MYO7A (USH1B), CDH23 (USH1D), PCDH15 (USH1F), USH1C(USH1C), and USH1G(USH1G). Three genes are involved in USH2, namely, USH2A (USH2A), GPR98 (USH2C), and DFNB31 (USH2D). USH3 is rare except in certain populations, and the gene responsible for this type is USH3A.Usher syndrome (USH) is an autosomal recessive disease characterized by hearing loss, retinitis pigmentosa (RP), and, in some cases, vestibular dysfunction. It is clinically and genetically heterogeneous and is the most common cause underlying deafness and blindness of genetic origin. Clinically, USH is divided into three types. Usher type I (USH1) is the most severe form and is characterized by severe to profound congenital deafness, vestibular areflexia, and prepubertal onset of progressive RP. Type II (USH2) displays moderate to severe hearing loss, absence of vestibular dysfunction, and later onset of retinal degeneration. Type III (USH3) shows progressive postlingual hearing loss, variable onset of RP, and variable vestibular response. To date, five USH1 genes have been identified:  Usher syndrome (USH) was first described by von Graefe in 1858 and is characterized by the association of sensorineural hearing loss, retinitis pigmentosa (RP), and, in some cases, vestibular dysfunction. Its heritability was established by Charles Usher, a British ophthalmologist , 3, abouUsher patients present progressive photoreceptor degeneration in the retina called retinitis pigmentosa, which leads to a loss of peripheral vision. This degeneration is predominantly attributable to rod dysfunction, although cones usually degenerate later in the course of the disease. Clinical symptoms may vary and include night blindness  with elevated dark adaptation thresholds, abnormal electroretinogram responses, visual field constriction, abnormal retinal pigmentation including peripheral bone spicules, arterial narrowing, and optic-nerve pallor, and predisposition to myopia and posterior subcapsular cataracts .The human inner ear consists of the cochlea, a snail-shaped organ which mediates sound transduction, and the vestibular labyrinth, which detects gravitational force and angular and linear accelerations. Both structures have specialized hair cells which convert mechanical stimuli into variations of intracellular potential, thus transmitting afferent nerve signals toward the brain. On the apical surface of these cells there is a mechanosensitive organelle, the hair bundle, which consists of precisely organized actin-filled projections known as stereocilia. In Usher syndrome patients, alteration in the morphogenesis and stability of stereocilia results in sensorineural hearing loss and may also cause balance defects .The majority of patients with Usher syndrome usually fall into one of three clinical categories . Of thesUSH1B-H, USH2A, C-D, and USH3A-B (hereditary hearing loss homepage: http://hereditaryhearingloss.org). Nine genes have been identified through the discovery of a mouse homolog or by positional cloning. There are five USH1 genes that codify known products: myosin VIIA (MYO7A), the two cell-cell adhesion cadherin proteins cadherin-23 (CDH23) and protocadherin-15 (PCDH15), and the scaffold proteins harmonin (USH1C) and SANS (USH1G). The three identified USH2 genes are USH2A, which codes for the transmembrane protein usherin (USH2A); the G-protein-coupled 7-transmembrane receptor VLGR1 (GPR98), and whirlin (DFNB31), another scaffolding protein. The USH3A gene encodes clarin-1, which exhibits 4 transmembrane domains. Mutations in any one of these genes cause primary defects of the sensory cells in the inner ear and the photoreceptor cells of the retina, both being the source of the clinical symptoms of USH.All subtypes are genetically heterogeneous and 12 loci have been described, namely, MYO7A causes DFNB2/DFNA11 [USH1C also causes DFNB18 [CDH23 causes DFNB12 [PCDH15 causes DFNB23 [DFNB31 also lead to DFNB31 [USH2A gene cause isolated RP [Many of these genes can also cause either nonsyndromic hearing loss (NSHL) or isolated RP. In fact, 2/DFNA11 , 14; USHs DFNB18 , 16; CDHs DFNB12 , 18; PCDs DFNB23 ; mutatioo DFNB31 , 21. Morlated RP .  Table Usher syndrome type I is the most severe form. USH1 patients suffer from severe to profound congenital and bilateral sensorineural hearing loss. These individuals are either born completely deaf or experience hearing impairment within the first year of life and usually do not develop speech.Constant vestibular dysfunction is present from birth; children manifest a delay in motor development and begin sitting independently and walking later than usual.Onset of retinitis pigmentosa occurs during childhood, resulting in a progressively constricted visual field and impaired visual acuity which rapidly proceeds to blindness. Anomalies of light-evoked electrical response of the retina can be detected by electroretinography at 2-3 years of age, which allows for early diagnosis of the disease.USH1B–USH1H) have been mapped and five causative Usher genes have been cloned: MYO7A, USH1C, CDH23, PCDH15, and USH1G, which are known to be implicated in USH1B, USH1C, USH1D, USH1F, and USH1G, respectively.Seven loci . These iewed in –27. CDH2families , 26, 28.ngements , 30. Theor 6%-7% , 26 and  Kingdom . Howeverentified . There apulation  but has  [PCDH15  gene, whFirstly, RP symptoms manifest later in USH2 patients than in their USH1 counterparts, for whom onset occurs during or after puberty.The degree of hearing impairment in patients diagnosed with USH2 increases from moderate in low frequencies to severe in high frequencies, tending to remain stable. Hearing loss is congenital but may be detected at later stages when it hinders communication. Vestibular function in Usher type II patients is normal.USH2A, USH2C-2D) have been proposed as being responsible for USH2, and three causative genes have been identified: USH2A (USH2A), GPR98 (USH2C), and DFNB31 (USH2D).To date, three loci  is variable though usually occurs by the second decade of life.Sensorineural hearing loss is postlingual and progressive and can appear between the first and third decade of life. In its initial stages, the degree of hearing impairment is similar to that seen in USH2, with major impairment seen in high frequencies. The progression rate is variable but, in most cases, hearing loss becomes profound. Nevertheless, hearing levels during the first stages of development are good enough to permit well-developed speech. Thus, successive audiometric examinations are needed in USH3 patients in order to obtain an accurate clinical diagnosis. The vestibular responses are also variable, with 50% of cases experiencing impairment.USH3A gene was initially described as being responsible for USH3 cases, recent studies have demonstrated that mutations in USH3A can also produce clinical forms of Usher that are similar to USH1 and USH2 [Although the and USH2 , 45. Ushand USH2 , 47.USH3B) was suggested by Chaïb et al. in 1997, although these findings have yet to be confirmed in [The existence of a second locus for this clinical type (irmed in .USH1B) is an actin-based motor protein; harmonin (USH1C), SANS (USH1G), and whirlin (USH2D) are scaffolding proteins [USH1D) and protocadherin 15 (USH1F) are cell-adhesion molecules [USH2A) and VLGR1 (USH2C) are transmembrane proteins with very large extracellular domains [USH3A) is a protein with four transmembrane domains [The proteins encoded by the identified USH genes belong to different protein classes. Myosin VIIA .In murine models, hair cells in the developing inner ear, known as stereocilia, maintain their cohesion by interstereocilia fibrous links and links with the kinocilium. There are several types of links depending on the stage of hair-cell development. In the mouse, transient lateral links appear at very early stages of stereocilia formation, but while other links appear at the base of stereocilia (ankle links), these lateral links diminish progressively throughout development. Later, ankle links diminish, and tip and horizontal links appear and are preserved in adulthood .The large extracellular domains of the cell adhesion proteins cadherin-23 and protocadherin-15 and the transmembrane proteins usherin and VLGR1 are part of these links. The proteins are anchored to the intracellular scaffolding proteins harmonin and/or whirlin, which connect, via myosinVIIa and possibly other interactome proteins, to the actin core of the stereocilia , 59, 60.The role of the different proteins in the links probably depends on the spatiotemporal stage of the links. It has been proposed that protocadherin-15 and cadherin-23 in the tip link play an essential role in triggering the mechanotransduction cascade . McGee eUsher proteins also take part in the transport of vesicles from the cuticular plate to the growing apical tip of stereocilia .Besides this, the presence of many of these proteins in the synaptic regions of inner and/or outer hair cells suggests that the Usher interactome might play a role in the neurotransmission of the mechanotransduction signal , 60, 63.There is evidence that myosin VIIa plays a role in the transport of opsin from the inner segment to the outer segment of the photoreceptors through the connecting cilium. Such evidence appears in studies in shaker-1, the mouse model defective for myosin VIIA, since shaker-1 accumulates opsin in the ciliary plasma membrane of photoreceptor cells , 65.Further studies have proven that both USH1 and USH2 proteins interact in the ciliary/periciliary region of cone and rod photoreceptors. The proteins usherin, VLGR1b, and SANS are associated with the periciliary ridge complex, which is thought to be the docking side for cargo loaded post-Golgi vesicles . In mammUsher proteins also localize in the photoreceptor synapse, as they do in the hair cells in the organ of Corti, where they could form a complex involved in the trafficking of the synaptic vesicles . HoweverIn the retinal pigment epithelium (RPE) the absence of myosin VIIa causes a significant decrease in phagocytosis of outer segment disks by the pigment epithelial cells , suggest(USH2A) is known to be responsible for isolated RP, which suggests that usherin plays a main role for the photoreceptor or that the rest of the Usher proteins are not essential in the photoreceptor function.Most of the USH genes are responsible not only for Usher syndrome but also for nonsyndromic hearing loss. To date, however, only one gene Usher syndrome is a clinically and genetically heterogeneous disorder which is important from a public health viewpoint because of the social isolation which Usher patients must endure. The first step towards correct diagnosis is proper differential diagnosis of the syndrome.Initially, USH manifests as a sensorineural hearing impairment, sometimes with vestibular dysfunction, with RO onset occurring later in life. Several syndromes may exhibit clinical signs which are similar to USH. Differential diagnosis should take into account the presence of endocrine abnormalities such insulin resistance, type 2 diabetes, hypertriglyceridemia, hepatic dysfunction, and/or renal failure, all of them would indicate Alström syndrome or the presence of obesity, mental retardation or cognitive impairment, and postaxial polydactly and hypogenitalism, which may be indicators of a Bardet-Biedl syndrome (BBS). If a family history of X-linked inheritance is observed, or if signs of dystonia or ataxia are detected, Mohr-Tranebjaerg syndrome should be suspected.Genetic tests could be a very powerful tool in differential diagnosis of USH patients. However, there are many factors that make the genetic study of this disease a complicated difficult one. As explained in this paper, the genes identified to date do not explain all the USH cases (this is true for BBS and Alström syndromes as well), and the variable nature of the proteins involved in USH and the complexity of the USH interactome make identifying novel genes a difficult task. This is due to genetic and allelic heterogeneity, which contribute to the low rate of mutation detection, together with the possible presence of large deletions, mutations in noncoding regions, or isoforms in low concentration only present in the affected tissues. Moreover, other complex inheritance forms could modify the phenotype and its expression, as recently shown by Ebermann et al. . All of Application of new technologies based on DNA chips could solve this problem; in fact, the recent creation of a specific microchip for this disease , 72 permGene characterization and mutation screening will unravel the functional aspects and allow a phenotype-genotype correlation to be established.Currently, there is no treatment available for Usher syndrome. The hearing-loss problem can be solved by the use of hearing aids and cochlear implantation, but the retinal problem remains unsolved. Therapeutic strategies to treat retinal degeneration target the specific genetic disorder (gene therapy), slowing or stopping photoreceptor degeneration or apoptosis  or even the replacement of lost cells  (reviewed in ). Howeve


This paper reviews the development process and discusses the key findings which resulted from our multidisciplinary research team's effort to develop an alternative digital hearing suitable for low-resource countries such as Thailand. A cost-effective, fully programmable digital hearing aid, with its specifications benchmarking against WHO's recommendations, was systematically designed, engineered, and tested. Clinically it had undergone a full clinical trial that employed the outcome measurement protocol adopted from the APHAB, the first time implemented in Thai language. Results indicated that using the hearing aid improves user's satisfaction in terms of ease of communication, background noises, and reverberation, with clear benefit after 3 and 6 months, confirming its efficacy. In terms of engineering, the hearing aid also proved to be robust, passing all the designated tests. As the technology has successfully been transferred to a local company for the production phase, we also discuss other challenges that may arise before the device can be introduced into the market. The World Health Organization (WHO) has estimated in 2005 that 278 million of the world population may have hearing difficulties . In ThaiIn principle, hearing aids are devices that amplify and customize incoming sound signals to meet specific requirements of each hearing impaired. For a digital hearing aid, the sound is picked up via its microphone(s) and converted into the digital form that is processed according to predefined DSP algorithms tailored for each patient's requirements. The modified signal is converted back to the analog form for the receiver to convert it into sound waves and send them directly into the ear. Through this process, precise amplification characteristics desired can be achieved. Today, there are various types of hearing aids  on the market, from body-aid (or pocket-type) to BTE (behind-the-ear), ITE (in-the-ear), ITC , or CIC . These readily available devices offer choices on both price and performance. It is believed, however, that there are still demands for alternative hearing aid devices that are not readily available on the market. This can particularly be true in developing countries, where desirable features such as cost-effectiveness, ease of use and maintenance, and fully digital processing capability are imperative.This paper discusses the development of an indigenous digital hearing aid targeted for rural usage. The result is a fully digital hearing aid that is designed for those with moderate-to-severe hearing loss. It comes in the shape of a pocket-type to meet the specific requirements for ease of use and robustness against extreme conditions of rural usage. The form factor also allows us to have an appropriate battery system that helps address the issue of maintenance cost. The hearing aid was systematically designed, engineered, and tested acoustically, functionally, and clinically. The design has now successfully been licensed out for local production.The paper is organized as follows: As hearing aids can vary vastly in terms of specifications and prices, it is imperative that the target for this project be the appropriate one. As guidelines for hearing aids and services for developing countries, the World Health Organization (WHO) has published a report in 2004 . Our appAnother important issue is the fitting range. As the majority of the elderly with hearing impairment fall into the class of having moderate to severe hearing loss, this would be the target group. It is important to design this early as it affects greatly the amplification characteristics needed. Our digital hearing aid, called the PDN-01B, is a pocket-type digital hearing aid. Even though it can be considered by some as old fashioned, the feedback that was gathered from users, especially those in the rural areas, suggested that there were still demands for a pocket-type device, which was considered more robust to extreme conditions and easier to operate. This is slightly different for the WHO recommendations, which prefer BTE to pocket-type. And we believe this was justified to pursue this pocket-type option based on our survey of actual needs of the local. The key was to provide a digital processing quality that would differentiate the hearing aid from the usual analog pocket aids, while offering the minimum cost of maintenance. The pocket-type device developed  is fullyIn engineering the device, we hoped to achieve the goals of cost-effectiveness, sound quality, and ease of use. The key components were the microphone, receiver, and the baseband processor (DSP). Although there are consumer grade parts available in the market, our design was based on using hearing-aid-specific parts, especially for these components. The main reasons for choosing these parts were to address the issues of current consumption that would affect its usage time before replacing the battery and the quality of the amplified acoustics. A hearing-aid-specific DSP is a low-voltage chip (1 Volt), which would offer superior current consumption over general DSP baseband counterparts . Also, being a system on chip  such as the hearing-aid-specific DSP usually implies lower power consumption when compared to equivalent systems built on discrete parts such as a DSP chip plus peripherals such as analog-to-digital converter (A/D) or digital-to-analog converter (D/A).In terms of the sound quality, it is also imperative to have as little as possible noise in the system as it affects the quality of the amplified acoustics. Also, using a relatively flat response receiver will make the overall transfer function of the system more precise. For these reasons, a low-noise microphone and a flat response receiver, designed specially for hearing aids, were preferred over consumer-graded counter parts. Doing so has a small disadvantage in terms of the cost of the device, but we expect that saving from lower cost of maintenance will be higher than the additional cost of the device itself. Our third objective was ease of use. By designing the device as a pocket-type with minimal use of peripherals such as sockets and buttons, we aimed to make the device easy to use and maintain. The number of buttons was also reduced further by the fact that the hearing aid was designed to be fitted via computer rather than needed built-in trimmers. The Thai language fitting software also provided easy and precise fitting.Based on the concept discussed, the PDN-01B digital hearing aid has key features as summarized in Amplification characteristics are arguably the most important features that will determine the usefulness of a hearing aid. Therefore, an approach to systematically and universally test and benchmark these devices via standardized tests is needed. Indeed, at present testing electroacoustics performance of hearing aids generally can be done to serve two purposes: (1) to verify that the instrument is functioning properly—according to the manufacturer's specifications and (2) to verify that the instrument is functioning appropriately—according to auditory needs of the wearer , 8. In tFor device development purposes, the measurement issues concerned here are with regard to the first purpose. Within the scope, the International Electrotechnical Commission (IEC) and American National Standards Institute (ANSI) are arguably the two world leading organizations for standardization of electroacoustic measurements in hearing aids. The most important sets of standards are the IEC-118 (IEC 60118) and ANSI-322 (ANSI S3.22) which cover most of the tests required. They are similar to a certain degree, and usually a hearing device certified for compliance with at least one of the standards should be sufficient.Among these standards, IEC 60118-0 measurement of electroacoustical characteristics  and IEC Our research team has set up our own electroacoustic testing facility that can support electroacoustic measurements according to the requirements set by the standards called the HA-TEST . It consApart from the needs to confirm its amplification characteristics, determined by electroacoustical measurements, another key test is to evaluate its real effects on wearer. This was done via a trial which aimed specifically to study users' satisfactions. The trial was carried out at Chiang Mai University, Thailand. It followed the prospective descriptive study approach, where descriptive statistic (both means and standard deviation) of the users' satisfaction was to be obtained from data analysis of handed-out questionnaires. The data was collected during the initial fitting, 1, 3, 6, and 12 months of followup. The candidates to this program needed to meet the criteria as stated in  The sample size was calculated for a single mean as follows:The questionnaire used in this trial was the universally accepted Abbreviation Profile of Hearing Aid Benefit (APHAB) by Cox and Alexander . It was The objective of this one-year-trial phase was also for the aids to undergo scheduled acoustical and engineering tests, especially to evaluate their reliability. These tasks were done when each user visited the hospital for his/her routine followups . They were (1) fitting and refitting, (2) acoustical measurements, and (3) component checking. The objective of the fitting and refitting was to check the ability of device to be refitted after the period of using. For each visit, the fitting data of each patient was recorded. The patient was either refitted with new set of parameters as determined appropriate by the audiologist or refitted with the same values if no adjustment was needed. Key engineering according to this part was to confirm that its ability to be refitted was intact. The second task was to check their acoustical performance. It was imperative that the hearing aid's amplification characteristics remained consistent. Although it was expected that some patients might need to adjust some fitting parameters during the followups, key acoustical parameters should remain within the intended rages. Here three parameters that can routinely be measured as part of IEC 60118-7 test that were the maximum output sound pressure level at input 90 dB SPL (Max OSPL90), the full-on gain (FOG), and lastly the total harmonic distortion (THD) were recorded. All measurements were performed onsite, using the FP35 Portable Hearing Aid Analysis available courtesy of the Rural ENT Foundation. The trial period was from October 2009 to July 2012. Overview of the completed trial is as illustrated in ease of communication ,reverberation ,background noise ,aversiveness of sounds .The data was collected and compared for pre- and posthearing aid usage to determine its benefit. Results analyzed are as shown in The result of users' satisfaction was determined via APHAB questionnaire. The APHAB questionnaire consists of 24 items. For each question, the hearing aid user is required to indicate at what level he/she agrees with the sentence; that is, A Always (99%), B almost always (87%), C generally (75%), D half-the-time (50%), E occasionally (25%), F seldom (12%), and G never (1%). The form was translated into Thai and used to evaluate the user's satisfaction of using the hearing aid in four main aspects that areIt can be seen that users were satisfied in terms of ease of communication (EC), reverberation (RV), and background noise (BN), with results found to be significant after 3 and 6 months. The relatively less-clear benefit in aversiveness of sounds (AV) is expected as amplification of unwanted noises is one of the most common problems faced when using a hearing aid. It is noted that similar results are obtained in Table 3 of   , among oThis paper discusses the development of an indigenous digital hearing aid targeted for rural usage. The result is a fully digital hearing aid that is designed for those with moderate-to-severe hearing loss. It comes in the shape of a pocket-type to meet the requirements for ease of use and robustness against extreme conditions of rural usage. The form factor also allows for appropriate battery system that helps address the maintenance cost issue. The PDN-01B hearing aid was systematically designed and tested acoustically, functionally, and clinically. The design has now successfully been licensed out for local production.


Lawsonia intracellularis in porcine cases of diarrhoea and the quantitative detection of Lawsonia intracellularis in faeces. A total of 156 pigs (10 to 70 days post weaning) with diarrhoea were randomly selected from 20 herds: The pigs were subjected to necropsy, histopathology, immunohistochemistry and faecal quantification of Lawsonia intracellularis by real time PCR.The study was designed to investigate correlation between histological findings of Lawsonia intracellularis excretion was significantly higher in pigs with gross lesions of proliferative enteropathy (median excretion: 5.92 log10 bacteria/g faeces) compared to pigs without gross lesions of proliferative enteropathy (median excretion: <3.3 log10 bacteria/g faeces) (P<0.001). Spearman’s correlation coefficient between the measureable PE lesions and L. intracellularis excretion was 0.50 (P<0.001). A significantly increasing trend in Lawsonia intracellularis excretion level for increasing proliferative enteropathy histopathology and immunohistochemistry scores was demonstrated . Spearman’s correlation coefficient between the histopathology scores and L. intracellularis excretion was 0.67 (P<0.001). Spearman’s correlation coefficient between the IHC scores and L. intracellularis excretion was 0.77 (P<0.001).The median Lawsonia intracellularis were correlated in pigs with diarrhoea. Overall the results suggest that clinically important levels for Lawsonia intracellularis excretion in faeces may be established. Such clinical threshold levels may be used in practice to confirm a diagnosis of Lawsonia intracellularis associated diarrhoea.The histological and quantitative PCR detection of  Escherichia coli, Brachyspira pilosicoli, Brachyspira hyodysenteriae, Salmonella spp. and Lawsonia intracellularis. L. intracellularis is the cause of proliferative enteropathy (PE) and considered one of the most important intestinal pathogens [L. intracellularis obtaining a correct microbiological diagnosis is complicated. Subclinical infection is common [L. intracellularis can be demonstrated by PCR in animals vaccinated by an avirulent live vaccine [L. intracellularis in faeces [L. intracellularis bacteria in faeces on a routine basis. This may be useful for confirmation of L. intracellularis as the microbiological cause in porcine diarrhoea. Correlation between disease severity and excretion load for a specific pathogen may be expected. However, such correlation cannot always be demonstrated [The antibiotic consumption in the agricultural industry has increasing concern in relation to development of antimicrobial resistance in both animals and humans . In Denmathogens . Clinicas common , L. intr vaccine  and the  vaccine . Developn faeces ,7-10 hasnstrated .L. intracellularis in porcine cases of diarrhoea and the quantity of L. intracellularis detected by qPCR in faecesIn this paper a quantitative real time PCR was used to investigate correlation between histological findings of A total of 20 outbreaks were examined (one outbreak in 20 different herds) and 160 pigs (mean days post weaning = 33) were euthanized during the herd visits. Four pigs were excluded as less than 1.0 gram of faeces had been obtained, providing a total of 156 pigs with diarrhoea for the statistical analysis.L. intracellularis was detected by qPCR in 25.6% of the pigs. Among the qPCR positive pigs 30.0% were below the dynamic range (4.3 log10 bacteria/g faeces) of the qPCR, 70.0% were in the dynamic range and no pigs were above the dynamic range (8.3 log10 bacteria/g faeces). The median excretion for all positive pigs was 5.44 log10 bacteria/g faeces.n=11) were 193 cm in extent (range: 17 to 600). Among the pigs without gross lesions, 20.3% were qPCR positives (median excretion: 5.03 log10 bacteria/g faeces). Among the pigs with gross lesions, 84.6% were qPCR positives (median excretion: 6.01 log10 bacteria/g faeces). Spearman’s correlation coefficient between the measureable PE lesions and L. intracellularis excretion was 0.50 (P<0.001). Association between the measureable PE lesions and L. intracellularis excretion in qPCR positive pigs are displayed in Figure L. intracellularis excretion was significantly higher in pigs with gross lesions (median excretion: 5.92 log10 bacteria/g faeces) compared to pigs without gross lesions (median excretion: below limit of detection) (P<0.001).Gross lesions of PE were demonstrated in 8.3% of the pigs. The mean gross lesions in pigs with measureable lesions n=1 were 1910 bacteria/g faeces). Among the pigs with histological lesions, 90.9% were qPCR positives . The test for trend demonstrated a significantly increasing trend in excretion level for increasing histopathology scores (P<0.001). Spearman’s correlation coefficient between the histopathology scores and L. intracellularis excretion was 0.67 (P<0.001). Association between the histopathology score and L. intracellularis excretion is displayed in Figure The Kruskal-Wallis equality of populations rank tests demonstrated an overall significantly difference between the median excretion levels for the histopathology scores . Among the IHC positive pigs, 87.1% were qPCR positives (median excretion = 5.83 log10 bacteria/g faeces). The Kruskal-Wallis equality of populations rank tests demonstrated an overall significantly difference between the median excretion levels for the IHC scores (P<0.001). The test for trend demonstrated a significantly increasing trend in excretion level for increasing IHC scores (P<0.001). Spearman’s correlation coefficient between the IHC scores and L. intracellularis excretion was 0.77 (P<0.001). Association between the IHC score and L. intracellularis excretion is displayed in Figure Borrelia burgdorferi[Mycoplasma genitalium[Brucella spp. [Helicobacter pylori[Mycobacterium leprae[Mycoplasma gallisepticum[Streptococcus pneumoniae[Brucella melitensis[Haemophilus influenzae[Legionella penumophila[Streptococcus pneumoniae[Haemophilus influenzae[Mycobacterium tuberculosis[Gardnella vaginalis and Atopobium vaginae[Routine q-PCR quantification of a number of virus infections is now well established in medicine and has recently been reviewed -14. Applrgdorferi, Mycoplaenitalium, Brucelllla spp. , Helicober pylori, Mycobacum leprae,20, Mycoisepticum, Streptoneumoniae, Brucellelitensis, Haemophnfluenzae and Leginumophila. Furtherneumoniae,26, Haemnfluenzae, Mycobacerculosis, Gardnelm vaginae.L. intracellularis in faeces was demonstrated in pigs with diarrhoea. These results are in accordance with a previous report of correlation between L. intracellularis bacteria load in mucosal scrapings and the severity of intestinal lesions [L. intracellularis excretion level was higher in pigs with PE gross lesions compared to pigs without gross lesions. This suggests that quantification by qPCR might be applied for examination of presence or absence of gross lesions of PE. The extent of PE gross lesions and L. intracellularis excretion levels were apparently not correlated. However, this association should be further investigated because of the low number of pigs with gross lesions in the current study.In the current study a positive correlation between disease severity in terms of pathological findings and quantitative detection of  lesions . In qPCRL. intracellularis excretion levels. One interesting aspect of the observed observations is that demonstration of L. intracellularis in faeces is not evident of ileitis but in case of ileitis high excretion levels of L. intracellularis can be expected. The applied study design did not take progression of L. intracellularis infection into consideration. The obtained results are only relevant for the association between gross pathology, histopathology, IHC and quantification of L. intracellularis by qPCR at the time of faecal sampling. Correlation over time and progression of L. intracellularis excretion during an infection should be performed in a longitudinal study design. Factors that potential could have influenced the results are the intermitted shedding of L. intracellularis previously described [L. intracellularis in faeces and/or intestinal tissue. Quantification of L. intracellularis in faeces has been reported to have an acceptable within day repeatability [L. intracellularis in faeces is not a major bias in the current study. Some of the qPCR negative animals were probably false negatives because of intermitted shedding caused by a L. intracellularis excretion level close to the qPCR’s limit of detection as previously described [L. intracellularis in the intestinal tissue.In contrast, increasing histopathology/IHC scores were correlated to increasing escribed  and non-tability  suggestiescribed . This asL. intracellularis. Therefore it seems very unlikely that other factors would have a confounding effect between these lesions and the faecal L. intracellularis excretion. However it is possible that interactions exists providing different absolute quantitative associations between L. intracellularis and the lesions depending on simultaneous infections or other factors. The number of observations in the current dataset did not allow for investigation of this aspect, which should be explored before clinical relevant threshold levels can be used in practice.Other factors including intestinal infections could potentially influence the reported associations. The reported lesions  are very specific for L. intracellularis is correlated in pigs with diarrhoea. Overall the results suggest that clinically important levels for L. intracellularis excretion in faeces may be established. Such clinical threshold levels may be used in practice to confirm a diagnosis of L. intracellularis associated diarrhoea, for monitoring of treatment effects or for prognostic purposes.The study has demonstrated that histological and quantitative PCR detection of L. intracellularis in pigs with different levels of histological findings of L. intracellularis was available for a formal sample size calculation. General sample size and power considerations for estimation of correlation and differences between groups for continuous outcome variables were performed using Stata IC version 11. Data previously collected for investigation of outbreaks of treatment-requiring diarrhoea in pigs 10–70 days post weaning was considered to contain a sufficient number of pigs for the current study. In brief, farmers from 32 selected herds were requested to notify the corresponding author at initiation of an outbreak of diarrhoea in pigs between 10 and 70 days post weaning. All herds were visited the day following notification and the farmer was not allowed to medicate any pigs before the pigs had been examined.No prior information concerning faecal excretion levels of o C. Necropsy and processing of faecal samples were performed the following day.To avoid any post antibiotic effect the outbreaks were not included if the pigs had received antibiotic medication in feed or water with-in the last 7 days. In each outbreak 8 pigs with diarrhoea was selected by random sampling for necropsy and collection of faecal samples. The pigs were euthanized in the herd, the abdomen was incised and intestinal specimens for histopathological examinations were collected. At the end of each herd visit the euthanized pigs and faecal samples were transported to the National Veterinary Institute, Technical University of Denmark. Pigs and faecal samples were stored at 4All parts of the study were performed in consistency to the Danish welfare legislation.The pigs were subjected to necropsy and evaluation of internal organs. Gross lesions of PE was graded as 0 = no lesions, 1 = thickened intestinal wall and/or mucosa hyperplasia, 2 = necrotic enteritis. Furthermore, the extension of PE gross lesions was measured.Intestinal samples were obtained within 5 minutes of euthanasia and immediately fixated in 10% neutral buffered formalin. The intestinal tissue samples were obtained from ileum , one random site from jejunum and from the mid-spiral region of colon.At necropsy further tissue samples for histology were obtained from jejunum  and from any intestinal gross lesions. All samples were embedded in paraffin wax and sectioned in 3 μm sections.All segments were stained by hematoxylin and eosin (H&E) for histopathology. Histological lesions of PE were graded on a scale from 0 to 4: Grade 0: no significant lesions; grade 1: up to 25% hyperplastic enterocytes, focal or multifocally, and reduction of the number of goblet cells; grade 2: 25 to 50% hyperplastic enterocytes, multifocally, and reduction of the number of goblet cells; grade 3: 50 to 75% hyperplastic enterocytes, multifocally, and reduction of the number of goblet cells; grade 4: More than 75% hyperplastic enterocytes, multifocally or diffusely, and reduction of the number of goblet cells.L. intracellularis as previously described [All segments were stained by immunohistochemistry for detection of escribed . ResultsAll faecal samples were subjected to faecal dry matter determination to confirm the diagnosis of diarrhoea in the individual pigs. The individual faecal samples were mixed with a spoon and faecal dry matter content was determined by drying to constant weight using a microwave oven as previously described . Only piL. intracellularis were included in each experiment. All qPCR experiments were run in duplicates as previously described [10 bacteria/g faeces and the dynamic range was 4.3 - 8.3 Log10 bacteria/g faeces.A suspension of 10% faeces in phosphate buffered saline (PBS) was prepared from each faecal sample. The individual faecal samples were mixed with a plastic spoon and 0.1 gram of faeces was suspended in 0.9 gram of PBS. The faeces suspension was stored at -80°C until further processing. Total DNA was extracted from the 10% faecal suspensions by using QIAsymphony extraction robot and QIAsymphony Virus/Bacteria Mini Kit . The protocol was Pathogen Complex 200, the processed sample volume was 200μL and elution was done in 110μL. Prior to DNA extraction the 10% faecal suspensions were pre-treated by bead beating in Tissuelyser  with stainless steel beads . The suspensions were centrifuged for 90 sek. at 10 000 rpm  and the supernatant was transferred to the QIAsymphony robot. One negative extraction sample of other bacterial cells and one positive extraction sample of P-values were calculated.Classification of individual pigs in relation to histological lesions of PE and IHC was performed before the statistical analysis. The highest grade from any intestinal segment in a pig defined the grade for that pig. To obtain more observations in each category, both histopathology and IHC grades were reclassified in the statistical analysis. In relation to histopathology, pigs without histopathological lesions of proliferative enteropathy were classified as score zero. Pigs with histopathology grade one or two were classified as score one and pigs with histopathology grad three or four were classified as score two. In relation to IHC, pigs observed to be negative by IHC were classified as score zero. Pigs with labeled antigen in lamina propria only  were classified as score one. Pigs with IHC grad four or five were classified as score two and pigs with IHC grad six or seven were classified as score three. Overall differences in the median excretion level between pigs with different scores of gross pathology, histopathology and IHC classifications was assessed by the Kruskal-Wallis equality of populations rank test. The overall trend in excretion levels for the different scores was assessed as previously described  and SpeaThe authors declare that they have no competing interests.All authors conceived and designed the study. KSP performed all clinical herd investigations. RG and TKJ performed all pathological and histopathological examinations. MS and ØA performed all PCR experiments. KSP conducted the statistical analyses. All authors participated in drafting the manuscript. All authors have read and approved the final manuscript.


C. elegans AWC sensory neurons to promote olfactory adaptation. After prolonged stimulation of these neurons, EGL-4 translocates from the cytosol to the nuclei of the AWC. This nuclear translocation event is both necessary and sufficient for adaptation of the AWC neuron to odor. A cGMP binding motif within EGL-4 and the Gα protein ODR-3 are both required for this translocation event, while loss of the guanylyl cyclase ODR-1 was shown to result in constitutively nuclear localization of EGL-4. However, the molecular changes that are integrated over time to produce a stably adapted response in the AWC are unknown. Here we show that odor-induced fluctuations in cGMP levels in the adult cilia may be responsible in part for sending EGL-4 into the AWC nucleus to produce long-term adaptation. We found that reductions in cGMP that result from mutations in the genes encoding the cilia-localized guanylyl cyclases ODR-1 and DAF-11 result in constitutively nuclear EGL-4 even in naive animals. Conversely, increases in cGMP levels that result from mutations in cGMP phosphodiesterases block EGL-4 nuclear entry even after prolonged odor exposure. Expression of a single phosphodiesterase in adult, naive animals was sufficient to modestly increase the number of animals with nuclear EGL-4. Further, coincident acute treatment of animals with odor and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) decreased the number of animals with nuclear EGL-4. These data suggest that reducing cGMP levels in AWC is necessary and even partially sufficient for nuclear translocation of EGL-4 and adaptation as a result of prolonged odor exposure. Our genetic analysis and chemical treatment of C. elegans further indicate that cilia morphology, as defined by fluorescent microscopic observation of the sensory endings, may allow for odor-induced fluctuations in cGMP levels and this fluctuation may be responsible for sending EGL-4 into the AWC nucleus.The Protein Kinase G, EGL-4, is required within the  In order to effectively shape behavior, neural circuits must integrate sensory information by collating input and placing this input in a context of previous or concurrent sensory information. This modulation of behavior by previous experience or against a background of current input is termed behavioral plasticity. All sensory systems, namely the visual, olfactory, mechanosensory, thermosensory, and auditory systems, are capable of exhibiting neuronal plasticity C. elegans is inherently attracted to a variety of volatile odors. Using a pair of head neurons, called the ‘AWC Right’ and ‘AWC Left’, C. elegans can sense and seek out attractive odors such as butanone, benzaldehyde, and isoamyl alcohol The nematode C. elegans will cease to seek out this odor. This decreased odor-attraction is termed odor adaptation After prolonged stimulation with a particular odor, We had previously shown that morphologically intact AWC cilia, as determined by fluorescent microscopic examination of the endings of the AWC neurons, are required for cytoplasmic localization of EGL-4 in an odor-naive animal cGMP signaling is an ancient and highly conserved feature of ciliary signaling. It has been noted that many eukaryotes that have lost cilia during evolution also lack cGMP signaling machinery C. elegans to sense various volatile attractive odorants The AWC neurons enable odr-1 mutant genetic background daf-11 and odr-1 mutants. Our laboratory has shown previously that the transgene GFP::EGL-4 is a fully functional molecule that can rescue adaptation defects in egl-4 null mutant animals odr-1(n1936) = 100% nuclear (as seen in daf-11(m47) = 100%; p<0.0005 for wildtype naive animals versus odr-1 and daf-11 naive mutant animals). One explanation of these results is that reducing cGMP levels may promote the nuclear entry of GFP::EGL-4.Multiple guanylyl cyclases are expressed in the AWC C. eleganspde-1 pde-5; pde-2; pde-3 we found that GFP::EGL-4 is constitutively cytoplasmic. In wildtype animals after prolonged benzaldehyde exposure 87% of animals display nuclear GFP::EGL-4 and after prolonged butanone exposure 85% of animals display nuclear GFP::EGL-4. In the PDE quadruple mutant, however, after prolonged benzaldehyde exposure only 2.5% of animals display nuclear GFP::EGL-4 and after prolonged butanone exposure only 1% of animals display nuclear GFP::EGL-4 . This further suggests that decreases in cGMP levels may direct the nuclear entry of GFP::EGL-4 and increases in cGMP may block the nuclear entry of GFP::EGL-4.To test this in another way we examined the localization of GFP::EGL-4 in PDE mutants. There are four known cGMP PDEs in pde genes in the PDE quadruple mutant  of 0.19 after prolonged benzaldehyde exposure. However, the PDE quadruple mutant displayed a CI of 0.85 after prolonged benzaldehyde exposure . Thus, chronically low cGMP (in guanylyl cyclase mutants) or high cGMP (in phosphodiesterase mutants) results in either constitutively nuclear or cytoplasmic EGL-4 respectively. Also, we did not observe adaptation defects in any of the pde single mutants we examined . Incubating animals with 1 mM or 5 mM IBMX and odor had no statistically significant effect on the nuclear entry of GFP::EGL-4 (p = 0.32 for 1 mM and p = 0.14 for 5 mM), and incubating with IBMX alone without odor had no effect on the localization of GFP::EGL-4 (data not shown). We also increased the time frame of IBMX exposure by pre-incubating animals in 10 mM IBMX for 45 minutes prior to the co-incident exposure of 10 mM IBMX with benzaldehyde for 80 minutes  changes in cGMP levels are required to trigger the nuclear entry of EGL-4, we blocked cGMP reduction acutely by treating EGL-4::GFP expressing animals with the PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX: a non-selective inhibitor of both cAMP and cGMP PDEs) during the period of prolonged odor exposure that induces nuclear translocation of EGL-4 and adaptation. In the negative control treatment, 92% of animals that were incubated with the odor benzaldehyde for 80 minutes without IBMX displayed nuclear GFP::EGL-4 ( minutes . Howeverhsp16.2::pde-3.1a) in odor-naive animals. Inducing the overexpression of pde-3 in wildtype, odor naive adult animals resulted in modest but significantly higher numbers of animals displaying nuclear GFP::EGL-4 . Taken together, these data argue that reducing or increasing cGMP levels can dynamically modulate the nuclear entry of GFP::EGL-4.To investigate further if dynamic changes in cGMP levels trigger the nuclear entry of GFP::EGL-4, we took the opposite approach and asked whether acutely decreasing cGMP levels in adults might be sufficient to send EGL-4 into the nucleus of a naive animal. Thus, we overexpressed the cGMP phosphodiesterase PDE-3 under a heat-inducible promoter , and are also defective in adaptation responses osm-1::OSM-1 into the genetic background of py825 osm-1::OSM-1, the nuclear GFP::EGL-4 phenotype is reduced to 8%. This is statistically indistinguishable from wildtype animals (p = 0.46). OSM-1 is an ortholog of the intraflagellar transport (IFT) complex B component, IFT172 osm-1 locus, we found that the py825 lesion is caused by a premature ochre stop codon in the tenth exon che-3::CHE-3 into the genetic background of py827 che-3::CHE-3. This is statistically indistinguishable from wildtype animals (p = 0.408). The gene che-3 encodes the isoform 1b of the dynein heavy chain (DHC) che-3 locus in py827 revealed that the lesion is defined by a 397 bp deletion that removes the final 54 bps of the final exon of the che-3 gene  animals expressing this transgene exhibited normal cilia  mutant animals by overexpressing the constitutively cytoplasmic GFP::EGL-4. In this scenario we observed no rescue of the ciliopathy  suppressed the constitutively nuclear GFP::EGL-4 phenotype of che-3 mutant animals (che-3(e1124) mutant animals display nuclear GFP::EGL-4 in 100% of cases. In che-3(e1124) transgenic animals expressing GFP::EGL-4(T276A), in which the cGMP binding site is mutated, the nuclear GFP::EGL-4 phenotype is reduced to 4%. This is statistically indistinguishable from the percent of naive wildtype animals displaying nuclear GFP::EGL-4 (p = 0.84), and also indistinguishable from the number of naive wildtype transgenic animals expressing the transgene GFP::EGL-4(T276A) (p = 0.09). Importantly, the cilia defects of che-3(e1124) were not altered by expression of this constitutively cytosolic form of EGL-4 (data not shown). These data suggest that the native cGMP binding sites within EGL-4 are required for nuclear entry of EGL-4, even in a cilia biosynthesis mutant background such as che-3(e1124), which normally exhibits a constitutively nuclear EGL-4 phenotype. Mutation of one of the cGMP binding sites within EGL-4 may disrupt proper cGMP occupancy that could normally direct autophosphorylation or heterophosphorylation events important for the nuclear entry of EGL-4. Alternatively, by interfering with the ability of EGL-4 to bind cGMP, we may have altered the conformation of EGL-4 in a way that inhibits its ability to translocate to the nucleus.We wanted to understand whether the nuclear localization of EGL-4 in mutants with defects in cilia morphology is dependent upon its ability to bind cGMP. We examined a mutation (T276A) within the low affinity binding site of EGL-4. Our decision to examine a mutation affecting the low affinity cGMP binding site was guided by previous studies into cGMP binding within PKGs. These studies have shown that there are two cGMP bindings sites (low affinity and high affinity) and they act allosterically to activate the kinase domain with a Hill coefficient of 1.6  animals . che-3(eC. elegansp = 0.01). They did, however, show evidence of cilia defects that were comparable to the EGTA-treated wildtype animals (data not shown). In fact, even without EGTA treatment a subset of PDE quadruple mutants exhibit a decreased AWC cilia fan shape size, and these animals still display cytosolic GFP::EGL-4. We also compared the percentage of cilia defective animals exhibiting nuclear GFP::EGL-4 after 100 mM EGTA treatment .Our data so far describes a model whereby the AWC cilia facilitate changes in cGMP levels, and these changes are interpreted by EGL-4 to determine its proper localization. Next, we tested if GFP::EGL-4 is cytosolic in the PDE quadruple mutant background even when cilia morphology is perturbed. Calcium has been shown to be critical in maintaining cilia morphology in reatment  between C. elegansp = 0.001 between wildtype animals expressing RAB-8[Q67L] and PDE quadruple mutants expressing RAB-8[Q67L]). Taken together, this suggests that elevated cGMP levels are epistatic to changes in cilia morphology in relation to the localization of EGL-4.Next we used a genetic manipulation to examine the epistatic relationship between elevated cGMP and cilia morphology in AWC with respect to the localization of GFP::EGL-4. Previously it has been shown that over-expression of a constitutively active form of the small GTPase RAB-8 resulted in cilia defects in the AWB neurons in The elaborate structure of the AWC cilia is exquisitely formed for function. The petal-shaped cilia provide a large membranous area within which sensory and transduction proteins are localized. This structure both concentrates and compartmentalizes sensory signal transduction components. In concert with intracellular signaling proteins, the factors within the AWC cilia provide a dynamic response range as well as functional plasticity to the AWC neuron.The protein kinase G EGL-4, which is found throughout the AWC cytoplasm including the cilia, is a central regulator of plasticity in the AWC neuron odr-1(null) mutants C. elegans genome Non-motile cilia, like that of the AWC, are developed and maintained by intraflagellar transport (IFT). IFT employs the anterograde motor, Kinesin, to traffic ciliary cargo to the growing end of the ciliary structure, and uses the retrograde motor, cytoplasmic Dynein 1b, to traffic cargo back to the base of the cilium Interestingly, in our previous studies, over expression of the guanylyl cyclase ODR-1 did not have the same effect as loss of PDE expression We also demonstrated that dynamic changes in cGMP levels in adults through over expression of the phosphodiesterase PDE-3 under a heat-inducible promoter or pharmacological treatment with IBMX both modestly affected the nuclear entry of GFP::EGL-4 in AWC. The discrepancy between the marked extent of our ability to alter nuclear EGL-4 levels by genetic means (genetic loss of PDEs), which resulted in a high percentage of cytoplasmic EGL-4 animals in the presence of odor, versus the modest block in nuclear translocation by acute inactivation of the PDEs by IBMX might indicate either that the pharmacological manipulations are less effective or that the PDEs are required for additional developmental events that allow EGL-4 to enter the AWC nucleus. Likewise, acute expression of a PDE from the heat-inducible promoter was only able to promote nuclear translocation in a small percent of the treated animals. This could indicate that the treatment did not adequately reduce cGMP levels or that additional factors, perhaps phosphorylation events downstream of Gα activation, are required to promote nuclear entry of EGL-4. Once again, cGMP-based imaging may help to distinguish between these possibilities.C. elegans, Liu et al. pde-1 pde-5; pde-2; pde-3 quadruple mutants but exhibited very slow recovery, suggesting that cGMP levels may be modulated independently of PDE activity. Perhaps AWC exhibits a similar cascade, whereby acute odor recognition is executed independent of PDE activity, and more long-term events such as olfactory adaptation are defined by PDE modulation of cGMP levels.Interestingly, chemotaxis was not affected by loss of the PDEs in contrast to adaptation, which was completely abolished. This indicates that dynamic fluxes of cGMP via PDE may be less important for acute odor recognition than adaptation. Although, there may be alternate factors or pathways required to modulate cGMP levels to allow for acute odor recognition. In a recent study of the photoreceptor cell ASJ in C. elegans may provide information on the temporal activity of cGMP in AWC, during both acute odor recognition and sustained odor stimulation.Though we have implicated fluctuations in cGMP as being important for directing EGL-4 localization and initiating olfactory adaptation, to develop a complete picture of how cGMP dynamics shape AWC mediated behaviors, we will need to develop temporal and spatial indicators of cGMP levels. Recently, several groups have reported their data on a cGMP-sensitive GFP molecule osm-1(py825), che-3(py827), dpy-6(e14) unc-3(e151), dpy-5(e61) unc-13(e1091), osm-1(pr816), che-3(e1124), py825; Ex[OSM-1], py827; Ex[CHE-3], egl-4(n479), N2; pyIs500, N2; Ex[(p)odr-3::NLS::GFP::EGL-4], py825; Ex[(p)odr-3::GFP::ΔNLS EGL-4], egl-4(n479); Ex[(p)odr-3::GFP::ΔNLS EGL-4], N2; Ex[(p)odr-3::GFP::EGL-4 (T276A)], che-3(e1124); Ex[(p)odr-3::GFP::EGL-4 (T276A)], daf-11(m47), daf-11(m47); pyIs500, odr-1(n1936), odr-1(n1936); pyIs500, pde-1(nj57) pde-5(nj49); pde-2(tm3098); pde-3(nj59); Ex, pde-1(nj57) pde-5(nj49); pde-2(tm3098); pde-3(nj59), pde-1(nj57) pde-5(nj49); pde-2(tm3098); pde-3(nj59); Ex[(p)ceh-36::RAB8[Q67L], N2; Ex[(p)ceh-36::RAB8[Q67L], unc-5(e53)IV; dpy-11(e224)V; lon-2(e678)X, dpy-5(e61)I; bli-2(e768)II; unc-32(e189)III, pyIs500; Ex. All strains were maintained on NGM plates that were seeded with E. coli strain OP50 according to standard protocol Bristol N2, pyIs500 animals were mutagenized using 50 mM of EMS as described previously L4 hermaphrodite py825 mutant was assigned to LGX by the following cross: py825 hermaphrodites were crossed with wildtype males and F1 male cross progeny were examined for the constitutively nuclear GFP::EGL-4 phenotype - all male cross progeny were mutant for the GFP::EGL-4 localization phenotype suggesting that py825 is X-linked. Three factor mapping with unc-3 dpy-6 animals mapped py825 very close to unc-3 . Complementation tests were performed with mutant animals harboring lesions in the chromosomal region of unc-3 and from this we found that py825 and osm-1(pr816) are allelic. The py827 mutant phenotype was assigned to LGI using the mapping strains MT464 and MT465 . Three factor mapping with unc-13 dpy-5 animals placed py827 very close to unc-13 . Complementation tests with animals harboring mutations in this chromosomal region were performed and it was found that py827 and che-3(e1124) are allelic. AWC cilia surface area was measured for osm-1(py825) and che-3(py827) (see osm-1(py825) lesion is caused by a premature ochre stop codon in the tenth exon changing a tyrosine (TAT) at amino acid position 496 to a STOP (TAA). The che-3(py827) lesion is defined by a 397 bp deletion that removes the final 54 bps of the final exon of the che-3 gene and can be genotyped using the following primer pair: jz827-F GACTCCTTCTCAACTACCAGCTAAACAATG and jz827-R CATCTGCGAGACGTACTGATAGAATACAAG.The 20 and examined using a TRITC filters under 40× magnification.The lipophillic dye DiD (Invitrogen) was eluted in 2.5 ml of dimethylformamide (DMF) to obtain a stock solution of 10 mg/ml. Worms were washed from OP50 seeded plates two times in M9 solution and exposed to DiD dye (1∶1000) on a rotator for 2 hours. The worms were then washed two times in M9 and allowed to recover on an OP50 seeded plate for 1 hour. The animals were then washed off the plate two times in ddHChemotaxis was performed as described previously pyIs500 worms were included for every translocation assay as a positive control using ≥75th percentile as the baseline for successful control assays and treatment assay inclusion. Between twenty and fifty animals were scored for each translocation assay and repeated on separate days three to five times.Four to five L4 animals were picked onto a 10 cm OP50 seeded plate and incubated at 25°C. Animals were washed from these plates and translocation assays were performed by exposing mutant animals containing the GFP::EGL-4 transgene to odor (adapted) or S-Basal (unadapted) for 80 minutes and then scoring the number of worms exhibiting EGL-4 in the AWC ‘ON’ nucleus after butanone exposure or in both AWC nuclei after benzaldehyde exposure under 40× magnification. Wildtype pde-1 pde-5; pde-2; pde-3 mutant animals containing the transgene GFP::EGL-4 (pyIs500) were incubated in 100 mM EGTA (pH 7.0) for 60 minutes and the subcellular localization of EGL-4 in AWC was examined as described. For IBMX (Sigma) treatment, GFP::EGL-4 (pyIs500) expressing animals were incubated with adaptation solutions containing various IBMX concentrations . Between 50 and 80 animals were counted for each assay, and the assay was repeated in three separate trials. For the heat-induced expression of pde-3.1a, ∼5 L4 pyIs500 (GFP::EGL-4) transgenic animals containing the hsp16.2::pde-3.1a transgene were grown at 15°C for ∼8 days, then washed 3 times in S-basal buffer and placed on an unseeded plate at 30°C for 2 hours. Then the subcellular localization of EGL-4 in AWC was examined microscopically as described. Between 30 and 50 animals were examined on each day for both the control and experimental populations and repeated in four separate trials on different days.For EGTA (Sigma) treatment, wildtype and odr-3::GFP::EGL-4, (p)odr-3::GFP::NLS::EGL-4, (p)odr-3::GFP::EGL-4ΔNLS and (p)odr-3::GFP::EGL-4(T276A) was described previously ceh-36::RAB8[Q67L] plasmid was generated by PCR amplifying the RAB-8[Q67L] cDNA with primers flanked with EcoRI and KpnI sites, the amplicon was digested with EcoRI and KpnI and inserted into a (p)ceh-36 containing plasmid in pPD95.75. The OSM-1 rescuing array was a gift from Lesilee Rose, and the WRM0637cB09 fosmid library clone was used for the CHE-3 rescuing array. The PCR fusion hsp16.2::pde-3.1a was made by PCR amplifying the heat shock promoter hsp-16.2 and pde-3.1a gene (from ATG start codon to ∼1 kb downstream of stop codon) from genomic DNA and fusing these products together by PCR fusion as described previously hsp16.2 was: hspF gatcaagagcatttgaatcagaatatgg and hspR pde-3 fuse ttttgatttaaaaaccttggagttcatagtgagatgattatagtttgaagatttctaat. The primer pair used to amplify the pde-3.1a product was: pde-3F atgaactccaaggtttttaaatcaaaaaaaa and pde-3R gaaaaagggcagttttgttgtgtatgc. These products were fused using the nested primer pair: hspF nest agttttttagatgcactagaacaaagcgtg and pde-3R nest gggcagttttgttgtgtatgcaaat. Rescuing pde transgenes were generated by PCR amplifying gDNA fragments containing ∼1.5 kb upstream of the start site and ∼1 kb downstream of the stop codon of the pde-1, pde-2, and pde-5 genes. In the case of pde-3 we used the fosmid WRM0623dE11 which contains part of the pde-3 locus. The PCR amplicons and fosmid were co-injected together at 5 ng/µl each and transgenic lines were assayed for rescue of the PDE quadruple adaptation behavior defect. The following primer pairs were used to amplify gDNA fragments from the pde-1, pde-2 and pde-5 loci: pde-2F ttatcagctgattatttcgttttagtgatagtg and pde-2R caccatgatctctaattataatcccgc; pde-1F acaaaaatgtgttcaactgatacaattttc and pde-1R acttgaactcggtgcggattccggaatc; pde-5F acgattcctacaaactacgaataccctac and pde-5R gttctttgaaccaaaaggaaggcatac.Construction of (p)Figure S1Chemotaxis response of the PDE quadruple mutant and the guanylyl cyclase mutants daf-11 and odr-1 to the AWC sensed odor butanone. “−” indicates unexposed animals and “+” indicates exposed animals. **Indicates p≤0.005 significant differences between chemotaxis index (CI) values between wildtype unexposed animals and odr-1 or daf-11 mutant unexposed animals. *Indicates p≤0.05 significant differences between wildtype odor-exposed CI values and pde quadruple mutant odor-exposed CI values.(TIF)Click here for additional data file.Figure S2Chemotaxis response of wildtype, PDE quadruple mutants, and PDE quadruple mutants expressing rescuing pde transgenes to the AWC sensed odor benzaldehyde. “−” indicates unexposed animals and “+” indicates exposed animals. *Indicates p≤0.05 significant differences between chemotaxis index values between PDE quadruple mutants expressing rescuing pde transgenes plus and minus odor.(TIF)Click here for additional data file.Figure S3Populations of GFP::EGL-4 (pyIs500) expressing animals were incubated with 10 mM IBMX for 45 minutes and then exposed to the odor benzaldehyde with 10 mM IBMX. The percentage of animals displaying nuclear GFP::EGL-4 were compared to populations exposed to odor alone for 80 minutes or populations exposed to no odor. *Indicates p≤0.05 significant differences.(TIF)Click here for additional data file.Figure S4Chemotaxis responses of wildtype animals and the mutants, py825 and py827 to the AWC-sensed odor benzaldehyde. “−” indicates unexposed animals and “+” indicates exposed animals. *indicates p≤0.05 significant differences between mutants and wildtype animals.(TIF)Click here for additional data file.Figure S5PCR genotyping of the 397 bp deletion lesion of py827. First lane: DNA marker; Lanes 2–4: py827 gDNA PCR product; Lanes 5–7: N2 (wildtype) gDNA PCR product. For each template (i.e. py827 and wildtype) the PCR reaction is performed in triplicate. Primer pairs used were: jz827-F GACTCCTTCTCAACTACCAGCTAAACAATG jz827-R CATCTGCGAGACGTACTGATAGAATACAAG.(TIF)Click here for additional data file.Figure S6Image of a wildtype animal after 60 mins treatment with 100 mM EGTA. The white dotted box indicates morphological defect in the fan-shaped AWC cilia after treatment.(TIF)Click here for additional data file.Figure S7Image of the AWC neuron in a transgenic animal expressing RAB-8[Q67L] under an AWC promoter. Expression of RAB-8[Q67L] in AWC causes defective cilia morphology.(TIF)Click here for additional data file.Figure S8The AWC cilia surface area of adult animals grown at room temperature was calculated using Volocity® software. Using the ‘measure objects’ tool the region of interest (ROI) was captured as indicated in image by a blue box and measured (µm2).(TIF)Click here for additional data file.Table S1The AWC cilia surface area of adult animals grown at room temperature was calculated using Volocity® software. The most distal (anterior) 5 µm region was selected as the region of interest (ROI) and this area was measured (µm2) using the ‘measure objects’ tool in Volocity®. Refer to p≤0.05 significant differences compared with wildtype animals using a two-tailed Student t-test.(DOC)Click here for additional data file.


GJB2 and SLC26A4 genes for the presence of mutations, screened for the mitochondrial DNA (mtDNA) A1555G mutation, and screened for congenital CMV infection in DNA isolated from dried newborn blood spots. Results were obtained from 364 children. We established etiology for 60% of children. One or two known GJB2 mutations were present in 82 children. Twenty-four children had one or two known SLC26A4 mutations. GJB2 or SLC26A4 changes with unknown consequences on hearing were found in 32 children. The A1555G mutation was found in one child, and CMV infection was detected in 28 children. Auditory neuropathy spectrum disorder was confirmed in 26 children whose DNA evaluations were negative. A secondary objective was to investigate the relationship between etiology and audiological outcomes over the first 3 years of life. Regression analysis was used to investigate the relationship between hearing levels and etiology. Data analysis does not support the existence of differential effects of etiology on degree of hearing loss or on progressiveness of hearing loss.Hearing loss is an etiologically heterogeneous trait with differences in the age of onset, severity and site of lesion. It is caused by a combination of genetic and/or environmental factors. A longitudinal study to examine the efficacy of early intervention for improving child outcomes is ongoing in Australia. To determine the cause of hearing loss in these children we undertook molecular testing of perinatal “Guthrie” blood spots of children whose hearing loss was either detected via newborn hearing screening or detected later in infancy. We analyzed the  Permanent childhood hearing loss, which occurs in 1–2 per 1000 live births GJB2  gene, the SLC26A4 (pendred) gene and in the mitochondrial DNA at position 1555 (A1555G mutation) and congenital cytomegalovirus (CMV) infection have been considered as the major causes of congenital hearing loss in developed countries Childhood hearing loss is an etiologically heterogeneous condition, caused by environmental factors and/or genetic factors. Despite this heterogeneity, mutations in the The study was approved by institutional ethics review boards .The LOCHI study commenced in 2005, with recruitment completed in 2007. All families with children born in Australia between 2002 and 2007 and who presented for hearing services below 3 years of age at Australian Hearing (AH) were invited to participate. Parents of children enrolled in the LOCHI study provided written, informed consent to participate. All participants were invited to give additional informed consent for molecular testing. Of the 451 eligible children, written consent was obtained for 387 children for molecular testing. There was no significant difference in hearing loss  between the consent group and the non-consent group (p = 0.605). A total of 364 Guthrie card samples were obtained from the custodian authorities of health records. The sample comprised 280 whose hearing loss was detected via UNHS, 64 who did not have access to UNHS and 20 whose screening status was unknown.In Australia, the hearing of newborns is screened using automated auditory brainstem response (AABR). Infants who do not pass hearing screening are referred for diagnostic audiological assessment. These include tympanometry, middle ear muscle reflexes, otoacoustic emissions (OAEs), click-evoked auditory brainstem response (ABR) testing, and ABR or auditory steady-state responses (ASSR) testing using frequency-specific stimuli. Following diagnosis, children are referred to AH for assessment of hearing and provision of amplification, at no cost to families. Electrophysiological tests results are converted to estimated behavioral thresholds for fitting of hearing aids The hearing thresholds of participants from inception to 44 months of age were retrieved from clinical records held at the AH national database, with written permission from parents. Hearing thresholds at 0.25, 0.5, 1, 2, and 4 kHz for each ear were obtained. These audiometric thresholds were used to characterize evolution of children’s hearing loss over the first 3 years of life as fluctuating, progressive, or stable. In accordance with the audiological protocol of AH, fluctuating hearing loss is defined as a change in hearing threshold of 15 dB or greater at any octave frequency between 0.5 and 4 kHz, but subsequently recover over the period of investigation. Progressive hearing loss is defined as a decrease in 10 dB or greater at two or more adjacent frequencies between 0.5 and 4 kHz or a decrease in 15 dB at one octave frequency in the same frequency range over the period of investigation. Longitudinal audiograms that do not demonstrate the changes specified above are labeled as stable.ANSD is characterized by a dysfunction in neural/brainstem transmission of auditory stimuli in the presence of normal cochlear outer hair cell function. The clinical presentation is an absent or abnormal ABR responses and the presence of otoacoustic emissions or cochlear microphonics.Information about enlarged vestibular aqueduct (EVA) was obtained from clinical records held at the AH national database. The diagnosis of EVA was based on CT scan. Radiographic imaging was not part of routine clinical service offered to children identified with hearing loss.et al. 2; 1 mg/ml Proteinase K; 1% Tween-20) at 56°C for 1 hour. Following incubation at 100°C for 7 mins. the samples were rapidly cooled on ice. The samples were centrifuged  and the supernatants frozen for >4 hours at −80°C.Lysates for molecular screening were prepared from Guthrie blood spots, using a modification of the method described by Barbi GJB2 (exon 2) and the splice site mutation in intron 1 (IVS1+1 G>A). Exon 2 PCR products were sequenced in both directions. The IVS1+1 G>A mutation was screened for by Hph1 digestion of a PCR product which spans the splice site. If a single mutation in GJB2 was found, connexin-30 deletions del(GJB6-D13S1830) and del(GJB6d13s1854) were screened for as described by Castillo et al.Lysates were screened for mutations in the coding region of SLC26A4 gene exons was PCR amplified. Following nested PCR, the products were analyzed by High Resolution Melt (HRM) on the Corbett Rotorgene 6000 . Primary PCR products were sequenced when HRM variants were identified.Each of the in silico approaches to assess if unreported changes in the GJB2 and SLC26A4 genes are likely to affect protein function: PolyPhen-2 We used A1555G mutation introduces a HaeIII restriction enzyme site. PCR products were digested with HaeIII and analyzed by agarose gel electrophoresis.The mitochondrial 12S rRNA et al. et al. The presence of CMV DNA was determined in a multiplex, quantitative, real-time PCR assay, using the CMV and CPOL probes published in Sanchez GJB2, two mutations for GJB2, one mutation for SLC26A4 gene, two mutations for SLC26A4 gene, CMV infection, enlarged vestibular aqueduct (EVA), conductive loss) as two-category predictors, and the interactions between etiologies and age. We used two-tailed tests for all analyses and set statistical significance at p<0.05. The statistical analysis was performed using Statistica  and R The results are summarized as means and percentages. To investigate the relationship between etiology and hearing thresholds at 3 years of age (Hypothesis 1), multiple regression analysis was carried out using the four-frequency average hearing loss  as dependent variable and etiology as a predictor variable. To determine the evolution of hearing loss, children were grouped according to the AH national protocols into those with fluctuating, progressive, or stable hearing loss. To consider the relation between progression of hearing loss and etiology (Hypothesis 2), a mixed-effects model was fitted using the longitudinal 4FA HL  hearing level) data as dependent variable, the fixed effects were age (continuous variable), the seven etiologies (one mutation for Cx26 and SLC26A4 mutation databases We use the wording “reported mutations” for DNA and protein changes classified as causative mutations in GJB2 or SLC26A4 gene were detected in 31 (9%) children. We did not detect mutations or ambiguous changes or evidence of congenital CMV infection in 198 (54%) children. These findings were supplemented by information retrieved from clinical records and parental reports. The cause of hearing loss remained unknown for 146 children (40%).GJB2 were present in 82 children. Of these, 48 (58.5%) had two known mutations and 35 (41.5%) children had one mutation. The inheritance is in all cases compatible with a recessive inheritance pattern. Details of mutations and the evolution of hearing loss for individual children are shown in GJB2. Of the children with two mutations of Cx26, one child passed hearing screening at birth, but was diagnosed with moderate hearing loss at 19.4 months of age. Another child was diagnosed with auditory neuropathy spectrum disorder (ANSD), and two children had EVA. Ambiguous changes in the GJB2 gene were detected in 24 children, 22 of whom have one mutation and the remaining 2 have two mutations.Reported mutations of SLC26A4SLC26A4 mutation, two of whom also carried one uncertain mutation and four others also carried GJB2 mutations. For 8 other children, one ambiguous change of the SLC26A4 gene was detected. Details of mutations and evolution of hearing thresholds are shown in Reported mutations of SLC26A4 gene are associated with Pendred syndrome  and non-syndromic enlarged vestibular aqueduct . Three children who carried one mutation of SLC26A4 also had EVA, and one child who carried two mutations of SLC26A4 also had EVA. EVA was also found for 4 children who had Cx26 mutations, 1 who had CMV infection, and 8 who had normal results for the four causes tested. The number of children who had EVA could not be ascertained because information was available only for those with imaging results recorded in their clinical files.Mutations in the A1555G mutation was detected in one child. The mutation appeared homoplasmic on agarose gel electrophoresis. The child has bilateral ANSD with profound hearing loss. It is not known whether the child had been exposed to aminoglycosides.The mtDNA SLC26A4 gene. Details of the evolution of hearing loss for children who are CMV positive are presented in Cytomegalovirus infection was detected in 28 children. Of these, one child passed UNHS but was diagnosed with meningitis and hearing loss at 5.1 months of age. A second child with congenital CMV infection also had one mutation of Cx26, and was referred for unilateral hearing loss after screening. One CMV positive child also carried two mutations of the In the regression analysis examining the relation between etiology and hearing threshold levels at 3 years of age, the overall p-level for the regression model was 0.27. GJB2, SLC26A4, A1555G and CMV infection, and established a likely etiologic diagnosis for 166 (45.6%) of the 364 cases tested. Of the causes identified, a genetic cause was present in 138 children (83.1%) and an environmental cause in 28 children (16.9%).The incidence of permanent childhood hearing loss is 1 to 1.2 per 1000 at birth increasing to about 1.5 by 3 years of age et al for infants at a hospital in the US showing that 6% (16 of 256 infants with hearing loss) were CMV positive through urine cultures It has previously been suggested that hearing loss attributable to congenital CMV may be progressive in >50% of cases GJB2 gene accounted for 76.1% of the genetic cases. This frequency of occurrence is higher than the 50% previously reported for congenital, autosomal recessive, nonsyndromic, sensorineural hearing loss in the white population GJB2 mutations was not significantly different from those without the mutations is consistent with previous reports Looking specifically at the genetic causes of hearing loss in our population (n = 138), mutations in the GJB2 gene was detected in 35 children. The pathogenic effect of these single mutations are uncertain, but we have attributed the hearing losses to thise GJB2 gene changes, because the proportion of children with hearing loss and one detectable recessive GJB2 mutation is significantly higher than that of the general population (approximately 1%) GJB2 gene of 24 children are also ambiguous. We have classified the V27I and E114G changes, when together, as a mutation in accordance with the Connexins and Deafness HomepageGJB2 protein is unable to form functional gap junction channels in silico analysis suggests that is unlikely to cause hearing loss. Although SIFT Sequence analysis predicts that it is “likely to affect protein function”, PolyPhen-2 and SIFT BLink predict it is benign Click here for additional data file.Table S2Genotype-phenotype correlation in children with SLC26A4 mutations and evolution of hearing loss. Children with SLC26A4 mutations with additional diagnostic information regarding enlarged vestibular aqueduct (EVA), auditory neuropathy spectrum disorder (ANSD) and congenital cytomegalovirus infection (CMV) are also indicated.(DOC)Click here for additional data file.Table S3Hearing thresholds at time of diagnosis and evolution in children with hearing loss attributable to congenital CMV infection.(DOC)Click here for additional data file.Table S4PolyPhen-2, SIFT BLink and SIFT Sequence predictions of novel or controversial amino acid changes on GJB2 and SLC26A4 gene product functions.(DOCX)Click here for additional data file.