Farm and rural youth have frequent exposure to hazardous noise on the farm and recreationally, and have an increased prevalence of noise-induced hearing loss. There is a lack of programs to prepare this high-risk population to use hearing conservation strategies.The purpose of this project is to test innovative hearing health education programs delivered to a large target group and to determine the effectiveness and sustainability of these programs in promoting hearing health among farm and rural youth. Specifically, this project includes: a) an interactive face-to-face informational program alone, b) an interactive face-to-face informational program followed by an Internet-based booster, and c) a no-intervention control. Sites will include selected affiliates of a major farm youth safety education organization. Data will be collected at baseline, 3, and 12 months. A linear mixed model will be used to compare the effectiveness of the three interventions over time. Descriptive statistics will be used to compare program costs and sustainability ratings.Outcomes of this project will provide knowledge necessary to implement quality and cost-effective services to farm and rural youth, a high-risk and underserved population, that can be implemented and sustained after the study is completed.NCT02472821 Registered 09 Jun, 2015.Clinicaltrials.gov  Noise-induced hearing loss is a highly prevalent, permanent, irreversible condition that disproportionately impacts the future quality of life of farm and rural youth, their families, and communities. An estimated 2 million children and adolescents younger than 20 years of age are exposed to farm hazards as farm residents, farm family workers, hired workers, children of migrant or seasonal workers, or farm visitors resided on farms in 2010  farm youNoise-induced hearing loss (NIHL) is permanent, irreversible, and insidious. Noise-induced hearing loss is characterized by loss of hearing in higher frequencies. The condition is permanent and incurable, and typically progresses slowly and insidiously with continued exposure to high levels of noise. Most people are unaware that they are affected until it is already moderately severe .of tinnitus, and have increased safety risks due to difficulty hearing warning sounds . The proposed study seeks to assess the sustainability of hearing health education programs (with and without Internet boosters) using Schell’s model.Schell and colleagues  have devIn addition, the intervention will be guided by the Model of Effects on Predictors of Intent to Use Hearing Conservation Strategies Intervention Fig. .Fig. 1InAlthough hearing conservation programs for farm and non-farm youth have been successful in changing hearing protector use, , 27–30 eThis project will result in identification of the effectiveness, costs, and sustainability estimates associated with each approach to hearing health education among farm and rural youth. Cost-effective and sustainable hearing health education programs are expected to have the greatest impact on reducing rates of noise-induced hearing loss, tinnitus, and other negative effects of high noise exposure, and improving quality of life in this high-risk and underserved group.This study maximizes opportunity for a sustainable impact on public health by building on a well-established educational program, Safety Day. Ongoing activities in the Safety Day (exclusive of this study) include recruiting, training, and administering consents; the study activities will incrementally increase activities in these areas, leading to a high level of efficiency.The proposed study tests an under-tested intervention approach: the booster. Previous randomized clinical trial (RCT) studies examining the use of boosters on hearing protector use , 33, 34 The proposed study addresses long-standing problems with hearing health education programs which have not been addressed by previous effectiveness studies, i.e., access and program sustainability. The proposed study also uses a previously-established, tested,  free-to-The proposed study uses a strong (RCT) scientific design. Information from this stronger study design will provide more generalizable evidence useful in the future development of hearing conservation programs and health policy, resulting in enhanced opportunity to impact the public’s health.A team of multidisciplinary members with diverse expertise has been formed to accomplish the study aims. Areas of expertise include: 1) development and testing of programs designed to increase use of hearing protection devices among farmers, 2) farm youth safety education, 3) analysis of hearing health education programs, 4) health program cost analysis, 5) assessment of public health program sustainability, and 6) Web hosting and user monitoring.The purpose of this project is to compare the effectiveness and sustainability of new programs designed to increase hearing conservation practices  among farm and rural youth, thereby reducing noise-induced hearing loss, and involves a partnership with the Progressive Agriculture Foundation. Specifically, the study uses a cluster randomized-control design to compare two programs (with a control) for effectiveness and sustainability: a community-based interactive youth educational program alone (Group A); and a community-based interactive youth educational program followed by an Internet-based booster (Group B). Both programs will be delivered through the Progressive Agriculture Foundation (PAF) Safety Day program.targeted to youth offered through Oregon Health Sciences University and partners. The DDVE will be used as a booster in the proposed study.Safety Days are one-day, hands-on workshops that teach farm children safe farm practices. Each year, Safety Days reach more than 98,000 children and adults in over 400 sites across the United States and Canada. These fun, interactive, and hands-on learning experiences result in increased knowledge about safety, as well as positive change in safety behavior –38. The The study aims to test alternative hearing health education programs, and determine their effectiveness and sustainability for farm and rural youth.. In addition, results of the pilot test suggested that overall, the pilot program was acceptable to school officials and students, instruments were reliable and valid, and the brief educational program is feasible.Intervention A consists of the interactive face-to-face hearing conservation program developed by the project team. It consists of discovery-based, developmentally appropriate activities designed to teach common farm and recreational sources of hazardous noise, effects of hazardous noise on ear structure and function, and age-appropriate ways to protect oneself, e.g., walk away, turn it down, and wear protection. The 20-minute lesson includes student involvement in demonstrations and visuals, demonstration and supervised practice in insertion of a child-safe hearing protection device, provision of a sample hearing protector, and elements relevant to the rural and farm community . In a preliminary test with 64 fourth grade students, comparison of pre- and post-test results showed changes of scores in the desired direction, and pre-post-test differences were statistically significantTraining in use of the face-to-face hearing health lesson plan will be provided to selected local safety program coordinators through face-to-face and virtual train-the-trainer sessions conducted by study personnel, and a written lesson plan; the Study Coordinator will also reach out to instructors by offering individualized training and consultation. Coordinators will prepare instructors, include the lesson in their local program, and complete surveys. The hearing health lesson includes use of easily accessible teaching materials, e.g., paper-based visual aids, locally-sourced noisy equipment (e.g. electric leaf blower), pipe cleaners, and a sound meter.www.dangerousdecibels.org/exhibit/virtual-exhibit/). The booster is developmentally appropriate, focuses on sources of sounds, consequences of noise exposure, and prevention of hearing loss. In a study testing the effectiveness of the DDVE site when used alone, students showed improvement in knowledge immediately after the intervention, although improvements were not sustained after 3 months. The Internet-based booster is a user-driven activity where students explore a variety of learning activities at their own pace. Highly interactive game-based activities include rating noise sources  as safe or dangerous, determining the best course of action  in a variety of noisy situations, and choosing a course of action when confronted with social non-support of use of hearing protection. A computer (with Macromedia Flash® and Shockwave®) and Internet connection  is required for participation.Intervention B consists of an Internet-based educational booster. The Internet-based booster reinforces Intervention A content through use of the Dangerous Decibels® virtual exhibit (DDVE), an existing online educational program consisting of eight activities , a face-to-face interactive lesson followed by an Internet-based booster (Group B), and a no-invention control . Local Safety Day programs will serve as clusters.Farm and rural youth, as well as Safety Day instructors and coordinators will be sampled. Inclusion criteria for youth participants include enrollment in grade 4, parental consent, English speaking, and attending a Safety Day event included in the cluster sampling. Inclusion criteria for coordinators include demonstrated ability to successfully coordinate a Safety Day program, interest in the study, opportunity to implement the lesson in the designated study period, and willingness to comply with study procedures. Site selection will be aimed at including a diverse study sample, from the perspective of race, ethnicity, and geography. Inclusion criteria for instructors include accepting a hearing health teaching assignment at a Safety Day event included in the cluster sampling.The study will use pre-existing Safety Day protocols for recruitment and consenting participants, with some modifications. Local Safety Day programs from diverse areas across the US will be offered the opportunity to enroll by a member of the study team. Recruitment of Safety Day instructors will be done in collaboration with the study team, who will collaboratively develop instructor recruitment strategies. Coordinators (and delegates) will recruit youth to participate.With parental consent, youth will select their preferred communication media , which will be used to communicate study activity reminders and surveys. The study coordinator will distribute reminders, and 3- and 12-month follow-up surveys by email, postal mail, or phone. Coordinators will access youth for 12 month follow-up as they return for Safety Day participation in the subsequent yearly meeting. The study team will support the PAF Safety Day trainers, coordinators, and instructors through site visits and frequent email and telephone contacts. Participating youth will be recognized with a $5 cash incentive for completing up to three (depending on study arm) surveys. Coordinators will be awarded checks on submission of study materials (surveys) as tokens of appreciation for their contribution to accomplishment of study goals .To reduce contamination of intervention effects across participants, randomization will occur at the level of local Safety Day programs. Participating Safety Day programs will then be randomly assigned to one of the three study conditions so that individuals within the same site receive the same intervention.Program process and outcomes will be measured by surveys completed by students, instructors, and coordinators. Instruments are described below, and instrument means, standard deviations, and alpha coefficients are displayed in Table After obtaining parental informed consent and soliciting the assent of child participants, pre- and post-intervention data will be collected from youths. Pre-test data will be collected via pencil-and-paper survey forms prior to the delivery of content. Three- and 12-month post-test data will be collected via the youth’s preferred communication medium following the booster intervention. Instrument means, standard deviations, and alpha coefficients are summarized in Table The 3-item, multiple-choice instrument measures youths’ use of hearing conservation strategies  during the previous 3-months. The instrument has a reading grade level of 6.9. A sample item from this instrument is, “During the past 3 months, how often did you wear earplugs or ear muffs when you were around loud sound?”If earplugs were around when I needed them, I would use them.”The 6-item, multiple-choice instrument measuring youths’ intent to use hearing conservation strategies . The insThe 12-item, multiple-choice instrument to measure youths’ knowledge of noise, its effects on hearing, and hearing conservation strategies was adopted from items used by Martin et al. to measure this concept among elementary students . A samplvery to not at all embarrassing).” The instrument has a reading grade level somewhat higher (11.3) than the other instruments, due to use of the parenthetical phrase “if no one else is wearing them” and the multi-syllabic word, embarrassing. However, in previous use and pretesting, 4th grade students responded to this item without difficulty.The 1-item, multiple-choice instrument to measure interpersonal factors influencing use of hearing conservation strategies among youth was adopted from a set of items used by Martin et al . The iteDose of intervention 2 will be measured as youth log into the DDVE site where they will enter identifying information which will allow for their time on the DDVE Web site to be tracked. Consistent with standard dose measurement methods, inactive time on site will result in stopping the time-on-site clock. The study team will monitor local Safety Day coordinators and instructors using phone calls and email messages timed to coincide with program planning, delivery, and follow-up to support delivery of the intervention as planned, and to accomplish submission of post-intervention surveys. Reach will be measured by counting the number of Safety Day youth (by site) who received the hearing health lesson. Recruitment will be measured using a coordinator survey item, “List and describe the youth recruitment procedures used.” Context includes the organizational factors, barriers, and facilitators to program delivery. Context will be measured using coordinator and instructor survey items, “What were the barriers to implementing the lesson?” and,”What were the facilitators to implementing the lesson?”Measurement of program process will include program fidelity, dose delivered, dose received, reach, recruitment, and context using instruments designed by the interdisciplinary study team for this purpose. The process evaluation plan was developed with consideration to system resources and program characteristics and context. Sample measurement items for the interactive Safety Day program follow. Fidelity will be measured by research team review of lesson audiorecordings for key points. Group C (control) site instructors will respond to a survey querying the inclusion of hearing conservation messages within the context of their sessions not specific to this topic . Dose of Intervention 1 will be measured using an instructor survey item, “How many minutes were spent on the lesson with each group of learners?” This 3-item instrument measures youths’ sex, age, and race/ethnicity.Personnel : We will estimate hearing education program personnel costs beyond those already incurred through previously established Safety Day programs, i.e., youth, coordinator, and instructor time.Instructional materials: We will value all materials used to implement the lesson , including lesson development costs and youth time on public-access or home-based computers.Travel: We will estimate travel costs incurred by coordinators, instructors, and youth to participate in pre-program training and the local Safety Day program.The cost of implementing the program will be based on data associated with program personnel, instructional materials, and travel incurred with the educational process.to a little or no extent and to a great extent.Schell’s  tool wild of .48 compared to the effect of the control treatment. This is the effect size on intention to use hearing conservation strategies we found in our preliminary study and is just under what Cohen defined as a medium sized effect. Other necessary inputs into Optimal Design were ranges of the average number of students per site and estimates of the intraclass correlation coefficient (ICC), a measure of the tendency of individuals in the same site to have similar (correlated) responses. To determine the number of sites needed, we examined numbers of site ranging from 20 to 80, and a value of the ICC of .10  to the no-intervention control treatment. A secondary component of Aim 1 is to compare two interactive hearing interventions on youth.site variable. More precisely, we will use random intercept model for the site variable. We assume the site variable follows a normal distribution with mean zero and is independent to the error terms in the mixed model. The hypothesis is that there will be a significant effect of hearing intervention on the use of hearing conservation strategies and the intervention group will have higher use of hearing conservation strategies than the control group. If the effect of intervention is significant, follow up descriptive statistics on mean intent by time by treatment group will allow us to describe the effects, for example to determine whether each treatment led to more improvement in intent to use than the control condition and how big those effects were. Further tests within this analysis will compare the effects of the two interactive hearing interventions on youth.The primary measure to assess effectiveness is the 6 item scale of intention to use hearing conservation strategies . The knowledge scale and the scale of interpersonal influences will also be examined. All individuals will be asked to assess these measures before intervention (baseline), and at 3 months and 12 months after the intervention. Since the cluster randomized design will be used to assign interventions and repeated measurements will be collected from the same individuals, linear mixed effects model will be used to investigate the effect of the interventions. The outcome variable is the scale of intention to use hearing conservation strategies. The important/main fixed effect is a categorical variable identifying the type of interventions. In addition, demographics variables will be included in the mixed model as fixed effects to increase the precision of estimates. The random effect in the model is the Costs in dollars  will be summarized using mean and standard deviation for each site. Costs in time from volunteers and students will be summarized using mean for each site. At site level, the difference in cost between three intervention groups are compared using one-way analysis of variance. If the effectiveness of intervention is significant, the costs to effective ratio will be calculated to quantify incremental cost of effectiveness.t-test or chi-squared test will be used to compare the difference between groups A and B. These testing results will help identify strengths and weaknesses of the Safety Day program (and the hearing conservation strategies within them) to pinpoint where efforts can be made to enhance the sustainability of the programs. Subsequently, a panel of Safety Day leaders and study team members will meet via teleconference to compare sustainability assessments of the two programs, and implications for program development.Safety Day coordinators (Groups A & B), program leaders, and research team members will complete the 40 item questionnaire rating 8 domains of sustainability. Two sample The study procedures include measures to protect children from coercion. Parental consent will be obtained prior to data collection; children who are not enrolled in the study will also receive the Safety Day intervention, but no data will be collected from them. Safety Day personnel are well-known local community members who are invested in the health and safety of youth in their own community, and often have a history of volunteering with the program for many years.81 In other reports, self-report and observations were highly correlated [The study effectiveness measures are based on participant self-report, which is vulnerable to social desirability bias. However, in a nationwide sample of farmers participating in a test of hearing conservation interventions, participants scored low on a standard measure of social desirability reporting.rrelated , 43.Successful recruitment and retention strategies used in previous longitudinal studies by McCullagh will be used in this project to maximize enrollment and reduce attrition; these strategies have resulted in consistently high study recruitment  and retention  across studies, and include attention to communication with participants, high regard for participant autonomy, careful recordkeeping of participant progress through the study, and fastidious attention to follow-up. For missing data that does occur, we will use multiple imputation to impute missing values while providing a theoretically appropriate indication of uncertainty assuming the data are missing at random . The samThe PAF Safety Day program is delivered in the US exclusively in English, and primarily to residential (non-migrant) youth. This is a significant limitation of the study, and an area for future program development.and national  educational benchmarks for science, physics, math, and health education.This translational study design facilitates the transition of the program from research study to full-scale implementation within the Safety Day program, should study results demonstrate satisfactory effectiveness, increasing access to hearing conservation services to an usually large audience in a very short period of time. The potential exists for adding additional learning activities for use in repeating the lesson in subsequent years to the same youth. Also, opportunities for distributing the program through other venues will be explored, e.g., NIDCD website, selected teacher associations , and selected Web-based lesson planning resources used by science, health, and elementary teachers, as the program includes many components of state Farm youth are exposed to farm noise hazards as farm residents, farm family workers, hired workers, children of migrant or seasonal workers, or farm visitors, and have an increased prevalence of noise-induced hearing loss. Although previous tests of programs to promote hearing conservation among farm youth have demonstrated increases in hearing protector use or intention, their impact has been limited by program scope, cost, and sustainability.This proposed study capitalizes on partnerships with existing infrastructures . These partnerships will serve to incorporate hearing health education into already existing systems designed to deliver health and safety educational programming to rural students. The interventions are aimed broadly at situation-appropriate hearing conservation strategies , and not limited to use of hearing protection devices. The proposed interventions are developmentally appropriate, use a highly interactive and discovery learning approach, were successfully pilot tested, and are ready for RCT.This RCT test of interventions is designed to determine the most cost effective and sustainable approach to increasing use of hearing conservation strategies among farm and rural youth. Further, given effectiveness of the interventions, a system is already in place to disseminate programs to reach a larger farm youth audience. Results of this study will inform future intervention studies, interventions aimed at farm youth, and interventions to increase use of hearing conservation strategies, as well as offer a base for developing programs for non-English speaking children.A strong team of researchers and the largest farm youth health and safety education program in the US have formed a partnership in an effort to address this important health problem in the interest of reducing hearing loss and tinnitus, and improving the quality of life of this high risk and underserved group.The study protocol was reviewed by the University of Michigan Health Sciences and Behavioral Sciences Institutional Review Board (HUM00077214) who determined that the proposed research conforms to applicable guidelines, State and federal regulations, and the University of Michigan’s Federalwide Assurance (FWA) with the Department of Health and Human Services (HHS). Parental consent will be obtained prior to data collection from youth; adult participants will be required to provide their own consent.


Congenital human cytomegalovirus (HCMV) occurs in 0.5–1% of live births and approximately 10% of infected infants develop hearing loss. The mechanism(s) of hearing loss remain unknown. We developed a murine model of CMV induced hearing loss in which murine cytomegalovirus (MCMV) infection of newborn mice leads to hematogenous spread of virus to the inner ear, induction of inflammatory responses, and hearing loss. Characteristics of the hearing loss described in infants with congenital HCMV infection were observed including, delayed onset, progressive hearing loss, and unilateral hearing loss in this model and, these characteristics were viral inoculum dependent. Viral antigens were present in the inner ear as were CD3+ mononuclear cells in the spiral ganglion and stria vascularis. Spiral ganglion neuron density was decreased after infection, thus providing a mechanism for hearing loss. The lack of significant inner ear histopathology and persistence of inflammation in cochlea of mice with hearing loss raised the possibility that inflammation was a major component of the mechanism(s) of hearing loss in MCMV infected mice. Congenital infection with human cytomegalovirus (HCMV) is the most common viral infection of the fetus and occurs in 0.5–2.0% of all live births in most regions in the world. Infection of the fetus can result in a spectrum of end-organ disease, including long term damage to the central nervous system (CNS). Although less than 10% of infected infants exhibit clinical evidence of end-organ disease, up to 10% of the total number of infected infants develop hearing loss. Mechanisms of disease leading to hearing loss are poorly understood because of the limited availability of pathological specimens and accessibility of the inner ear. Existing small animal models fail to recapitulate many features of this infection of the inner ear. In this report we describe a mouse model in which newborn animals infected peripherally with murine CMV develop hearing loss following hematogenous spread of virus to the inner ear. Hearing loss occurs in 30–50% of animals and characteristics of hearing loss in infants with congenital HCMV infection, including delayed onset of hearing loss, progressive hearing loss, and unilateral hearing loss were present in infected mice. Our findings suggest that host derived inflammatory responses and not direct virus-mediated cytopathology are responsible for hearing loss. Findings from this study provide insight into potential mechanisms of hearing loss in infants with congenital HCMV infection. Human cytomegalovirus (HCMV) is the largest human herpesviruses and an important pathogen in immunocompromised hosts ,2. Althond trimester human fetus, this model has allowed study of the effects of CNS infection on brain development, including auditory development that takes place postnatally in the mouse. In this report, we describe findings in this model that are consistent with the loss of spiral ganglion neurons and/or inflammation in the stria vascularis in infected mice as mechanisms of hearing loss. In addition, our results also suggested that virus-induced inflammation is likely a major component of mechanisms leading to hearing loss in MCMV infected mice.The mechanism(s) of disease leading to hearing loss following intrauterine HCMV infection are not understood. In addition, hearing loss can develop months after the newborn period in about 50% of infected infants and be progressive in other infants, suggesting that ongoing virus replication could contribute to hearing loss ,12. Stud1–104 copies of MCMV DNA per mg of cochlear tissue could be detected in 100%, 77%, and 50% of cochlea at PNd14, 4 mo, and 12 mo, respectively  and mock infected controls using 2 modalities, (1) Distortion Product Otoacoustic Emission (DPOAE), and (2) Auditory Brainstem Evoked Response (ABR). DPOAE was used to measure otoacoustic emissions from the inner ear in MCMV and mock-infected mice with DPOAE thresholds defined as the lowest stimulus intensity at which an otoacoustic emission was detectable. DPOAE thresholds were measured at 4 frequencies  and although thresholds were high for both infected and mock-infected mice, we observed an increased DPOAE threshold at only a single frequency (8 kHz) in MCMV infected mice .Auditory brain stem response (ABR) testing served as our standard measure of auditory function. ABR sound pressure levels (SPL) thresholds were assigned as the lowest intensity stimulus at which measurable ABR waveforms were detected as depicted in recordings from a mock and MCMV infected mouse . In thisIn a significant number of infants congenitally infected with HCMV hearing loss can be unilateral, thus overall hearing function can be estimated by determining auditory function for the best and worst ear ,12. FurtAn important clinical feature of hearing loss associated with congenital HCMV infection is delayed onset hearing loss and/or progressive hearing deficits in a significant number of infants ,12. SeriABR thesholds were noted in about 50% of Smith MCMV-infected mice with documented hearing loss . SignifiTo investigate the influence of virus inoculum on progressive hearing loss, hearing was analyzed as a function of time post infection in mice given 50 and 200PFU of BAC cloned MCMV. There was an increase in ABR thresholds between PNd42 and 78 in mice given both 50PFU and 200PFU as compared to mock infected mice . ImportaThe findings that, (i) virus persisted in the inner ear for over four months , (ii) anTo determine if further histologic analysis of the inner ear in mice with increased ABR thresholds could point to mechanisms of disease in the auditory system, cochlear tissue from a group of mock and MCMV infected animals with and without hearing loss were analyzed. No obvious differences in cochlear histology were found between mock infected, infected mice with normal hearing, and infected mice with abnormal hearing . BecauseEven though the development of hearing loss in MCMV infected mice was related to the magnitude of the intial viral inoculum, the lack of a direct relationship between cochlear viral DNA and increased ABR thresholds raised the possibility that MCMV-induced inflammation and not direct viral cytopathic effects, could be responsible for hearing loss in MCMV infected mice. This mechanism was also suggested by both limited cytopathology in the cochlea  and the Although the association of hearing loss with congenital HCMV infection has been recognized for over 4 decades, the pathogenesis of this sequelae of congenital HCMV infection remains poorly understood. Early studies of temporal bones from a small number of infants and older children with congenital HCMV infections described viral antigens and viral particles in the inner ear, inflammatory cellular infiltrates in the inner ear, and degeneration, calcifications, and fibrosis of structures in the inner ear in older children . More reDeficits in hearing in MCMV infected mice were documented primarily by findings of increased ABR thresholds. Although our findings in DPOAE testing suggested normal hair cell function in infected animals, elevated stimulus thresholds in our DPOAE protocol could have obscured abnormal test results. However, the lack of cytopathology in the Organ of Corti, increased ABR thresholds, and the loss of spiral ganglion neurons suggested that hearing loss in this model was consistent with lesions in the auditory system other than hair cell loss. In contrast to our results, two recent studies utilizing intracranial (IC) inoculation of newborn mice with MCMV to produce hearing loss documented hair cell loss ,19. ThesOther small animal models of CMV associated hearing loss have been reported, including congenital infections in guinea pigs (gpCMV). Although this model includes hematogenous spread of virus to the inner ear, it is also associated with modest and often inconsistent inner ear infection, hearing loss in animals without inner ear infection, and confounding issues associated with the severity of maternal infection required to reproducibly achieve intrauterine transmission . This laLarger initial inoculums of MCMV resulted in increased amounts of virus in the inner ear and an increase in the incidence of hearing loss suggesting a direct relationship between virus replication in the cochlea and hearing loss; however, by PNd 42 there was no correlation between virus in the cochlea and hearing loss suggesting that persistent virus replication in the cochlea was not directly related to hearing loss. Thus, it could be argued that only the quantity of virus present in the cochlea early in infection was related to the development of hearing loss. Possible explanations that could account for these findings include the increased likelihood of infection of specific cell types in the inner ear following infection with larger inoculums or, alternatively, inoculum dependent induction of cochlear inflammation early in infection that was not reflected in levels of virus replication at later time points. Thus, larger virus inoculums resulted in increased virus replication and as we observed, a corresponding increase in expression of some proinflammatory cytokines and chemokines early in infection . These eObservations in other small animal models of hearing loss as well as findings in human temporal bones are consistent with a mechanism(s) of virus-induced inflammation; however, specific lesions and the pathways leading to hearing deficits have not been well described. Virus infected cells and mononuclear cells in the spiral ganglion are common to rodent models as well as tissue from infected fetuses, findings suggesting that disruption of spiral ganglion function could contribute to hearing deficits in MCMV infected mice. The loss of spiral ganglion neurons following MCMV infection in our model are similar to findings reported in a more limited analysis of a murine model of CMV associated hearing loss . Thus, lA decrease in the number of spiral ganglion neurons provided a potential mechanism of hearing loss in infected animals secondary to loss of afferent input into cochlear nuclei during this critical time of auditory development ,36. StudThe finding of virus infected cells and CD3+ mononuclear infiltrates in the stria vascularis suggested additional mechanisms of hearing loss in MCMV infected mice. This region of the inner ear is critical for maintaining the unique electrolyte composition of the extracellular fluid within the Organ of Corti that contributes to electrical potential of the endocochlear fluid, the endocochlear potential ,47. HairIn summary, we have described a model of hearing loss in mice inoculated early in the postnatal period with MCMV that closely recapitulates key aspects of hearing loss associated with congenital HCMV infection. Our studies have documented the dependence of viral replication in the cochlea on the initial viral inoculum size and in turn, the level of expression of proinflammatory molecules in the cochlea as well subsequent increases in the ABR thresholds on the level of virus replication during this early time in auditory development. Although virus infected cells could be detected in various regions of the inner ear, significant cytopathology or tissue damage was absent arguing that the mechanism(s) of hearing loss following this infection could have a significant inflammatory component.The University of Alabama-Birmingham Institutional Animal Care and Use Committee (IACUC) guidelines were followed for all animal experiments and breeding. These guidelines are in strict compliance with the NIH (OLAW Assurance Number—A32555-01) guidelines. Animals were euthanized with carbon dioxide under regulated flow. Following carbon dioxide, cervical dislocation was implemented to ensure animals were completely euthanized prior to carrying out experimental protocols, according to the UAB Animal Resource Program (ARP) guidelines. An approved protocol (APN09024) was obtained from IACUC (UAB) for all experiments involving animals and animal tissues carried out at UAB. All procedures involving animals and their care conducted at the Faculty of Medicine, University of Rijeka were approved by the Veterinary Department of the Ministry of Agriculture, Croatia —Assurance no. A5905-01 (effective until 10/31/2019) and were conducted in compliance with institutional guidelines, as well as with national  and international Directive 2010/63/EU of The European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes, Guide for the Care and Use of Laboratory Animals, National Institutes of Health, The National Academies Press, Washington, DC) laws and policies.Pathogen-free BALB/c mice were obtained from a colony propagated from animals purchased from Charles River Laboratories. All breeding and experiments were done in accordance with the guidelines of the University of Alabama at Birmingham Institutional Animal Care and Use Committee and with the University of Rijeka-Croatia animal use policies. Auditory testing including ABR and DPOAE (see below) measurements were performed in anesthetized animals (ketamine (75 mg/kg) and medetomidine (1 mg/kg) delivered i.p.). Animals were maintained at 37°C during all testing and monitored for movement.Virus stocks ) were propagated in mouse embryonic fibroblasts. In some experiments, a virus that has been determined by sequence analysis and characterization in-vivo to be wild type Smith virus was recovered from an infectious BAC clone of MCMV kindly provided by Dr. Ulrich Koszinowski (University of Munich) . Virus rDPOAEs were measured using Tucker Davis Technologies  System III equipment and software using published protocols . StimuliABRs were also measured with the TDT System III system, using tone pips at 4, 8, 16, and 32 kHz (90 dB to 20 dB in -10 dB steps), as well as broadband click stimuli (90 dB to 20 dB in -5 dB steps). Signal was detected using 3 stainless steel needle electrodes , amplified, band-pass filtered , and averaged over 750 sweeps. The ABR threshold was defined as the lowest intensity signal at which an ABR waveform was detectable. We adapted protocols from several sources including material provided during a training course in audiometric testing at Jackson Labs  and published protocols .th section). Immunohistochemical (IHC) staining for the IE-1 protein (pp89) encoded by MCMV IE-1 was performed as described previously [Animals were exhaustively perfused initially with phosphate buffered saline  and then with 4% paraformaldehyde (PFA) as described . Cochleaeviously . Anti-CDeviously .Nucleic acids were isolated from cochleae using several protocols depending on downstream applications. In the initial experiments, nucleic acids were isolated using a QIAGEN M48 robot and MagAttract RNA Tissue Mini M48 kit as per the manufacturer’s instructions . In latter experiments, RNA and DNA nucleic acids were isolated using SQ Total RNA Kit and HP Total RNA Isolation Kit  with minor modifications. Quantitative real-time PCR was performed by amplification of a fragment of immediate-early 1 (IE1) exon 4 using the primers forward, 5'-GGC TCC ATG ATC CAC CCT GTT A-3' and reverse, 5'-GCC TTC ATC TGC TGC CAT ACT-3'. The probe (5'-AGC CTT TCC TGG ATG CCA GGT CTC A-3') was labeled with reporter dye FAM and quencher dye TAMRA. To quantify viral genome copy number, serial dilutions (log10) of a plasmid with the cloned IE1 exon 4 PCR product was used to create a standard curve. This assay has a sensitivity of between 50–100 copies of MCMV DNA. For the detection of inflammatory network genes, the Mouse Antiviral Response PCR Array  and for proinflammatory chemokines and cytokines (PAMM-150Z) were used according to manufacturer's protocol. Quantitative real-time PCR was conducted using TaqMan Gene Expression master mix (Qiagen). Samples were run in triplicate using a two-step amplification protocol on the Applied Biosystems 7500 Real-Time PCR System  of life and 50ng reverse transcribed and assayed using PCR arrays as described in Materials and Methods. Number of animals are listed in parentheses and PCR results compared to RNA prepared from cochlea of mock infected animals . Results are expressed as fold increase over signal from cochlea obtained in mock infected animals with statistical measures of differences shown as (TIF)Click here for additional data file.S1 FigNewborn mice infected with 200 PFU of tissue culture derived Smith strain MCMV, or mock infected, were sacrificed on postnatal day (PNd) 11 and after perfusion with PBS and fixation, the inner ears were removed, decalcified and embedded in paraffin as described in Materials and Methods. After sectioning, de-paraffinization, sections were stained with hematoxylin and eosin (H&E) and photographed (x40). Panel C and F represent digitally enlarged images of Panel B and E respectively.(TIF)Click here for additional data file.S2 FigAfter extensive perfusion, temporal bones from mice infected with 200PFU Smith MCMV were harvested on PNd7, fixed and embedded in paraffin as described in Materials and Methods. Sections were cut and stained with H&E . Boxed areas demonstrated large cell with characteristics of MCMV infected cell. Other sections were reacted with anti-MCMV IE-1 antibodies and developed with HRP anti-mouse IgG followed with diaminobenzidine to produce brown color in infected cells . Scale bars shown in lower right corner.(TIF)Click here for additional data file.S3 FigCochlea from mock infected and MCMV infected mice were harvested on PNd7, decalcified, fixed and embedded in paraffin as described in Materials and Methods. MCMV infected cells were stained with anti-IE-1 antibodies and then developed with HRP anti-mouse IgG and diaminobenzidine (brown). This section reveals infected cell in marginal layer of stria vascularis. Panel A at 20x  and panel B at 100x . Panel C is digitally enlarged image of panel B.(TIF)Click here for additional data file.S4 Fig○; n = 25) and MCMV infected  mice. Mice were tested at PNd42 as described in Materials and Methods. (*) indicates significant difference in response at 8 kHz.DPOAE revealed a significant differences at only one frequency between mock infected ((TIF)Click here for additional data file.S5 FigAfter extensive perfusion, cochlea from mice between PNd42-60 were harvested, decalcified, fixed and embedded in paraffin as described in Materials and Methods. Following sectioning and staining with H&E, individual sections were viewed by blinded observer with experience in cochlear histology. Sections from 10 different cochlea were submitted for review and four representative sections are presented this figure. Samples 127 and 133 were from mock infected animals and samples 147 and 211 were from infected animals. The ABR thresholds for these ears are displayed at the bottom of each panel.(TIF)Click here for additional data file.S6 FigAfter extensive perfusion, cochlea from mock infected and MCMV infected mice were harvested on PNd7, decalcified, fixed and embedded in paraffin as described in Materials and Methods. Following sectioning, tissue was reacted with anti-Tuji 1 followed by HRP conjugated anti-mouse IgG and developed with diaminobenzidine(brown). Note the decreased density of Tuji 1+ cells in section from infected mouse .(TIF)Click here for additional data file.


Mycoplasma hyopneumoniae (M. hyopneumoniae) was assessed in neonatal piglets. To determine whether CMI and AMI responses could be induced in piglets with MDI, piglets with high and low levels of maternal M. hyopneumoniae-specific immunity were vaccinated against M. hyopneumoniae at 7 d of age. Piglet M. hyopneumoniae-specific antibody, lymphoproliferation, and delayed type hypersensitivity (DTH) responses were measured 7 d and 14 d post vaccination.Passively acquired maternal derived immunity (MDI) is a double-edged sword. Maternal derived antibody-mediated immunity (AMI) and cell-mediated immunity (CMI) are critical immediate defenses for the neonate; however, MDI may interfere with the induction of active immunity in the neonate, i.e. passive interference. The effect of antigen-specific MDI on vaccine-induced AMI and CMI responses to M. hyopneumoniae-specific MDI failed to show vaccine-induced AMI responses; there was no rise in M. hyopneumoniae antibody levels following vaccination of piglets in the presence of M. hyopneumoniae-specific MDI. However, piglets with M. hyopneumoniae-specific MDI had primary (antigen-specific lymphoproliferation) and secondary (DTH) M. hyopneumoniae-specific CMI responses following vaccination.Piglets with M. hyopneumoniae-specific CMI was not subject to passive interference by MDI. Further, it appears that both maternal derived and endogenous CMI contribute to M. hyopneumoniae-specific CMI responses in piglets vaccinated in the face of MDI.In this study neonatal  Infectious disease is a major contributor to morbidity and mortality among infants, children, and other young animals. Ideally, strategies should be practiced at an early age to confer protection from infectious disease-related morbidity and mortality experienced later in life. Vaccination is a commonly used disease intervention strategy; however, there are many issues that complicate vaccinating neonates including vaccine safety and vaccine efficacy. Vaccination regimens may be unsuccessful in stimulating protective immunity in neonates due to both ontogenic immune immaturity and passively acquired maternal derived immunity (MDI) interfering with active immune development.Escherichia coli[Mycoplasma hyopneumoniae (M. hyopneumoniae) [M. hyopneumoniae[Bordetella bronchiseptica[MDI is a critical contributor to the neonatal immune response. MDI is passively transferred to neonates across the placenta and via colostrum and milk in humans and mice -3 but onchia coli,8, Mycopumoniae) , Transmiumoniae) , and Porumoniae) . Howeverneumoniae, Bordetehiseptica, Pseudorhiseptica, Swine Ihiseptica,16, and hiseptica. FurtherTrichinella spiralis and Coccidioides immitis antigens, as evidenced by antigen-specific proliferation and nematode load, and DTH, respectively, has been demonstrated in neonatal mice born to vaccinated dams [M. hyopneumoniae vaccinated dams are transferred to piglets and participate in the in vivo neonatal CMI response to M. hyopneumoniae upon antigen challenge [Maternal CMI that is transferred to neonates across the placenta or in colostrum clearly participates in the neonatal CMI response. Transfer of functional tuberculin-specific immune cells, as evidenced by delayed-type hypersensitivity (DTH), has been demonstrated in infants from vaccinated mothers . Transfeted dams ,20. Furtted dams . We havehallenge . Still, M. hyopneumoniae[M. hyopneumoniae-specific cells participate in the immune response following M. hyopneumoniae antigen challenge in piglets [M. hyopneumoniae vaccination with CMI but not AMI responses when vaccinated in the face of M. hyopneumoniae-specific MDI in a field setting. Piglets with M. hyopneumoniae-specific MDI failed to show vaccine induced AMI responses. In contrast, piglets with M. hyopneumoniae-specific MDI and vaccinated against M. hyopneumoniae developed primary and secondary M. hyopneumoniae-specific CMI responses.CMI is critical in the immune response to neumoniae-26 and m piglets . This stM. hyopneumoniae-specific antibodies  ratios compared to before vaccination (p = 0.0001) and compared to sows not vaccinated as part of this study , and there is no evidence of transplacental infection with M. hyopneumoniae. Prior to colostrum ingestion piglets did not demonstrate M. hyopneumoniae-specific immunity, either AMI .Newborn piglets are naïve to I Figure A or CMI I Figure B. FollowM. hyopneumoniae vaccine was administered when piglets were 7 d of age. Piglets were vaccinated at 7 d of age in an attempt to stimulate endogenous immunity in the presence of transferred antigen specific MDI. M. hyopneumoniae-specific antibody levels did not differ among piglets from nonvaccinated sows relative to piglet vaccination status 7 days post vaccination  from 7 dpv to 14 dpv; however, vaccinated piglets from vaccinated sows (Vs Vp) had lower M. hyopneumoniae-specific antibody S:P ratios at 14 dpv compared to 7 dpv (p = 0.05). Vs Np piglets had greater M. hyopneumoniae antibody S:P ratios compared to Vs Vp piglets 14 dpv  were detected from BMC across piglets at both 7 dpv and 14 dpv (data not shown). Vaccination in the presence of MDI (Vs Vp piglets) resulted in earlier M. hyopneumoniae-specific proliferative responses (7 dpv) compared to all other groups of piglets  and none responded to saline at both injection times . M. hyopneumoniae-specific DTH lesions were detected in some piglets across all treatment groups at both time points  compared to all other treatment groups at 7 dpv  and Vs Vp piglets (p = 0.01)  14 dpv. The mean orthogonal diameter of M. hyopneumoniae-specific DTH lesions was significantly larger at 14 dpv compared to 7 dpv for Ns Vp piglets only.To assess piglet s Figure . Vs Vp pv Figure B. The mes p = 0.0 and Vs VMaternal derived antibodies and lymphocytes acquired across the placenta or postnatally in colostrum or milk are critical participants in the neonatal immune response. Neonatal vaccination has been practiced across a variety of antigens in an effort to confer protection from specific pathogens during the period in which the neonatal immune system matures and maternal antibodies wane. However, MDI may interfere with immune priming and the generation of memory responses following neonatal vaccination.M. hyopneumoniae vaccination when vaccinated in the face of maternal M. hyopneumoniae-specific immunity. To determine whether CMI and AMI responses could be induced in piglets with MDI, piglets from M. hyopneumoniae vaccinated and nonvaccinated dams were vaccinated against M. hyopneumoniae at 7 d of age. Piglet M. hyopneumoniae-specific antibody, lymphoproliferative, and DTH responses were measured 7 dpv and 14 dpv. Vaccination of piglets from vaccinated sows did not induce AMI responses in those piglets; there was no rise in M. hyopneumoniae S:P ratios following piglet vaccination. In contrast, evidence from both antigen-specific proliferation and DTH testing demonstrates that M. hyopneumoniae-specific CMI priming and anamnestic responses are induced following vaccination of piglets with M. hyopneumoniae-specific MDI. Taken together, neonatal piglet M. hyopneumoniae-specific CMI responses induced by vaccination are not (at least wholly) inhibited by passive interference with M. hyopneumoniae-specific MDI.This study was conducted to determine whether neonatal piglets respond with AMI and/or CMI responses to M. hyopneumoniae-specific AMI responses within 14 dpv. These results are in agreement with Hodgins et al. [M. hyopneumoniae antibody titer 9 weeks post vaccination in piglets vaccinated at 14 d of age in the presence of high levels of antigen-specific MDI [M. hyopneumoniae antibody S:P ratios in piglets vaccinated in the face of MDI is most likely due to passive interference; however, this cannot be concluded with certainty in the current study as a group of vaccinated piglets lacking M. hyopneumoniae-specific maternal immunity was not included. However, piglets with low levels of maternal M. hyopneumoniae-specific AMI at the time of vaccination did produce M. hyopneumoniae-specific antibodies following vaccination, indicating that vaccination can induce AMI responses in 7 d old pigs dependent upon the level of maternal antigen-specific AMI in agreement with [M. hyopneumoniae vaccination with IgM responses; however, the ELISA test used lacked isotype specificity and IgG likely overshadowed any IgM response in all samples tested. While including a group of SPF piglets may have resulted in more clear interference results, SPF piglets are not representative of most commercial pigs. Further, as M. hyopneumoniae vaccination is one of the most commonly used vaccines across swine farms [M. hyopneumoniae S:P ratios were greater in Vs Np piglets compared to Vs Vp piglets at 14 dpv; this difference may be attributable to inter-animal variation.In the present study, there is no evidence that piglets vaccinated in the face of MDI developed active s et al.  who showific MDI . The lacent with . It is pne farms , the datin vitro studies. For example, antigen specific proliferative responses and cytokine production have been demonstrated in infants vaccinated against measles virus [in utero[M. hyopneumoniae vaccination with antigen specific CMI responses, and unlike previous studies, we show evidence of CMI stimulation in vivo. DTH testing is a valuable measure of CMI since the DTH test is an in vivo test and DTH responses are by definition anamnestic responses.There is a plethora of data demonstrating passive interference by maternal immunity with neonatal AMI responses following vaccination; however, reports assessing CMI responses following neonatal vaccination in the face of MDI are limited. Not only is there a paucity of information regarding the neonatal CMI response to vaccination in the face of MDI, the information that does exist is largely restricted to es virus , and neoes virus ,30 and les virus  followin[in utero,33. The In agreement with the present study, Bouma et al.  showed tin vivo but do not pose a risk of infection. Some DNA vaccines induce immune responses in neonates when administered in the face of MDI [Neonatal vaccination in the face of passive immunity does not always result in CMI responses. The capacity for neonates with MDI to generate CMI responses following vaccination may depend on their age and immune maturation at vaccination. Further, the concentration of maternal AMI at the time of vaccination may play a role in the neonatal immune response since the level of interference may be dependent on the level of maternal AMI in the neonate . For exae of MDI ,40,41 whe of MDI ,42,43. Fe of MDI .M. hyopneumoniae in the face of M. hyopneumoniae-specific MDI (Vs Vp piglets) exhibited greater M. hyopneumoniae specific CMI responses 7 dpv compared to all other groups. Therefore, CMI priming and anamnestic responses at 7 dpv depended upon piglet vaccination and transferred antigen-specific MDI. Capozzo et al. [Differences in neonatal responses to vaccination may also be due to the presence of functional maternal lymphoid cells at the time of vaccination. Various studies have shown that transferred maternal cells augment the neonatal response to nonspecific ,46 and so et al.  showed ts Vp piglets compared to Ns Np piglets 14 dpv, it is reasonable to conclude that piglet derived T cells are contributing to the neonatal response to M. hyopneumoniae antigen. In addition to neonatal cells responding to vaccination, we suggest that the CMI responses observed at 7 dpv are also due to the functional activity  of transferred antigen-specific maternal lymphocytes that are restimulated via neonatal vaccination. We have previously shown that maternal lymphocytes primed during vaccination are transferred to and are functional in piglets [s Vp piglets were greater than those of Ns Vp piglets. Further research is required to determine if these responses are protective.Capozzo et al.  suggeste piglets . Here weEvidence suggests that maternal microchimerism  persists into adulthood ; howeverM. hyopneumoniae-specific AMI and CMI is transferred to and detectable in piglets. Further, transferred maternal M. hyopneumoniae-specific CMI is functional and works in concert with vaccine stimulated endogenous M. hyopneumoniae-specific CMI to respond to M. hyopneumoniae in the piglet. We also demonstrated that vaccination was sufficient to prime a 7 day-old piglet’s immune system. Piglets developed M. hyopneumoniae-specific CMI responses when vaccinated in the face of MDI. In conclusion, vaccination of neonatal pigs against M. hyopneumoniae in the face of antigen-specific MDI results in CMI priming and anamnestic CMI responses following subsequent exposure to M. hyopneumoniae antigen.In the present study, we showed that maternal M. hyopneumoniae  at days 17 and 45 of gilt acclimatization by farm staff; the total acclimatization period was 110 days. Randomly selected piglets from the farm were tested for M. hyopneumoniae by PCR following nasal swabbing prior to starting the present study [M. hyopneumoniae-specific PCR testing was performed using a previously described protocol [M. hyopneumoniae DNA, indicating that M. hyopneumoniae was not circulating on the farm.This study was approved by the University of Minnesota’s Institutional Animal Care and Use Committee and all animals were cared for and housed under the University of Minnesota’s Institutional Animal Care and Use Committee guidelines. At all times during the study, animals were housed at a commercial facility  known to be porcine reproductive and respiratory syndrome virus negative. According to the facility’s protocol, replacement gilts were vaccinated against nt study . M. hyopprotocol . All swaM. hyopneumoniae , an adjuvanted bacterin. Respisure-One®, labeled for single dose administration in healthy swine one day of age or older, was given (2 ml intramuscular) at 5 and 3 weeks prior to the anticipated farrowing date.Animals were chosen to participate in this study based on breeding date and on gilt and first parity sow status. An equal number of gilts and first parity sows (from here known as sows) were randomly stratified into one of two groups: boosted or primed, based on previous on-farm vaccination. Boosted animals were vaccinated as part of this study and are referred to as “vaccinated” while primed animals were not vaccinated as part of this study and are referred to as “nonvaccinated.” Experimental vaccination was against s Vp); (2) sow vaccination, piglet nonvaccination (Vs Np); (3) sow nonvaccination, piglet vaccination (Ns Vp); and (4) sow nonvaccination, piglet nonvaccination (Ns Np).Farrowings were monitored and piglets were ear-tagged at birth. To ensure piglets received both maternal CMI and AMI, no cross-fostering was practiced among study animals . Eighty Blood collection of sows occurred at 5 weeks prepartum and 3 weeks postpartum. Blood was collected from the jugular vein into EDTA Vacutainer® tubes . Colostrum was collected from all sows within 2 hrs of farrowing. Teats were scrubbed with alcohol wipes  and gloves were worn to minimize sample contamination. Colostrum (25 ml) was collected manually from all functional teats.Blood was collected from piglets before colostrum ingestion, 24 hr after colostrum ingestion, and at 14, and 21 days of age. Blood was collected via jugular venipuncture into sterile EDTA Vacutainer® tubes. Blood sampling times are expressed in terms of days post vaccination (dpv).Mononuclear cells were isolated from sow and piglet blood via Ficoll density centrifugation as described  with modM. hyopneumoniae. Cells were stained with the membrane stain carboxyfluorescein diacetate succinimidyl ester  and washed with RPMI supplemented with 10% FBS, 100 U penicillin G per ml, and 100 μg of streptomycin per ml to stop the reaction. Cells were resuspended in RPMI and plated in duplicate at 5×105 cells/well in 200 μl in round bottom 96-well plates. CMC and BMC were stimulated with 10 μg/ml M. hyopneumoniae antigen as described [M. hyopneumoniae antigen was prepared as described [M. hyopneumoniae at passage 15 was harvested by continuous flow centrifugation at 70,000 x g and resuspended in Tris-sodium chloride buffer. M. hyopneumoniae was inactivated by one freeze-thaw cycle and then by sonic disruption. Nonstimulated and ConA  stimulated cultures served as negative and positive controls, respectively. Experimental, negative, and positive controls were analyzed for each animal.A dye-dilution method was used in order to evaluate the lymphoproliferative response to escribed . The M. escribed ; M. hyopM. hyopneumoniae-specific proliferation data are described as the percent M. hyopneumoniae-stimulated proliferation - nonstimulated proliferation.Following 5 d incubation, cells were transferred to sterile Facs tubes and washed. Proliferation was analyzed by flow cytometry using a Facs Caliber flow cytometer . Non-stained, non-stimulated cells and stained, non-stimulated cells were used to establish a baseline for the proliferation assay. Event acquisition was set for 10,000 events in a region encompassing the CFSE-positive quadrant. Results were analyzed by BD Cellquest™ Pro software . in vivo measure of the CMI response. The DTH assay for M. hyopneumoniae was performed as originally applied [M. hyopneumoniae antigen used in the DTH assay was the same antigen as used in the in vitro proliferation assay. M. hyopneumoniae antigen  was injected intradermally in 10 piglets per group at 14 d of age and a second set of 10 piglets per group at 21 d of age. PHA  and physiological saline (0.1 ml) were used as positive and negative controls, respectively. Injections were performed in the inguinal region. Injection sites were clearly marked with livestock paint. The DTH injection sites were assessed immediately and 36 h post injection. DTH lesion diameters were measured with digital calipers; DTH data are shown as mean orthogonal diameter of induration. Piglets were subject to DTH testing only once to avoid the possibility of the DTH antigens influencing subsequent DTH test results.DTH testing was used as an  applied  with modM. hyopneumoniae-specific antibodies were measured in all blood and colostrum samples employing Idexx ELISA kits  as described [M. hyopneumoniae ELISA kit is isotype non-specific. Positive and negative M. hyopneumoniae serological status was determined based on optical density (OD) of the sample to positive ratio (S:P); S:P = (sample OD - negative control OD)/(positive control OD - negative control OD). All samples were run in duplicate and sample means were used to determine the final S:P ratio. S:P ratios ≥0.4 were classified as positive; S:P ratios <0.4 were classified as negative.escribed . The IdeM. hyopneumoniae-specific antibody S:P ratio in sow sera before and after vaccination, the difference between M. hyopneumoniae-specific antibody S:P ratios in sera and colostrum of vaccinated or nonvaccinated sows, and the difference between M. hyopneumoniae-specific proliferation by CMC isolated from vaccinated or nonvaccinated sows was analyzed by the student t-test. The effect of piglet vaccination on M. hyopneumoniae-specific AMI and CMI responses  was analyzed via two-way ANOVA with piglet vaccination and sow vaccination being the two factors. Comparisons found to be significant by ANOVA (p < 0.05) were analyzed by Tukey’s HSD method. Student t-test was used to analyze the difference in mean M. hyopneumoniae-specific DTH lesion orthogonal diameter and the difference in M. hyopneumoniae-specific antibody S:P ratios between 7 dpv and 14 dpv among piglets of the same group. Statistical analysis was performed using GraphPad Prism 5 .Normality of data was evaluated prior to statistical analysis and approximated to be normal. The difference in M. hyopneumoniae: Mycoplasma hyopneumoniae; MDI: Maternal derived immunity; PCV2: Porcine circovirus type 2; PHA: Phytohemagglutinin; S:P: Sample to positive; SIV: Swine influenza virus; TGE: Transmissible gastroenteritis virus; CMC: Colostral mononuclear cells; Ns Vp: Nonvaccinated sow, vaccinated piglet; Vs Vp: Vaccinated sow, vaccinated piglet; Vs Np: Vaccinated sow, nonvaccinated piglet; Ns Np: Nonvaccinated sow, nonvaccinated piglet.AMI: Antibody-mediated immunity; BMC: Blood mononuclear cells; BVDV: Bovine viral diarrheal virus; CMI: Cell mediated immunity; conA: Concanavalin A; CSF: Classical swine fever; dpv: Days post vaccination; DTH: Delayed type hypersensitivity; None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.MB conceived the study, participated in its design, sample collection, immunoassays and data analysis, and drafted the manuscript. KT participated in study design and sample collection, coordinated the farm site, and carried out immunoassays. TWM participated in study design and its coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Lactococcus and Pseudomonas were enriched in preneoplastic tissue, whereas Enterococcus, Bacillus, and Solibacillus were reduced. However, both PCoA and cluster tree analyses showed similar microbiota structure between adenomatous and adjacent non-adenoma tissues. These present findings provide preliminary experimental evidence supporting that colorectal preneoplastic lesion may be the most important factor leading to alterations in bacterial community composition.Recent reports have suggested that the gut microbiota is involved in the progression of colorectal cancer (CRC). The composition of gut microbiota in CRC precursors has not been adequately described. To characterize the structure of adherent microbiota in this disease, we conducted pyrosequencing-based analysis of 16S rRNA genes to determine the bacterial profile of normal colons  and colorectal adenomas (CRC precursors). Adenoma mucosal biopsy samples and adjacent normal colonic mucosa from 31 patients with adenomas and 20 healthy volunteers were profiled using the Illumina MiSeq platform. Principal coordinate analysis (PCoA) showed structural segregation between colorectal adenomatous tissue and control tissue. Alpha diversity estimations revealed higher microbiota diversity in samples from patients with adenomas. Taxonomic analysis illustrated that abundance of eight phyla  was significantly different. In addition,  Colorectal cancer (CRC) is one of the leading contributors to cancer-related deaths. It is the fourth most commonly diagnosed cancer worldwide, with more than 1 million new cases diagnosed annually, and its incidence has increased rapidly in recent years in Chinaet al. reported that the genus group Bacteroides-Prevotella is over-represented in CRC patients compared with normal subjectset al. demonstrated a decreased abundance of butyrate-producing bacteria in CRC patientsFusobacterium may be associated with CRC8Firmicutes, Bacteroidetes, and Proteobacteria was observed, compared with non-adenoma subjects, in adenoma biopsiesThe human gastrointestinal tract is colonized by complex and diverse communities of commensal microorganismsIn this study, we performed a microbiome analysis of colorectal mucosal biopsies from healthy volunteers and polyp patients  to elucidate bacterial changes that might induce or accompany early oncogenic transformation of CRC using a deep sequencing platform.t-test; **P < 0.05; ***P < 0.01), demonstrating a significantly higher diversity found in patients with colorectal adenomas compared with healthy control tissue. The characteristics of each sample are shown in In total, 1180268 usable sequences were obtained from 82 samples using pyrosequencing. From these, 1138898 high-quality sequences were selected, with an average of 13889 sequences per sample. In total, 2083 OTUs were delineated at a 97% similarity level. The values of Good’s coverage of all libraries were above 99%. The rarefaction curves did not approach a plateau with the current sequencing, but the Shannon diversity estimates of all the samples were stable, suggesting that although new phylotypes would be expected with additional sequencing, most diversity had already been captured . Other diversity estimators of community are shown in Nineteen phyla were revealed from 31 colorectal adenomatous tissues and 20 healthy tissues. The bacteria flora analysis showed that Firmicutes was the most predominant phylum, contributing 88.6% and 53.7% of the tissue microbiota in healthy volunteers and colorectal adenoma patients, respectively. The second and third most dominant phyla in colorectal adenoma patients were Proteobacteria (30.1%) and Bacteroidetes (10.8%). However, there was a clear decrease in both of these phyla  in healthy volunteers compared with patients with colorectal adenoma patients . MetastaBeta diversity of each group was calculated through cluster tree analysis and PCoA and Non-metric multidimensional analysis(NMDS). Cluster tree analysis indicated that the bacterial structure was obviously separated between colorectal adenomatous and healthy tissues, and the bacterial composition of the same group was more similar . PCoA baLactococcus was the predominant genus in colorectal adenomatous tissue, and the content was much higher than in healthy tissue. The second dominant genus in colorectal adenomatous tissue was Pseudomonas, whose content in healthy tissue was less than 0.1%. Conversely, the dominant genera in healthy tissue were Enterococcus and Bacillus, the contents of which were much higher than those in colorectal adenomatous tissue. In addition, another dominant genus, Solibacillus, in healthy tissue was virtually absent in colorectal adenomatous tissue  is the most important factor contributing to changes in structure, considering that controls were matched for age and gender, had similar diets, and received no recent medication. In other words, the development of adenomas may contribute to microbiota imbalances. Mutually, local microbiota disturbances may further promote precancerous lesions. Disturbance in the balance between adenomas and the microbiota, whether causal or consequential, may still be a problem and requires further study to elucidate their relationship. In particular, we found that increased relative abundance of potential opportunistic pathogens such as blautia was reduced in patients with adenomas which was consistent with the previous report in CRC patientsAkkermansia, or Fusobacterium which have been reported to correlate with CRC between healthy controls and advanced adenoma samples921Akkermansia is known to break down mucin, while mucin degradation has been linked to intestinal inflammation and can facilitate colonization of intestinal pathogensClostridium, Roseburia), or Faecalibacterium or Bifidobacterium whereas a previous study showed depletion of butyrate-producing bacteria, Faecalibacterium or Bifidobacterium in CRC patients7212425In our study, we found et al. observed changes in rectal mucosal bacterial communities of adenoma patients as well as in healthy controls, and hypothesized that rectal mucosal bacterial composition may reflect the presence of adenoma bacterial communitiesPrevious studies have indicated that there are obvious differences in community composition between cancerous tissues and surrounding areas among late-stage CRC patients. These findings led to a bacterial driver-passenger model for colorectal cancerSolibacillus, a new member of the genus Bacillus, with little information available concerning the role of probiotics on colorectal carcinogenesis. PCoA analysis suggested that colorectal pre-neoplastic lesion is the primary factor leading to changes in the adherent bacterial structure. However, we also found no differences between adenomatous and adjacent mucosal microbiota composition, which indicates that local mucosal epithelium transits to a pro-inflammatory microenvironment that is conducive to colorectal tumorigenesis. Identifying the composition of adherent bacterial communities in the precancerous colon is an important step in our development of effective diagnostic, preventive, or therapeutic strategies.In conclusion, by comparing the intestinal microbiota composition between adenoma patients and healthy individuals, we have defined a structural imbalance in the gut microbiota, represented by an increased incidence of opportunistic pathogens in patients and reduction of Thirty-one patients with colon adenomas, aged 27–67 years, were selected from the Shanghai General Hospital from Jan 1 to Jun 30, 2014. All patients were of independent genetic background. Exclusion criteria included pregnancy, colitis , polyposis (>100 polyps), previous colon resection, or colorectal cancer. Twenty healthy volunteers were selected in the Shanghai General Hospital as controls after a routine physical examination. No subjects were taking medications at the time of sample collection or had used antibiotics in the past 3 months before sample collection . All sam® Soil DNA Kit  according to manufacturer’s protocols. Samples were placed into bead tubes with 500 mg of glass beads. Add 1 ml Buffer of SLX Mlus to the samples. Incubate at 70 °C for 10 min. Bead beating at 6.5 m/s for 5 min using a FastPrep-24 bead beater  . Total DNA was eluted in 60 μL of Elution buffer. The amount of DNA was determined by NanoDrop2000 (Thermo Scientific). Integrity and size of DNA were checked by 1% (wt/vol) agarose gel electrophoresis in 0.5 mg/ml ethidium bromide. All DNA samples were stored at −20 °C until further processing. The V3-V4 region of the bacteria 16S ribosomal RNA gene from each sample were amplified using the bacterial universal primer 338F 5′-barcode-ACTCCTACGGGAGGCAGCA-3′ and 806R 5′-barcode-GGACTACHVGGGTWTCTAAT-3′® FastPfu DNA Polymerase, TransGen BioTech, Beijing, AP221-01), and 10 ng of template DNA. PCR was performed on Thermal Cycler  with following procedure, 95 °C for 3 min, followed by 25 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s and a final extension at 72 °C for 5 min. The PCR products were evaluated using 2% (wt/vol) agarose gel electrophoresis.Microbial genomic DNA was extracted from each samples using the E. Z. N. A.http://support.illumina.com/downloads/16s_metagenomic_sequencing_library preparation.html). Briefly, Samples were multiplexed using a dual-index approach with the Nextera XT Index kit  according to the manufacturer’s instructions. The final library was paired-end sequenced at 2 × 250 bp using a MiSeq Reagent Kit v2 on the Illumina MiSeq platform.Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit  according to the manufacturer’s instructions and quantified using QuantiFluor™ -ST . Purified amplicons were pooled in equimolar. Index PCR and sequencing according to the Illumina MiSeq 16S Metagenomic Sequencing Library Preparation protocol  and chimeric sequences were identified and removed using UCHIME. The taxonomy of each 16S rRNA gene sequence was analyzed by RDP Classifier (http://rdp.cme.msu.edu/) against the SILVA 119 16S rRNA database using a confidence threshold of 70%. OTUs that reached 97% similarity were used for alpha diversity estimations, which included diversity , richness (Chao I), and Good’s coverage and rarefaction curve analysis using Mothur .The high-quality sequences were assigned to samples according to barcodes. Operational taxonomic units (OTUs) were clustered with 97% similarity cutoff using UPARSE .


Self report questions are often used in population studies to assess sensory efficacy and decline. These questions differ in their validity in assessing sensory impairment depending on the wording of the question and the characteristics of the population. We tested the validity of the self-report questions on hearing efficacy  used in The Irish Longitudinal Study on Ageing (TILDA).We tested sensitivity and specificity, positive and negative predictive values of each question against the Whispered Voice Test, a relatively easy to administer and cost effective alternative to the standard audiometric test.In this population the question ‘Is your hearing (with or without a hearing appliance)/ Excellent/Very Good/Good/Fair/Poor?’ showed the best diagnostic value in relation to the other questions (sensitivity 55.56% and specificity 94.67%). The question ‘Can you use a normal telephone?’ was deemed ineffective because of a very poor sensitivity (5.56%) and was proposed for exclusion from subsequent waves of TILDA.We showed that this validity check was useful to select the questions that most effectively assess hearing deficits and provided crucial information for the subsequent waves. We argue that longitudinal studies using self-reports of sensory efficacy would benefit from a similar check. Hearing loss is one of the most common chronic conditions affecting the ageing population with a reported prevalence of between 20% and 40% among adults aged over 50-3 or eveIn large survey studies it is often difficult to introduce objective hearing tests, such as the pure tone audiometric test, due to time constraints, costs and compliance. Therefore self reports are used to assess hearing , the exThe primary objective of this study was to examine the accuracy of different self reported hearing loss questions relative to a more objective hearing test, the Whispered Voice Test, when assessing hearing in a population study of ageing in Ireland, the Irish longitudinal study on Ageing (TILDA). Several studies have shown the Whispered Voice Test to be one of the best simple tests in identifying hearing impairment with respect to sensitivity and specificity when compared with audiometric testing,25 even For the purpose of this study, data from the pilot wave of TILDA were analysed which included both subjective and objective measures of hearing loss. We tested the specificity and sensitivity of a series of self rated questions used in TILDA in relation to the Whispered Voice Test in order to determine what questions were most effective in detecting hearing deficits for the purpose of inclusion/exclusion from the subsequent main waves.The sample studied here was derived from the second pilot of TILDA which was conducted between 2009 and 2010 and included 291 individuals. There is no enumeration of individuals in Ireland that could be used as a sampling frame, however a list of household addresses was available, and so sampling was initially conducted at the household level. The sample for this pilot study was selected using a RANSAM sampling system. The samInitially each respondent participated in a computer based questionnaire in their home which was carried out using Computer Assisted Personal Interview (CAPI). This questionnaire covered a broad range of categories from various aspects of physical health (including sensory abilities among which self-rated hearing questions were administered), functional ability, cognitive and psychological well-being, to socio economic information.Five hearing related questions with multiple choice answers were included in the questionnaire. Questions were designed to detect a range of functional deficits including self assessed hearing deficit and different aspects of functional use of hearing such as following a conversation and using a telephone. For international comparability these were modelled on self report questions from similar studies such as ELSA, SHARE and LASA. The questions and answers (multiple-choice) are listed below:1. Do you use any of the following appliances to help with your hearing? Also used in ELSA/HRS/SHARE. Answers: Hearing Aid ; Hearing Aid (some of the time); Amplifier; None of the above2. Is your hearing with or without a hearing appliance? Also used in LASA, answers: Excellent; Very Good; Good; Fair; Poor3. Can you follow a conversation with one person? Also used in LASA, answers: No Difficulty; Some Difficulty; Much Difficulty; No I cannot4. Can you follow a conversation with four people? Also used in LASA, answers: No Difficulty; Some Difficulty; Much Difficulty; No I cannot5. Can you use a normal telephone? Also used in LASA, answers: No Difficulty; Some Difficulty; Much Difficulty; No I cannotOnce each participant had completed the CAPI interview, they were asked to take part in a general health assessment in a separate session at the health assessment unit in Trinity College Dublin. Each assessment was conducted by qualified, specially trained research nurses who followed standard operating procedures for all tests and measurements. The health assessment included a comprehensive cardiovascular, cognitive, anthropometric, gait and balance, grip strength measurements and sensory assessment as well as the Whispering Voice Test. On average each assessment took between two and two and half hours to complete.In accordance with TILDA protocol, the participant was asked to sit with his/her back to the nurse throughout the test. The nurse stood at a distance of 0.6 m from the participant and each ear was tested separately. During the examination, the non-tested ear was masked by the nurse gently occluding the auditory canal with a finger and rubbing the tragus in a circular motion. Starting with the better functioning ear (as determined by the participant), the nurse whispered a combination of numbers and letters (e.g. 4-K-2) to the participant who had to repeat the combination back to her. The nurse was trained to exhale prior to whispering to ensure that the whispering was as quiet as possible. A maximum of six combinations were whispered to each ear and the respondent was required to repeat 3 sequences for each ear correctly in order to pass. Two research nurses administered the test in this pilot. They were extensively trained to fully adhere to the Standard Operating Procedure protocol in administering and scoring the test.Analyses were carried out with PASW SPSS 18. The result of the better ear for the Whispered Voice Test was used in the analysis. The sensitivity, specificity and positive and negative predictive values of each question were tested. For the purpose of this analysis the following cut-off points were established for the self report questions: in question 1, a response indicating the use of any hearing appliance resulted in a fail. In question 2 a participant was deemed to have failed if they gave a response of ‘fair’ or ‘poor’. In questions 3; 4 and 5 participants passed the test if they responded with ‘no difficulty’, all other responses were considered a fail. These cut-offs optimised the sensitivity and specificity values in relation to the objective hearing assessment based on Receiving Operator Curve (ROC) analysis.Two hundred and ninety one respondents 100%) completed the CAPI questionnaire, 168 (57.7%) participants attended the subsequent health assessment and successfully completed the Whispered Voice Test, 8 respondents (2.75%) were unable to take part or complete the Whispered Voice Test and 115 (39.55%) did not have a health assessment. The majority of participants who were administered the Whispered Voice Test was younger (58% under 65 years of age) and female (56%). Of the 168 who were administered the Whispered Voice Test, 150 respondents (89.3%) passed the test and demonstrated no hearing impairment, and 18 respondents (10.7%) failed . Table 00% complThe sensitivity and specificity values as well as the positive and negative predictive values for the self-rated hearing questions were then calculated Table . SensitiThe question, “Is your hearing (with or without a hearing appliance)/Excellent; Very Good; Good; Fair; Poor?,” was the most accurate in detecting hearing loss among participants, with 10 participants self-reporting poor hearing (true positive) out of the 18 who failed the Whispered Voice Test  and only 8 participants out of 150 passing the Whispered Voice Test while self-reporting poor hearing . This test therefore demonstrated the highest sensitivity and specificity values (55.56% and 94.67% respectively). Accordingly the positive and negative predictive values for this question were relatively high within this set of questions. For the self report question “Do you use any of the following appliances….?,” the false negatives were 17 out of 18 participants failing the Whispered Voice Test and the false positives were 6 out of 150 participants passing the test. Accordingly the sensitivity was low (5.56%) while the specificity was far higher (96%). The PPV was only 14% and the NPV 89%. The question regarding a conversation with one person produced 14 false negatives out of 18 people failing the Whispered Voice Test and 8 false positives out of 150 passing the test. The specificity was high (94.67%), however, the sensitivity was poor (22.22%), as indicated by the greater likelihood of false negatives. Accordingly the PPV was low (33%) and the NPV was high (91%). Sensitivity and specificity values from the question on following a conversation with 4 people were relatively convergent with our findings from ‘Is your hearing (with or without a hearing appliance)/Excellent; Very Good; Good; Fair; Poor?’ (50% and 84.67% respectively). False negatives were 9 out of 18 participants failing the Whispered Voice Test and false positives were 23 out of 150 participants passing the Whispered Voice Test. The sensitivity was higher than for the question on following a conversation with one person (50% vs. 22%), implying that fewer respondents with hearing loss were incorrectly diagnosed as being healthy according to this question. The specificity value was lower than that of the question on conversation with one person implying that a higher proportion of participants without hearing impairment could be misdiagnosed with hearing loss. Accordingly the PPV was lower for this question than for the question on following a conversation with one person (28% vs. 33%). The question “Can you use a normal telephone…?’ showed ceiling effects leading to a relatively large number of false negatives (17) and 1 false positive. The sensitivity value was extremely low (5.56%) and the specificity value was high (99.3%).Answers to five questions on different aspects of participants’ hearing abilities were compared with the results of the Whispered Voice Test. The Whispered Voice Test was not included in the main waves of TILDA due to time constraints; therefore this pilot study is of relevance in order to understand to what extent the self-report questions included in the main waves capture physiological hearing deficits (in the speech frequency range). The Whispered Voice Test is appropriate to determine hearing loss within the 30–40 dBL range although its use as an objective measure of hearing has limitations relative to the audiometric test especially due to the way it is administered,26. CareTwo questions enquired about the ability to follow a conversation, either with one or four people. Following a conversation requires cognitive abilities as well as good hearing (see e.g.), this iThe efficacy of the question on using a telephone in capturing hearing deficits here is questionable, in fact it is plausible to think that respondents interpreted the question more in relation to the instrumental skills required by the use of the telephone than in relation to hearing if we consider that only 2 people reported difficulties in the use of a telephone, while 12 reported difficulties in following a conversation with one person and 32 with four people (both questions assessing relatively similar use of hearing to the use of a telephone).Following our investigations, the original question set was revised for use in subsequent TILDA waves. The question, ‘Can you use a normal telephone…?’ was omitted for its lack of sensitivity and substituted with the question, ‘Do you feel you have hearing loss…?’ that was shown in the literature to have both high sensitivity and specificity values. The modifications have been introduced from Wave 2. The question regarding the use of hearing aids, despite showing very low sensitivity values was maintained in order to keep track of respondents using corrections for their deficits.Limitations of this study are the relatively small sample and the lack of audiometric test to be able to compare the self ratings with both the Whispered Voice Test and the standard audiometric test. In addition, in conducting the Whispered Voice Test wax in the ears temporarily limiting hearing abilities was not checked for. Clearly the self reported measures described here cannot substitute more objective assessments in terms of diagnosis of hearing loss and it should be taken into account that The Whispered Voice Test has limitations as an objective measure of hearing.Sensory decline, in particular hearing and vision, is linked to decline in functional abilities and cognition e.g., therefoIn sum the validation of self-reported hearing questions used in population studies is useful in determining whether an actual hearing deficit is captured. The Whispered Voice Tests is a viable instrument to assess the validity of these self report measures when the audiometric test cannot be used because of economic or time constraints (but see). In theThe authors have no financial or non-financial competing interests to declare.WKG designed the study and conducted the analysis. WKG and AS drafted the manuscript; HC and RAK designed the TILDA pilot II and critically revised the manuscript. All authors read and approved the final manuscript.


When insects communicate by sound, or use acoustic cues to escape predators or detect prey or hosts they have to localize the sound in most cases, to perform adaptive behavioral responses. In the case of particle velocity receivers such as the antennae of mosquitoes, directionality is no problem because such receivers are inherently directional. Insects equipped with bilateral pairs of tympanate ears could principally make use of binaural cues for sound localization, like all other animals with two ears. However, their small size is a major problem to create sufficiently large binaural cues, with respect to both interaural time differences , but also with respect to interaural intensity differences (IIDs), since the ratio of body size to the wavelength of sound is rather unfavorable for diffractive effects. In my review, I will only shortly cover these biophysical aspects of directional hearing. Instead, I will focus on aspects of directional hearing which received relatively little attention previously, the evolution of a pressure difference receiver, 3D-hearing, directional hearing outdoors, and directional hearing for auditory scene analysis. In insect taxa which use acoustic signals for intraspecific communication, the recognition of the acoustic signals is crucial for their fitness, since it enables species identification and prevents hybridization  component of near-field sound.The problem with the task of directional hearing is obvious, however, with tympanate hearing; it is basically a biophysical one associated with the small size of most insects. Insects equipped with bilateral pairs of tympanate ears could principally make use of binaural cues for sound localization, like all other animals with two ears. However, their small size and hence interaural distance result in only minute interaural time differences (ITDs). At the same time acoustic theory predicts that significant diffraction for the establishment of reasonable interaural intensity differences (IIDs) occurs only when the ratio of body size to the wavelength of sound exceeds a value of 0.1  acoustic inputs which conduct pressure waves also to the internal side of the tympanum. Thus, the force driving the tympanal membrane is the difference between the external and internal pressures. Since the internal pressure component travels either through some body tissue, air sacs or in tracheal tubes, various degrees of attenuation or amplification and phase shift can occur relative to the external component. Theoretically, evolutionary modifications of anatomical structures (see below) resulting in the proper amplitude and phase shifts between internal and external pressure components could create highly directional ears despite unfavorable ratios of body size to the wavelength of sound. Since the internal sound conduction can depend on frequency, insect ears can act as pressure difference receivers at low frequencies and as a pressure receiver at higher frequencies. The locust (grasshopper) ear is such a case , or only 1.45 µs (parasitoid fly). These values of ITDs are so strongly constrained by body size and ear separation, that it had been accepted for a long time that such small ITDs cannot be used for neuronal processing of sound direction. Mörchen  thus sugChorthippus biguttulus males for ITDs was also in the order of 1 ms: when both stimuli were presented at equal loudness, but one speaker was leading the other by only 0.5 ms this resulted in significant turns towards the leading side (von Helversen and Rheinlaender Ormia fly below), the onset of binaural arrival of receptor activity at these synapses  may be quite important for directional coding.The hypothesis of the dual mode of directional coding was examined using dichotic stimulation of the tympana in the locust  at the midline enhancing the time delay in the internal interaural transmission , cross-section and length of tracheal branches , their relative position to one another, the size of the acoustic vesicle and biomechanical properties of the medial septum Fletcher . CurrentTeleogryllus oceanicus, they may not be essential for spatial hearing  or on ear asymmetries in owls  in the avoidance response of a flying cricket is just to fly away from the ultrasound stimulus, but most likely not d Robert  with theLeptophyes punctatissima,  as well, since their microhabitats are trees and bushes, where biologically important sounds may arise from positions above and below the receiver, in addition to different locations in azimuth. Rheinlaender et al.  used theA further attempt to investigate spatial hearing in the same katydid species was made using a walking compensator . The only remaining information is the number of afferents being activated, as a result of their absolute sensitivity and/or tuning (Hardt IIDs have to be translated into binaural discharge differences of neurons to be processed by the auditory pathway. Simultaneous recordings of the activity of a pair of directionally-sensitive interneurons allowed Kostarakos et al.  determinng Hardt .Since the pioneering work by Morton  and WileChorthippus biguttulus, the directional dependence of the activity was monitored first in the laboratory under free field conditions, and then in various natural habitats of the species. On gravel and in sparse vegetation, the general patterns of directionality were quite similar to those in the free sound field of the laboratory, although the amount of IIDs calculated from the nerve activity was reduced. Strongest differences were found in dense vegetation, both with respect to the maximum IIDs and the location of the minima in nerve activity, which not always occurred on the contralateral side, as is typical for the free sound field.A similar attempt was used to study the directionality in various types of habitat for short-horned grasshoppers (Gilbert and Elsner G. bimaculatus, I will compare the localization performance of this species under outdoor conditions with the one in the laboratory.Field crickets live and communicate on the ground, which may create specific problems for communication. Kostarakos and Römer  examinedG. bimaculatus on an open-loop trackball system was much higher (Schöneich and Hedwig O. ochracea (Mason et al. Apparently, this performance is different in the various behavioral paradigms used for quantifying the acuity of localization. Data from compensated walking on a closed-loop trackball system, as well as orientation in a Y-maze indicated that field crickets are unable to discriminate the side of sound incidence within a frontal “area of uncertainty” covering ±25° in azimuth (Weber et al. In their natural grassland habitat female crickets reliably approach the target speaker broadcasting a standard model of the calling song in a no-choice trial, although for the shortest possible straight path females walked a considerable detour (on average about 1 m for the shortest straight path of 2 m; Hirtenlehner and Römer The distorted directional cues under outdoor conditions are also evident when we compare results of two-choice experiments conducted in the lab and field (Hirtenlehner and Römer As evident from the previous sections, the issue of directional hearing is always strongly associated with the two important tasks for a receiver, namely to use directional information for approaching towards a mate, or in the case of a predation, to initiate appropriate escape responses away from the predator. We often forget, however, that directional hearing also contributes to auditory scene analysis, by separating sound sources according to their spatial location, as well as in reducing the effect of masking, by so-called spatial release from masking.T. viridissima revealed that the problem for a receiving insect may be quite complex, requiring them to discriminate the individual calls of up to four nearby males and more than ten others within hearing range, some of which are quite similar in amplitude (Römer and Krusch For auditory scene analysis, a receiver groups and segregates all incoming sounds either according to their similarity or their differences in a number of sound parameters, one of which is their location in space Bregman . SignaliOne result was that the auditory world of the katydid is sharply divided into two hemispheres, with a pronounced separation along the longitudinal body axis. Even with an angular separation of only 7.5° off the midline axis, two sound signals are better represented in the activity of the respective ipsilateral neuron. The strength of separation is given by the peripheral directionality of the ear, and by enhancement as a result of lateral inhibition. This mechanism, referred to as spatial release from masking, can also improve the detection and discrimination of signals in noise when the masker is spatially separated from the signal Klump . Again;