) seems to perform better than CVT(3) when the Manhattan distance between the donor and host genomes is between 0.12 and 0.22, while CVT(3) is slightly better than CVT(4) when the Manhattan distance is between 0.22 to 0.31. The results are reasonable since when the donor and host genomes are relatively close, relative long kmers are needed to separate the transferred fragments from the background. On the other hand, when the donor and host genomes are relatively far apart, relatively short-mers are more discriminative. For the last group of donor genomes with large distances between the donor and host genomes, all the seven methods perform decently well with CVT(3), We divided the donor genomes into three groups separated by horizontal lines in Table F1-score, we also plotted the precision-recall curves for three donor genomes S. sonnei, B. abortus, and C. coli in Figure F1-scores were observed.In addition to the comparison of the different methods based on the optimal E. coli K12. Optimal F1 score of different methods using different window sizes over these 8 genomes are shown in Table F1 score increases as the window length increases from 3 to 8 kbp. But CVT(3) is the most robust with different window sizes and its performance suffers less with the decrease of window size compared with other methods.We further studied the influence of window size on different methods as the performances of alignment-free methods always reply on the sequence length that should be long enough to represent the genomic signature. Besides 5 kbp window size with 500 bp sliding step, we also checked the performance of different methods based on 3 kbp window size with 300 bp sliding step and 8kbp window size with 800 bp sliding step by using the same evaluation approach. Among the 20 artificial genomes that have been generated to study the influence of the genetic distance between the donor genome and host genome, we chose 8 of them in which donors have different order level from that of E. coli to B. abortus and K. pneumoniae, respectively. The complete results are given as Tables To see the robustness of our results on the relative performance of the different alignment-free HGT detection methods with respect to host genomes, we changed the host genome from CVT(3), CVT(4), Eu(5), Ma(5), and d2(5). Similarly, we also evaluated the different methods based on the optimal F1-score as shown in part (b) of Table F1-score are essentially the same as that based on optimal accuracy. In addition, we also plotted the precision-recall curves of the different methods based on this data set and the resulting figures are shown in Figure CVT(3), We next applied the various dissimilarity measures to identify genomic islands generated from HGT for the 118 genomes described in the “Materials and Methods" section. We still chose 40 thresholds as in our simulation studies for each method, and calculated the optimal accuracy that is the highest accuracy one method can achieve under certain threshold. The results are shown in part (a) of Table CVT, CVT(4),e et al. , SIGI-HME. faecalis V583, a genomic region that contains 7 genes (EF2293-EF2299) conferring vancomycin resistance to E. faecalis has been known to have been horizontally transferred , CVTree(k = 4), k = 5), Eu(k = 5), and d2(k = 5) for more detailed comparisons including influence of different factors and their performance on real data sets.We first generated simulated artificial genomes with HGT by using CVTree, d2. In terms of word length, k = 3 or k = 4 seems to perform well in both our simulation and real data analysis.As a conclusion, we evaluated the performance of kmer-based alignment-free methods with different dissimilarity measures, kmer length and Markov order on both artificial genomes and real data sets. Our results suggest the background adjusted dissimilarity measures, Although kmer-based alignment-free methods for HGT detection are more time and memory efficient than alignment-based methods and they do not depend on genome annotation or evolutionary tree, they also have limits. First of all, their performances depend on the evolutionary distance between host and donor genomes. Our study showed alignment-free methods are suitable for HGT detection when host and donor genomes are in different order levels. In addition, the size of sliding window is the smallest length of HGT that can be detected by the kmer-based alignment-free methods, so they are not suitable for identifying HGT smaller than 5 kbp. Furthermore, they are not likely to detect HGT that occurred in the very distant past, as these sequences transferred from the donor genome will ameliorate to reflect the DNA composition of the host genome over time (Lawrence and Ochman, CVTree with word length of 3, Therefore, alignment-free methods are not aimed to replace alignment-based methods in all cases. Instead, they are complementary as each has unique advantages in different scenarios and they also tend to find complementary sets of HGT regions (Tamames and Moya, KT implemented and carried out the computational analyses and wrote the paper. YL provided software for calculating alignment-free dissimilarity measures. FS led the project and finalized the paper. All authors agree to the content of the final paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hearing disorders are reported in thalassemia patients treated with deferoxamine. This study aimed to assess hearing loss in Iranian thalassemia major patients treated with deferoxamine.2 index were used to assess the heterogeneity of the studies. The data were analyzed using Comprehensive Meta-Analysis software version 2 and p<0.05 was considered significant.This review article was designed based on PRISMA guidelines. To review the literature, two researchers studied national and international databases including Iranmedex, Magiran, Medlib, SID, Scopus, PubMed, Science Direct, Web of Science and Google Scholar without time limit until May 2017. Cochran's Q test and IA total of 17 articles involving 1,835 Iranian thalassemia major patients treated with deferoxamine were included in the meta-analysis. The overall prevalence of hearing loss was estimated 27.3% : 19-37.6). The prevalence of sensorineural, conductive and mixed hearing loss was estimated 10.6% (95% CI: 5.7-18.8), 14.6% (95% CI: 10.5-20.6) and 9.1% (95% CI: 5.6-14.6), respectively. No significant differences were noted regarding the relationship hearing loss and mean serum ferritin (P=0.29) and average daily deferoxamine (P=0.30). Meta-regression model showed an increased significance in the prevalence of hearing loss based on the year of studies (p<0.0001).There is a high prevalence of hearing loss in Iranian thalassemia major patients treated with deferoxamine. Therefore, periodic hearing assessments and regular check-ups after the initiation of chelation therapy are necessary. Thalassemia is a common disorder in Southeast Asia caused by genetic defects in the synthesis of hemoglobin chain leading to chronic hypochromic microcytic anemia . These pA simple review of the documents indicated that the prevalence of hearing loss in patients with thalassemia major treated with DFO varied in Iran -13 and nStudy protocol: This review study was designed based on the recommendations of PRISMA  non-random sample size for prevalence estimate; (2) non-Iranian patients; (3) patients not treated with DFO; (4) being irrelevant to the topic; and (5) review studies, letters to the editor, case reports.Search strategy: To review the literature, researchers studied the national and international databases, including Iranmedex, Magiran, Medlib, SID (Scientific Information Database), Scopus, PubMed, Science Direct, Web of Science and Google Scholar without time limit until May 2017. To maximize the comprehensiveness of the research in the databases, we used MeSH keywords including Prevalence, Epidemiology, Hearing Loss, Sensorineural, Drug-Related Side Effects, Deferoxamine, Ferritin, Iron Overload, Chelation Therapy, Thalassemia and Iran and then combined our search with operators AND/OR in English databases. PubMed combination search is shown in appendix 1. References were also evaluated to find further studies.Quality assessment: Researchers examined the selected articles using STROBE checklist , the association between hearing loss and ferritin , the association between hearing loss and age  and for the association between hearing loss and average daily DFO use (mean daily DFO use in case and control groups). we asked specific questions or ambiguities from the authors of the articles via email.Statistical analysis: The binomial distribution was used to calculate the variance of each study for the prevalence of hearing loss in patients with thalassemia major. To study the relationship between hearing loss and ferritin, average daily DFO use and age, odds ratio (OR) and the standardized mean difference (SMD) were used. Cochran's Q test and I2 index were used to assess heterogeneity of the studies. In this study, the heterogeneity was found to be 92.5% for the prevalence of hearing loss, which was a high heterogeneity . So, the random effects model was used in the meta-analysis. To investigate the effect of ferritin (≥3000 vs. <3000) on hearing loss, random effects model was used due to high heterogeneity and for other variables, fixed effects model was used due to low heterogeneity -21. To fSearch results: 280 relevant studies were found in the initial systematic review by two authors and 140 studies were excluded due to duplication. After screening the titles and abstracts, 96 studies were excluded because of their irrelevance to the topic. Finally, 17 studies with appropriate quality entered the meta-analysis : 10.0-14.9). Details of the studies that entered the meta-analysis are shown in Overall prevalence of hearing loss: The rate heterogeneity in this study was 92.5% (p<0.0001). In 14 studies including1,422 Iranian thalassemia major patients treated with DFO, the overall prevalence of hearing loss was estimated 27.3% (95% CI: 19-37.6). The lowest prevalence of hearing loss was reported in a study conducted in Kerman (1.8%) while the highest prevalence was reported in a study conducted in Yazd (63%) (zd (63%) .Subgroup analysis of hearing loss prevalence based on thresholds: The prevalence of hearing loss based on the thresholds 15, 20 and 25 dB HL of sound intensity in audiometry was 26.2% (95% CI: 5.3-69.4), 36.8% (95% CI: 21.8-54.6) and 8.6% (95% CI: 0.4-68.4), respectively and the subgroup difference was not significant (P=0.64) ((P=0.64) .Subgroup analysis of hearing loss prevalence based on provinces: The prevalence of hearing loss based on provinces was shown in Prevalence of sensorineural, conductive and mixed hearing loss: The prevalence of sensorineural, conductive and mixed hearing loss was estimated 10.6% (95% CI: 5.7-18.8), 14.6% (95% CI: 10.5-20.6) and 9.1% (95% CI: 5.6-14.6), respectively  and hearing loss was 1.93 (95% CI: 1.24-3.02) and was statistically significant (p=0.004) . The relMeta-regression model: Meta-regression model was used to assess the relationship between sensorineural, conductive and overall prevalence of hearing loss and year of studies and the p-value was <0.0001, which was statistically significant . The prevalence of sensorineural, conductive and mixed hearing loss was estimated 10.6%, 14.6% and 9.1%, respectively. The subgroup analysis of hearing loss prevalence based on provinces and thresholds of sound intensity was conducted and the difference was statistically significant (p<0.01). In other countries, the prevalence of sensorineural and conductive hearing loss in patients with thalassemia major treated with DFO were reported to be 3.8 - 57% -38 and 4According to thalassemia prevention program , there are 20,000 patients with thalassemia receiving blood transfusions and iron chelation in Iran. DFO and deferiprone are considered as iron chelators in Iran, while DFO is more common and deferiprone is less common due to the complications such as agranulocytosis and the need for the routine evaluation of white blood count (WBC) . In a syThe limitations of this study include: 1. Low sensitivity of national databases for combined search; 2. Failure to provide statistics on the prevalence of hearing loss according to gender; 3. The limitation of the number of studies regarding the relationship between hearing impairment and age, and the mean ferritin that may affect the results.In conclusion, the prevalence of hearing loss in patients with thalassemia major treated with DFO in Iran is high. Potential lesions causing hearing impairment may be present in any part of the auditory pathway. Therefore, periodical hearing assessments and regular check-ups after onset of chelation therapy are necessary. No conflict of interest in this article.