TM inertial device. Accelerations/decelerations were recorded. The number of accelerations and decelerations, intensity category, and type were recorded. These variables varied between quarters  and playing positions . The shorter but more intense accelerations took place in the last quarter, due to the tight results of the matches. Besides, players in the Guard positions performed more accelerations and their intensity was greater than that of other positions. An acceleration profile was established for the quarters of a basketball game, and was shown to depend on the playing position, being different for Guards, Forwards and Centers in U-18 women’s basketball players.The ability of a player to perform high-intensity actions can be linked to common requirements of team sports, and the ability to accelerate can be an important factor in successfully facing the opponent. The aim of this study was to determine the acceleration and deceleration profiles of U-18 women’s basketball players during competitive matches. This study categorized accelerations and decelerations by playing position and quarter. Forty-eight U-18 female basketball players from the same Spanish league participated in this study. Each player was equipped with a WimuPro Basketball is an intermittent high-intensity sport characterized by high aerobic and anaerobic demands. A major amount of energy depends on the aerobic pathway (aerobic glycolysis) ,2. HowevPrevious research has reported on different analysis load models for training and competition, mainly through internal load analysis  and Time Motion Analysis . However, global positioning systems (GNSS), especially in football, are increasingly used to evaluate the external load . NeverthPhysical stress in training and competition is divided between internal and external load. External load represents the variables manipulated to induce internal stress and include acceleration and deceleration movements . ExternaThe aim of this study was to analyze the number and duration of accelerations and decelerations, determining the profiles of U-18 women’s basketball players during competitive matches by an inertial device. Therefore, it was hypothesized that the acceleration’s length and intensity will decrease as the game progresses.Forty-eight U-18 women’s basketball players from the same Spanish league participated in this study. The participants belonged to four different teams that participated in the final tournament. Each team played three matches (n = 144). Players were categorized according to their playing position . All players and trainers were informed about the research protocol, requisites, benefits, and risks, and their written consent was obtained before the start of the study to conform to the Code of Ethics of the World Medical Association (Declaration of Helsinki), which was approved by the Ethics Committee of the University of Extremadura (no. 67/2017).2).Quantitative variables: The number of accelerations/decelerations was recorded. The variables analyzed in each acceleration/deceleration were Duration (ms), Start speed (km/h) and Acceleration peak (m/s2); A2 (High: 2.5–4 m/s2); A3 (Sprint: >4 m/s2) and D1 (Low: −1–−2.5 m/s2); D2 (High: −2.5–−4 m/s2); D3 (Sprint: >−4 m/s2).Qualitative variables: Accelerations and Decelerations were classified according to intensity into three ranges: A1 . The WIMUProTM inertial device, UWB system and Software come from the same organization .Each player was equipped with a WIMUProng field . The ANTTM inertial devices were synchronized to the UWB system through the ANT + technology. Each player was equipped with the inertial device 20 min before the start of the match. In this way, there was a period of familiarization during the warm-up. Once the match started, total and live times were calculated using the SVIVOTM software; with total time referring to all of the time that a player was on court, including all stoppages in play, but excluding breaks between quarters and times out. Live time corresponds to the time when the game clock was running and the player was on the court and also short moments in which the player was active during out-of–bounds.For the analysis of the competition, the UWB system was calibrated 1 h before the start of the games, and the WIMUProp, is important or not, in practice. Statistical analyses were performed using SPSS v.21 software . Statistical significance was set at p < 0.05. For MBI calculations spreadsheets designed with this purpose were used [The distribution of the data was checked with the Kolmogorov-Smirnov test , to seleere used .p = 0.700). The second quarter is where the greatest number of accelerations was performed, and the last quarter is where the greatest number of decelerations was performed.The number of accelerations and decelerations, and intensity category and type varied between quarters. The number of accelerations and decelerations declined from the first and third quarters to the second and fourth quarters   but they were not statistically significant , being higher in the last quarter compared to the rest . There were statistically significant differences in the intensity of the accelerations and decelerations between quarters . The decelerations were longer in the first period  than the second period too  with Almost certain magnitude-based inferences . The accelerations start speed was greater in the first period  than the second period  but significant differences were found in the comparison with the third quarter . The deceleration’s start speed was greater in the first period  than the second period too  with Almost certain magnitude-based inferences . The minimum acceleration peak was achieved in Q3 (2.34 m/s2) compared to Q1 and Q2   than the second period  with  = 99.5) .Almost certain” values were identified as the most important effects. “Almost certain” effects were observed in the length of accelerations and decelerations, being substantially higher in the first period (Q1 and Q2) compared to the second period (Q3 and Q4). In the start speed, there were important effects in decelerations, being higher in the first period compared to the rest of the periods.MBI’s were used to evaluate the effect statistic ; the resp = 0.001). The Center position performed less accelerations and decelerations per minute than Guards and Forwards  . In terms of intensity ranges, the Guard position showed a greater time percentage in ranges A3 and D3, while Centers were the players who spent more time in the ranges of less intensity A1 and D1 , being higher in Guards compared to the rest.The number of accelerations and decelerations, and intensity category and type varied between playing positions  among playing positions. There were differences in the lengths of accelerations with a low effect between Center and perimeter players , being greater in Guards and Forwards. The maximum speed peak and start speed were higher in Guards (ACC = 4.55 m/s2 and 2.70 km/h) than the rest of the playing positions with almost certain magnitude-based inferences (p = 0.001 and MBI = 99.8–99.9). In the variables of deceleration, no important effects were observed  probably due to fatigue; however, the acceleration peak and high start speed values were found in the last quarter. In women’s basketball competition, Delextrat et al. 8 ms prob,18. In cDifferences according to playing position are mainly due to the specialization of players and the demands of the competition ,23. In mMonitoring competition and reporting data individually appears to be essential for designing specific training sessions for the competitive demands of each player . The ideAn acceleration profile has been established for the first, second, third, and fourth quarter of a basketball game, as well as playing position, obtaining an acceleration profile in Guards, Forwards and Centers in U-18 women’s basketball players. An understanding of these differences would make it possible to design training sessions that are adequate for competitive demands. Therefore, specificity and individualized training principles have been shown to be important to assess performance and acceleration profile. As a future prospect, it would be vital to increase the sample, collecting a greater number of matches from different categories to observe the differences in the acceleration profiles in formative stages.


Usher syndrome, the most common form of inherited deaf-blindness, is unlike many other forms of syndromic hereditary hearing loss in that the extra aural clinical manifestations are also detrimental to communication. Usher syndrome patients with early onset deafness also experience vision loss due to progressive retinitis pigmentosa that can lead to legal blindness in their third or fourth decade.USH2A and ADGRV1) in cases initially referred for isolated vision or hearing loss.Using a multi-omic approach, we identified three novel pathogenic variants in two Usher syndrome genes , predicted to create a premature stop codon near the N-terminus of ADGRV1. Ophthalmological examination of the sisters confirmed typical retinitis pigmentosa and prompted a corrected Usher syndrome diagnosis. In an unrelated clinical case, a child with hearing loss tested positive for two novel USH2A splicing variants  and RNA studies confirmed that both pathogenic variants cause splicing errors. Interestingly, these same USH2A variants are also identified in another family with vision loss where subsequent clinical follow-up confirmed pre-existing hearing loss since early childhood, eventually resulting in a reassigned diagnosis of Usher syndrome.In a multiplex hearing loss family, two affected sisters, the product of a second cousin union, are homozygous for a novel nonsense pathogenic variant in USH2A splicing pathogenic variants, these solved USH2A patients may now be eligible to be enrolled in therapeutic trials.These findings provide empirical evidence to increase Usher syndrome surveillance of at-risk children. Given that novel antisense oligonucleotide therapies have been shown to rescue retinal degeneration caused by  Approximately 30% of inherited hearing loss is syndromic and is classically characterized by overt clinical features, such as distinctive craniofacial and eye abnormalities, and joint problems as in Stickler syndrome   and fluorescein angiography of the retina.For Family R2100, hearing loss (HL) is present in three sibships with varying audioprofiles, including two sisters who are the product of a consanguineous union  of a child diagnosed at 3 months (following newborn hearing screening), who presents by age 3 with mild to moderate bilateral sensorineural HL  was offered through a research study on hereditary vision loss. In the clinical case of the child who failed newborn hearing screening (Family R4110), the family was offered targeted screening . To validate variants of interest and check for co-segregation with RP and/or HL trait in the families, genomic DNA was amplified using custom primers and sequenced in both directions using standard touchdown PCR protocols . Sequence traces were analyzed using Mutation Surveyor Software .We initially screened the proband of Family 2100 for deafness alleles previously identified in the NL population and submitted representative audiograms to Audiogene, a program comparing these to average audiograms of 34 deafness loci . As thisFor variants of interest that reside within canonical +/− 1 or 2 splice sites, we conducted in silico analyses using Alamut Visual , a program that provides a splicing alteration report by linking to the MaxEnt, NNSPPLICE, SplicSiteFinder, and GeneSplicer algorithms.In order to experimentally validate splicing predictions, we extracted total RNA from B-cell lymphocytes using standard TRIzol-based methods  and prepared cDNA libraries with the Superscript III First Strand Synthesis System . RT-PCR was carried out with primers that spanned candidate splicing regions, followed by TOPO TA-Cloning Kit for Sequencing with One Shot TOP10 Chemically Competent E. coli  according to the manufacturer’s protocol. RT-PCR products were Sanger sequenced and then analyzed using Mutation Surveyor Software .ADGRV1 c.17062C > T; p.Arg5688*) is a nonsense variant associated with USH2C  population. According to ACMG guidelines, the ADGRV1 nonsense variant should be classified as pathogenic as it meets the following criteria: PVS1, PM2, PM3, PP1, PP3.In the step-wise analysis of hearing loss Family 2100, the proband  in three sibships with varying audioprofiles, including two sisters who are the product of a consanguineous union. On the basis of the pending molecular diagnosis of Usher syndrome and the serious prognosis, the clinic contacted the sisters in order to request a visual examination  of the USH2A variants, the genetic testing results are of limited value.The comprehensive gene panel that was offered to the clinical case of the 3-year-old child diagnosed with isolated hearing loss at 3 months  and his brother (PID II-3) are homozygous for UHS2A c.5777-1G > A, their nieces (PIDs III-1 and III-2) are compound heterozygotes . Using patient-derived cells, Sanger sequencing of cDNA confirmed that USH2A c.5777-1G > A causes the skipping of exon 29 leading to an in-frame deletion (p. Glu1926_Ala1952del) in an affected individual (PID III-2) compared with a control sample  [Cascade sequencing revealed that both P1, PP3) .Fig. 3SeUSH2A account for cases recruited or referred as isolated hearing or vision loss in two families in this study. Clinical evidence suggests that the two novel USH2A pathogenic variants result in congenital moderate to severe HL, and RP in the pre/post-pubertal period; findings similar to that of previously reported pathogenic variants in USH2A [USH2A c.5777-1G > A and c.10388-2A > G pathogenic variants are sufficient to cause USH2A and therefore are USH2A-specific. This finding is consistent with the allelic hierarchy model of USH2A alleles, which suggests that certain alleles are USH2A-specific and others RP-specific, and the presence of at least one RP-specific allele causes isolated RP with normal hearing [Two novel pathogenic variants in in USH2A . Several hearing . Given oADGRV1 . Visual examination secondary to molecular analyses confirmed typical RP (late third and fourth decade) in addition to hearing loss (first decade) and prompted a corrected USH diagnosis. This is consistent with previous reports of ADGRV1 pathogenic variants associated with early onset of hearing loss with delayed visual impairment [ADGRV1 c.17062C > T lies downstream (3′) to this calx-β motif. This variant is rare, and to our knowledge, has only been reported once before when it was identified in 1/31 French non-USH2A patients [ADGRV1 pathogenic variants have been shown to cause dominant audiogenic epilepsy [ADGRV1 c.17062C > T do not present with audiogenic epilepsy.Similarly, we find that two sisters with hereditary hearing loss, the product of a second cousin union, are homozygous for a nonsense pathogenic variant in pairment , 20, mospairment , and thepatients . In addiepilepsy , 24. HowClinically, USH2 should be suspected in patients with bilateral, congenital, sensorineural, mild to severe hearing loss, normal vestibular function, and post-pubertal RP, most often in the second decade , 26. VisADGRV1 c.17062C > T variant from each of them, establishing that we are detecting two disease alleles in trans and confirming the recessive pattern for Usher syndrome.To determine variant pathogenicity, clinical best-practice guidelines, such as ACMG  and EuroUSH2A (50%) mutations are the most common with ADGRV1 mutations being less frequent (5%) in patients with both visual and hearing impairments [In a recent meta-analysis including all of the known genes causing usher syndrome, airments . In patiRecognition of syndromic forms of both hearing and vision loss, especially Usher syndrome, is important given the major impact of these types of sensory losses on the acquisition of speech in children and quality of life for adults. In this report, USH was not considered in these cases until genetic testing was performed. Close collaboration between local clinics and molecular genetics researchers was necessary to fully categorize three novel USH variants as pathogenic using ACMG criteria. Accurate molecular diagnosis of patients is essential to provide new opportunities for patients and their families to enroll in therapeutic trials.


We previously showed that the regio- and stereoselectivity in terpene-forming reactions are determined by the conformations of the carbocation intermediates, which reflect the initial conformation of the substrate, geranylfarnesyl diphosphate (GFPP). However, it remains unclear how the initial conformation of GFPP is controlled, and which part(s) of the GFPP molecule are important for its fixation inside the substrate-binding pocket. Here, we present the first detailed analysis of the inherent atomic mobility in carbocation intermediates during sesterterpene biosynthesis. We identified two methyl groups as the least mobile of all the carbons of the carbocation intermediates in the first half of the cyclization cascade. Our analysis suggests that these two methyl groups are critical for the preorganization of GFPP in the biosynthetic pathways leading to sesterfisherol and quiannulatene. Terpene synthases are thought to have four main roles: (i) triggering the biosynthetic cyclization cascade by the elimination of pyrophosphate or by protonation; (ii) preorganization of the substrate to generate the reactive conformation; (iii) protection of reactive intermediates from water; and (iv) termination of the reaction by deprotonation or hydration. We previously established the importance of conformation in the carbocation cyclization cascade , focusinAlthough many terpene cyclases are known –10, it iThere have been many theoretical studies of terpene-forming reactions –18, and For the analysis of inherent structural mobility, we firstly carried out IRC calculations using GRRM11 with Gaussian 09, obtaining 2609 plots for quiannulatene formation and 2640 plots for sesterfisherol formation. We divided the whole biosynthetic process into three phases; (I) 5/12/5 tricycle formation, (II) conformational changes and hydrogen shifts, and (III) ring rearrangements . Then, wIM1 to IM2 involves C1–C15 and C14–C18 bond formations, the displacements of C6–C12, and C22 are also large, indicating that these regions, though they are distant from the reaction centre, are relatively flexible and not tightly fixed by the enzyme. As we reported previously, the initial conformation of GFPP, in particular the orientation of six methyl groups (C20–C25), is critical. Therefore, we focused on these methyl groups. While the C20, C21 and C23 methyl groups are quite static in phase I, the other three methyl groups are relatively flexible, which suggests that C20, C21 and C23 could be key determinants of the affinity for the enzyme’s binding pocket. The C24 and C25 methyl groups are the most mobile moieties, and the dramatic displacements of these two methyl groups are consistent with a previous report that the counterclockwise rotation of the isopropyl moiety is necessary for 1,5-hydrogen shift to occur (TS_2-3a in quiannulatene biosynthesis and TS_2-3 in sesterfisherol biosynthesis) [Based on our inherent mobility analysis, C1–C5, C15, C16, C20, C21 and C23 are static during 5/12/5 tricyclic formation in the biosynthesis of both quiannulatene and sesterfisherol . Interesnthesis) .3 carbon due to 1,2-hydrogen shift (TS_4-5). After two successive 1,2-hydrogen shifts, four-step conformational changes take place in quiannulatene biosynthesis, in which C24 and C25 are highly mobile. On the other hand, C8, C9 and C21 are quite mobile in sesterfisherol biosynthesis. These different mobility trends result from the different types of conformational change required to achieve regioselectivity in the following ring rearrangement reactions.In phase II, different trends of inherent mobility are seen between quiannulatene and sesterfisherol biosynthesis. As shown in While C23 is still static in phase III, C20 is relatively flexible, which might serve to decrease the affinity for the enzyme’s binding pocket. Interestingly, although different types of ring rearrangement reactions occur in each pathway, the same inherent mobility trends were observed. As shown in In order to clarify the influence of the conformational preorganization  of GFPP All calculations were carried out using GRRM11 –25 with File 1Coordinates and energies of quiannulatene biosynthesis.File 2Coordinates and energies of sesterfisherol biosynthesis.File 3Software for inherent mobility analysis.File 4How to use in house software to calculate inherent atomic mobility.


Citrus taxa—Citrus medica, Citrus bergamia, and Citrus medica cv. Salò—cultivated in the Cosenza province, Southern Italy, and to evaluate their antioxidant and antibacterial activity, performed against Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus, and Pectobacterium carotovorum. Furthermore, we assessed the inhibitory effect of the extracts on bacterial capacity to form biofilm, and on the metabolic activity of the cells present therein. The results indicated that such extracts could find new potential applications in the field of natural antioxidant and anti-bacterial agents in pharmaceutics, agriculture, and food fields. The aim of this paper was to study the polyphenols of peel and pulp of three  Citrus, belonging to the Rutaceae family [Citrus medica L., the cedar, was introduced to Italy by Hebrews, who promoted its cultivation on the Calabrian and Amalfi coasts and Garda Lake [C. medica L. are generally divided into two groups: “acidic” and “sweet” cedars. The first one, like the typical species, have red-purple flowers and buds and acidic pulp; the latter have white flowers and a sweeter pulp. Among the acidic cedars there are C. medica cv. Diamante, and the so-called “Hand of Buddha” . The Corsican and the C. medica cv. Salò are among the sweet cedars. Finally, other cultivars can be considered as hybrids between lemons and cedars; they produce fruits resembling cedar due to the size and thickness of the peel, quite suitable for candy manufacture, while the appearance is similar to a lemon.In recent decades, the international market has been characterized by increased consumer demand for products with improved quality and safety features. Fruit and vegetables contain very precious metabolites with antioxidant activity, which also represent a defense strategy of the plant organism in fighting biotic attacks from pathogenic microorganisms and from chemical-physical stresses due to environmental conditions. Several studies have shown that continuous treatment with conventional antibiotics lead to the development of bacterial resistance . Biofilme family . In partrda Lake ,5. The dCitrus species, such as cedar, cedar of Salò, and bergamot. Cedar (Citrus medica L.) is also known because it is considered the progenitor of many of the Citrus fruits present in the world. The fruit, a large 20–30 cm, is yellowish, oval, or almost round, sometimes with a slight protuberance at the peduncle and a little pointed at the opposite side. Its peel is very rough and exceptionally thick and represents up to 70% of the fruit, so, even after removing the seeds and the film between the segments, only 25%–30% of the cedar is edible. In Calabria, cedar is cultivated and worked mainly in the coastal area of the high Tyrrhenian coast of the province of Cosenza, called “Riviera dei Cedri”. Cedar is used in the food industry for consumption as a table fruit, and for the preparation of soft drinks and candy fruit, but mostly it is consumed in the pharmaceutical industry for the production of the essential oil. Cedar of Salò  is used for the production of essence and in the production of commercial juices. It gives, unlike cedar, a completely clear juice, so that many agronomists choose a total abandonment of the cultivation of cedar in favor of those of cedar of Salò. Bergamot (Citrus bergamia Risso) produces a yellow citrus fruit, which—according to some scientists—could be considered as a subspecies of bitter orange or a hybrid derived from bitter orange and lemon. Such products are some of the symbols of excellence of the Calabrian agricultural tradition and represent a great resource for the benefit of the local economy. Bergamot is used almost exclusively for the production of the essential oil, obtained from its peel, and the pulp is considered a merely by-product. On the contrary, the pulp and its juice are a rich source of polyphenols, in particular flavonoids, with attraction for pharmaceutical fields, while searching for new forms of nutraceuticals with beneficial properties for human health [Citrus consumption, is associated with a reduced cancer incidence [Citrus have also antiviral and anti-inflammatory activities [Citrus species act as potent antibacterial agents against several Gram-negative and Gram-positive bacteria [Helicobacter pylori [The Calabria region is famous for the production of different n health . The contivities ,9,10. Thtivities ,12,13. Tbacteria ,15. Flavbacteria . Furtherr pylori . HoweverCitrus taxa cultivated in the above-mentioned “Riviera dei cedri” and to evaluate their antibacterial activity. In particular, we assessed the inhibitory effect of the bacterial capability to form biofilm and to limit the metabolic activity of biofilm cells, in order to exploit new potential applications of such products, with novel challenges in the field of natural anti-bacterial agents.Therefore, the aim of this paper was to study the chemical composition of the extracts obtained from juice and pulp of three Total polyphenol content (TPC) of the ethanolic extracts of peel and pulp of bergamot, cedar, and cedar of Salò ranged between 148.98 µg/g (pulp of cedar) and 1002.3 µg/g of fresh product . By the whole, the cedar of Salò exhibited—both in peel and in pulp—the highest amount of polyphenols, reaching even 1 mg/g of fresh product .This indicates the high healthy valence of this product, whose traditional cultivation has been lost over the centuries and is now, at least in Italy, limited to few areas, such as its original location, Calabria, and to the area surrounding the Lake of Garda, and which could represent a product with wide potentialities for the Italian agri-food economy. Similarly, bergamot possesses noticeable nutritional challenge: peel and pulp, in fact, showed 748 and 208 µg gallic acid equivalent (GAE)/g of TPC, respectively, indicating that the consumption of just one fruit, which weight ranges generally between 80 and 200 g, can provide the body with a considerable amount of polyphenols. Concurrently, cedar, although containing the lowest amount of total polyphenols both in peel and in pulp, represents proportionally the richest source of total polyphenols among the three taxa analyzed .Citrus could be considered higher than that found in other Citrus species [C. medica 123.1 mg of total polyphenols in 100 g of the mesocarp. However, this discrepancy may be due, more than to the different cultivation area, to the climatic conditions, which are known to influence the metabolism of these molecules within the product.The amount of polyphenols detected in the peel of the three  species . Polyphe species , who det50 exhibited by its peel extract was just 3965 mg. Cedar, on the other hand, seemed a much weaker antioxidant agent, so much so that it takes almost 20 mg to inhibit by 50% the activity of 1 mL of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. Peel of bergamot showed a good antioxidant activity, although not equal to that exhibited by the peel of cedar of Salò, with an EC50 value of 6.47 mg. The values found for bergamot peel, but above all for that of cedar di Salò, can suggest their use as new sources of natural antioxidants, usable as functional ingredients in the food industry. The powder obtained from the entire bergamot fruit has shown, after all, an interesting antioxidant activity in both in vitro and ex vivo studies, which have also shown its ability to limit the endothelial alterations by reducing the resulting stress on the endoplasmic reticulum [Citrus samples is slightly different. In this case, both the bergamot and the Cedar of Salò pulps exhibited almost the same efficacy in inhibiting 50% of the activity of the DPPH radical. Once again, cedar, also with regard to the pulp, showed a doubly weaker antioxidant activity (A strong correlation (Correlation-coefficient = 0.96) between the total polyphenol content and the antioxidant activity exhibited by the peel of all three samples analyzed was observed . From theticulum . While sactivity . HoweverCitrus always contained more total phenolic acids than flavonoids. In some cases, for example in the pulp of cedar and cedar of Salò, such an amount was almost five times higher with respect to that of flavonoids. The parts of the three Citrus analyzed showed a different amount of phenolic acids. Peel of bergamot exhibited a content of total phenolic acids, which was more than double if compared to what was found in the pulp. Indeed, the peel of Salò cedar contained almost three times more acid phenols than the pulp. Cedar represented an exception, as previously indicated. Its peel had a much lower content of acid phenols (28.67%) than those found in the pulp. Cedar showed a lower content of phenolic acids. Indeed, its profile was characterized by the absence of chlorogenic, caffeic, and p-coumaric acids. On the other hand, ferulic acid was detected only in the pulp. Cedar of Salò showed all the phenolic acids identified. In general, we found a greater quantity of individual polyphenols in the peel with respect to the pulp. In some cases, such as that of rutin, the quantity of the same molecule found in the peel was almost 6 times higher than that found in the pulp . The presence of such an important concentration of rutin in the peel of cedar  is very significant. Rutin  has been extensively studied due to its anti-inflammatory [C. aurantium L. [Citrus analyzed, cedar of Salò showed the most variegated profile and almost all the known standards available were found both in the pulp and above all in the peel. In cedar of Salò, it is to highlight above all the abundance of the two hydroxycinnamic derivatives, chlorogenic and ferulic acids. The latter was the most abundant metabolite present in the peel (295 µg/g of fresh product), representing 62% of the total phenolic acids. The presence of such a high amount of ferulic acid was in agreement with other studies [Citrus fruits), could indicate a high degree of ripeness of the analyzed products. Apigenin, in fact, in the process of biosynthesis of phenylpropanoids, originates from naringenin, which on the one hand leads to the formation of apigenin and on the other gives rise to genistein [The three ammatory , antibacammatory , anti-deammatory , and canammatory  activitiammatory ,28. Rutiammatory . In othentium L. . Among t studies ,31. Two enistein . Moreoveenistein . The preP. carotovorum and Ps. aeruginosa), by the polyphenolic extract obtained from the bergamot pulp . In the case of L. monocytogenes, they inhibited 92.02% of the biofilm, just with 1.2 mg; in addition, 6 mg of such extract inhibited up to 90% of the biofilm formation by Staph. aureus. Such capability also corroborated with the data concerning the inhibitory action on viability of the cells present inside the biofilm, which was always more effective with respect to that exhibited by polyphenols of peel against E.coli, L. monocytogenes, and Staph. aureus  and Ps. aeruginosa (52.5% inhibition). The test with MTT showed that the extracts of bergamot peel were not only able to inhibit the formation of biofilm, but also to act on the metabolism of the cells therein, They were active against all pathogens tested, although with different effectiveness. Polyphenols of the pulp of bergamot did not exhibit a wider spectrum of action; they were almost incapable of constraining the biofilm formation by . aureus b. At theP. aeruginosa, the inhibitory activity exhibited by cedar peel was much stronger than that exerted by the pulp of cedar at the same quantities of TP used in the experiments  compared to untreated bacterial cells.The data acquired showed that the polyphenolic extracts obtained from the two portions (peel and pulp) of the three f action . The pref action . Moreovef action  and feruf action , are ablruginosa . Here, tp-coumaric, gallic, and chlorogenic acids, rutin, catechin, quercetin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 3--2,5-diphenyl tetrazolium bromide (MTT), dimethylsulphoxide (DMSO), sterile Luria-Bertani broth, tetracycline, ciprofloxacin, HPLC-grade ethanol, 4-methyl-2-pentanol, ethanol, and acetonitrile were purchased from Sigma-Aldrich . Apigenin was purchased from Extrasynthese . Ultrapure water was produced by a Milli-Q system . Caffeic, ferulic, Citrus medica L. (cedar), C. bergamia Risso (bergamot), and Citrus medica cv. Salò  were collected in 2015 in S. Maria del Cedro , Southern Italy. Professor Vincenzo De Feo identified the plants and provided to store voucher specimens in the Herbarium of the Medical Botany Chair at the University of Salerno. Fruits of Biochemical characterization involved the analysis of the total polyphenol content, the antioxidant activity, and the qualitative–quantitative profile of polyphenols by ultra-pressure liquid chromatography.v/v) for 24 h in the dark at 4 °C. Pulp was homogenized with a domestic homogenizer and, after centrifugation for 5 min at 3000 rpm , the juice was recovered and centrifuged again for 20 min at 6000 rpm. Then, it was mixed with two volumes of 100% ethanol, and left at 4 °C for 24 h.Peel was removed from fruits, carefully avoiding the recovery of the albedo, pounded, and left in contact with ethanol 100% .The antioxidant activity of the extracts was determined by evaluating their capability to inhibit the activity of the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH test) . The ana18 column, 1.7 μm, 2.1 mm × 100 mm. The mobile phase consisted of 7.5 mM acetic acid (eluent A) and acetonitrile (eluent B). The analyses were conducted at a back-pressure value between 6000 and 8000 psi. The DAD detector was programmed to a scanning range between 210 and 400 nm, with a resolution of 1.2 nm. The injection volume was 5 μL. Quantification of the polyphenols was determined through calibration curves obtained from the polyphenol standards mentioned above.The analysis of the polyphenolic profile was carried out following the method of Fratianni et al. . An ultrStaphylococcus aureus ATCC 25923, Listeria monocytogenes ATCC 7644, EHEC, Escherichia coli DSM 8579, Pseudomonas aeruginosa DSM 50071, and the phytopathogen Pectobacterium carovotorum DSM 102074 were used as test bacterial strains. We previously stored the strains at −30 °C in sterile Luria Bertani (LB) broth  supplemented with 20% sterile glycerol . Bacteria were thawed and added (inoculum 2%) to LB broth. S. aureus, L. monocytogenes, E.coli, and P. aeruginosa were grown for 18 h at 37 °C and 80 rpm . P. carotovorum was grown at 30 °C and 80 rpm. Then, fresh cultures were used as inoculum  and grown in the conditions above described.7 cells/mL) . Sterile DMSO and ciprofloxacin (prepared dissolving 1 mg/mL in DMSO) were used as negative and positive control, respectively. Microtiter-plates were prepared in triplicate and incubated at 37 °C for 24 h. The lowest concentration at which a color change occurred (from dark purple to colorless) indicated the MIC value.Values of the Minimal Inhibitory Concentration (MIC) were calculated using the resazurin microtiter-plate assay ,47. Two-S. aureus, L. monocytogenes E. coli, P. aeruginosa, and P. carovotorum were formed in flat-bottomed 96-well microtiter plates following the method of O’Toole and Kolter [7 cells/mL)  using Luria-Bertani broth. Then, 10 µL of the diluted cultures were distributed in each well, and different volumes of the extracts and Luria-Bertani broth were added, to reach a final volume of 200 µL/well. Microplates were completely covered with parafilm, to avoid the evaporation of samples with relative loss of volume and incubated for 48 h. Planktonic cells were removed and the attached cells were gently washed twice with sterile physiological saline solution. Then, 200 µL of methanol/well, which was left for 20 min to fix the sessile cells, was added. The methanol was discarded, and the plates were left under laminar flow cap until complete dryness of samples (at least 30 min). The staining of the adhered cells was obtained by adding 200 µL of 2% w/v crystal violet solution to each well that was left for 20 min. Wells were gently washed and left to dry. A volume of 200 µL of glacial acetic acid 20% w/v was added to allow the release of the bound dye. The absorbance was measured at OD = 540 nm (Varian Cary). Evaluation of the biofilm inhibition was calculated (percentage) with respect to that of control (cells grown without the presence of the extracts). Triplicate tests were done, and the average results were taken for reproducibility.The biofilms of d Kolter  using suThe effect of the extracts from bergamot, cedar, and cedar of Salò on the metabolic activity of cells present in the biofilm was evaluated through the MTT colorimetric test, modifying the method of Kairo et al.  as follo


The self-expanding metal stent (SEMS) has been used in malignant colorectal obstruction as a bridge to surgery or for palliative treatment. We report a case of obstructive descending colon diverticulitis in-stent restenosis, which is difficult to distinguish from colon cancer.A 48-year-old man presented with abdominal pain. Computed tomography demonstrated a segment of thickened wall in the descending colon with near-complete obstruction. Colonoscopy revealed stenosis over the entire circumference of the descending colon. Biopsy revealed no malignant findings. SEMS was inserted to improve bowel obstruction. However, bowel obstruction did not improve, and stent-in-stent insertion was performed. Because colon cancer could not be completely excluded, left hemicolectomy with lymph node dissection was performed. The pathological examination revealed colon diverticulitis and no malignant finding.Benign colorectal diseases can present with symptoms similar to those of obstructive colorectal cancer. As a result, it was benign colorectal stenosis, but it was thought that resection should be considered when the malignant lesion is suspected. The use of SEMS in benign colorectal disease has been reported a few. Reinsertion of the stent for restenosis, in bridge to surgery, was also considered useful from the viewpoint of avoiding emergent operation and reducing complications.For colon stenosis suspected of malignant disease, it was thought that resection by inserting SEMS should be taken into consideration even for benign disease as a result. This procedure has been used in malignant colorectal obstruction as a bridge to surgery or palliative treatment . However2A 48-year-old man complained of abdominal pain, nausea and vomiting. There was no significant past medical history. Diffuse abdominal distension and left abdominal tenderness were found on physical examination. Leukocytosis was detected in laboratory findings. Contrast-enhanced abdominal computed tomography showed marked thickening and circumferential stenosis in the descending colon . Colonos3A transanal decompression tube, emergency colostomy, and Hartmann’s operation have been used for treatment of malignant colorectal obstruction. Since 1991, colorectal stents have been used in the treatment of malignant stenosis . SEMS isRecently SEMS has been used for obstruction or stenosis in benign colorectal disease . HoweverThe use of SEMS in the setting of colon cancer is associated with tumor ingrowth rates of 3–46% . On the 4For colorectal obstruction difficult to distinguish between benign and malignant, it seemed reasonable to resect the lesion even after inserting stent in stent. SEMS bridging to surgery is an effective treatment for any colorectal obstruction and should be introduced positively.The authors declare they have no conflict of interests.This study has not received any funding.This case report was approved by the committee of our institute.Written informed consent was obtained from the patient.Ryo Ohta – Author, Editing of manuscript.Ryota Sakon – contributor.Manabu Goto – contributor.Yuji Tachimori – contributor.Koji Sekikawa – contributor.NA.Corresponding author; Ryo Ohta.Not commissioned, externally peer reviewed.


The lack of an appropriately trained global hearing-care workforce is recognized as a barrier to developing and implementing services to treat ear and hearing disorders. In this article we examine some of the published literature on the current global workforce for ear and hearing care. We outline the status of both the primary-care workforce, including community health workers, and specialist services, including audiologists, ear, nose and throat specialists, speech and language therapists, and teachers of the deaf. We discuss models of training health workers in ear and hearing care, including the role of task-sharing and the challenges of training in low and middle-income countries. We structure the article by the components of ear and hearing care that may be delivered in isolation or in integrated models of care: primary care assessment and intervention; screening; hearing tests; hearing rehabilitation; middle-ear surgery; deaf services; and cochlear implant programmes. We highlight important knowledge gaps and areas for future research and reporting. They caThe primary-care workforce for ear and hearing health may include community health workers, primary care nurses or primary care physicians, any of whom who may screen for disease and provide preventive or medical care. In most low-resource settings, there are inadequate numbers of doctors to provide primary hearing care, and in several countries nurses or community health workers fill this workforce gap. Community health workers have been defined as those who work predominantly in the community rather than in a health facility and have received some formal training in the tasks they perform, but do not have a certificate or degree-level education.Secondary or specialist care is traditionally delivered by audiologists who can test hearing and provide hearing aids; ear, nose and throat specialists who may offer medical or surgical treatment (surgery particularly for chronic suppurative ear disease); speech and language therapists who may assist adults and children with disabling hearing loss; and teachers of the deaf who can provide educational support to children with severe hearing loss.3–9,11,12 In addition the tasks undertaken by such specialists may encompass a variety and variable complexity of roles. In the United Kingdom of Great Britain and Northern Ireland, for example, training pathways exist for audiometrists (who perform basic diagnostic hearing tests), hearing aid dispensers (who supply and fit hearing aids), health-care science practitioners (who provide a range of diagnostic tests and treatment) and clinical scientists (who provide tests and treatments that includes complex cases). In other countries, the names and roles of the workforce in audiology are similarly disparate.Surveys conducted in the last few years reveal that in many low- and middle-income countries specialist workers in ear and hearing care are either sparse or non-existent .3–9,11,1Two parallel strategies can be pursued to fill the workforce gap in ear and hearing care. The first strategy is local capacity development, through the training and development of additional workers in the field of ear and hearing health. Even though locally delivered training may be preferred,In this article we assess opportunities for developing human resources in ear and hearing health. We present some of the relevant literature on training and development, and ask what has been shown to be effective or ineffective, and where there are opportunities and knowledge gaps. We identified relevant articles through a review of articles indexed in the PubMed® online database, using search terms “Training AND Audiology OR Otology OR Teacher Deaf OR Speech Language Therapy” as well as a search of the grey literature. We also look at examples of task-sharing and assess relevant outcomes reported from such initiatives. We structure the article by seven components of ear and hearing care that may be delivered in isolation or in integrated models of care: (i) primary-care assessment and intervention; (ii) screening; (iii) hearing tests; (iv) hearing rehabilitation; (v) middle-ear surgery; (vi) deaf services; and (vii) cochlear implant programmes .With an appropriately trained workforce, assessment and intervention for some ear and hearing disorders can potentially be undertaken in primary care. There are descriptions of programmes to train community health workers in such skills, and some anecdotes of success.Primary ear and hearing care training resource,Aboriginal and Torres Strait Islander ear health manualChinese ear and hearing care training manual.The World Health Organization’s (WHO’s) Other studies suggest that community health workers can acquire sufficient practical skills to assess and manage patients.Further research on methods and outcomes of training is needed to provide evidence on if, how and to what extent training of community health workers enables them to undertake independent assessment and management of patients with ear and hearing disorders. Consequently, evidence will also show to what extent task-sharing may be possible in hearing care.Achieving this higher level of ability in community health workers may be ambitious. It will likely require support from experts, both in delivering the training and in providing supervision in the initial stages of independent practice. In nations where such experts are available within the country, current technology may enable remote education and supervision.Screening for hearing loss may include mass population screening for neonatal hearing loss or targeted screening of people classified as high-risk based on their geographical location or patient-specific factors .Automated devices have simplified several screening protocols. In the majority of European or other high-income settings, screening for neonatal hearing loss is undertaken by a nurse (or sometimes an audiologist) using automated otoacoustic emission or auditory brainstem response devices.In many high-income countries, audiologists provide most of expertise in hearing assessment and rehabilitation, but task-sharing in this field has been described. A literature review found several studies reporting that community health workers were able to perform pure tone audiometry, the basic test of adult hearing, but details of the training given or the accuracy of the results were lacking in such studies.The fitting of hearing aids is also open to task-sharing. A survey of 62 countries globally in 2008 found that in 12 countries ear, nose and throat specialists undertook hearing tests, and in 11 countries they fitted hearing aids.Provision of more complex audiology services requires a specialized workforce and longer training. In Bangladesh, a trial of accelerated training of community health workers over two weeks to carry out play audiometry (a method to test hearing where young children are asked to respond to sound by performing a task) proved unsuccessful.Further studies are needed in this field, particularly those reporting methods and outcomes from the training of non-specialist workers to perform basic tests of hearing and to provide hearing rehabilitation services.The two operations for treating suppurative ear disease are tympanoplasty and mastoidectomy, both of which involve complex microsurgery on the temporal bone of the skull. Training in ear surgery is difficult, even in resource-rich environments. Standardized educational programmes exist in countries such as Australia, Canada, the United Kingdom and the United States, which typically comprise 5–6 years of specialist training in ear, nose and throat surgery,Targeted training in ear surgery may be available in low- and middle-income countries, but is often not possible due to a lack of in-country expertise, relevant equipment or (due to logistic or sometimes religious constraints) cadaveric material for rehearsal. Alternative simulation using plastic bones or virtual tools is possible, although inferior for learning outcomes and not without cost.An alternative is to rely on visiting specialists to deliver training. In many low- and middle-income countries, specialists from high-resource settings visit on trips (missions) to provide a surgical service to the local patient population, and many will try to incorporate training into such trips. However, a recent survey from the United States of ear, nose and throat surgeons involved in global health found that the majority focused on missions, but that only 35% (125 out of 362) had been to the same hospital more than once, and that 93% (187 out of 202) went for no more than two weeks.Coordinated missions can, however, be fruitful. A collaboration between ear, nose and throat departments in three North American medical schools  delivered in-country training in Uganda through frequent missions and a structured training programme.Another model is to provide prolonged in-country training by a visiting resident specialist. In Cambodia, two local ear, nose and throat trainees were trained by visiting ear, nose and throat specialists from the United Kingdom.It is uncertain if those with lower levels of background training could also perform ear surgery. Clinical or medical officers, who are not doctors, have been trained to deliver surgical care, such as laparotomy or caesarean section, in many countries,The existing literature suggests that the acquisition of skills in ear surgery is achieved via a focused, coordinated and consistent mentorship approach, taking place over several months or years. Future programmes for training in ear surgery should look to incorporate such ideals.Deaf services typically involve teachers of the deaf, and speech and language therapists. There are few published data on the methods or effectiveness of training for teachers of the deaf,,,Many countries have reported the existence of a training programme for speech and language therapists. In several countries in South America, and in some areas of India, audiology, and speech and language therapists are one profession, with a subspecialization.While the guiding principles of speech and language therapy or deaf education are universal, the specific nature of the training will be determined by the local culture and language (whether spoken or signed). Such differences means that sharing of training resources or learning across linguistic regions or borders may be difficult or even inappropriate. Outside assistance may be helpful to guide the set-up of services, but such initiatives need to remain sensitive to local needs.,Cochlear implants are surgically implanted devices for those affected by severe hearing loss. Implant programmes should ideally incorporate comprehensive pre-implant assessment and post-implant rehabilitation and support, which requires appropriately trained audiologists, ear, nose and throat surgeons, and teachers of the deaf or speech and language therapists. Anecdotally, cochlear implants have been performed in many low- and middle-income countries without such a comprehensive team, leading to concerns that such programmes may not be optimal. In general, data is lacking on outcomes from cochlear implantation in low- and middle-income countries, although data from several countries in South America and Asia show that in most locations implant use rates are over 95%.Other issues compound the challenges to human resources and training outlined above. One is the lack of specialist equipment, such as audiometers for performing hearing tests;Training, development and task-sharing are strategies that can be used to counter the significant human resource gap for ear and hearing health. However, the existing literature inadequately addresses or documents the potential for these strategies.In terms of training, most existing studies with community health workers have reported outcomes in terms of knowledge acquisition. We have not found any studies that evaluated whether community health workers may be able to independently assess and manage ear and hearing disorders in primary care. In the more complex tasks of undertaking hearing tests, hearing rehabilitation, middle-ear surgery and provision of deaf services, only a handful of studies describe training or task-sharing and of these few report outcomes.Future studies should therefore report not only models of training, but also short and long-term outcomes of training, including the effects on delivery of care. Such reporting will inform those trying to emulate or translate such training to other contexts, and so help to optimize workforce development in ear and hearing health.


Hearing loss is one of the most important disabilities in neonates. Delay in the detection of hearing loss leads to impaired development and may prevent the acquisition of speech. We aimed to determine the risk factors associated with hearing loss in neonatal patients aged more than 6 months with a history of hospitalization in Neonatal Intensive Care Unit (NICU).In this case-control study, screening for hearing loss was carried out on 325 neonates aged 6-12 months referred to Pediatric Neurology Office of Vali-e-Asr Hospital, Tehran, Iran up to 2011. Hearing loss was confirmed using Auditory Brainstem Response screening test (ABR). The prevalence of mildly and moderately hearing loss in neonates was determined as 3.6%. The most significant risk factors for hearing loss in neonates were neonatal icterus associated with phototherapy, respiratory distress syndrome (RDS) and lower Apgar score.It seems to quantitative auditory system screening using ABR is necessary for all neonates; because rehabilitation support such as speech therapy and hearing training in this age period is more effective than older ages.  One of the valuable human senses is hearing that facilitate communication between people through speaking . HearingThe majority of neonatal hearing losses are sensorineural , but acca) family history of hearing impairment, b)intrauterine infections such as cytomegalovirus (CMV), toxoplasma etc., c) neonatal specific disorders as severe hyperbilirubinemia that needs exchange transfusion, d) persistence pulmonary hypertension (PPHN) associated with mechanical ventilation and e) after birth infection like bacterial meningitis . By 1998According to American Pediatrics Academy (APA), the effective test for neonatal hearing screening should detect hearing loss more than 30 dB and be accurate in lower than 3-month neonates; two electrophysiological tests have these properties: Auditory Brainstem Responses (ABR) and Otoacoustic Emission (OAE) . ABR mea"Hearing loss causes delays in development of speech and language, and those delays then lead to learning problems, often resulting in poor school performance" . NowadayWe aimed to evaluate hearing loss in 6-12 months of neonates with a history of NICU admission.This case-control study was conducted for further investigations of hearing loss in 6-12 months neonates referred to Pediatric Neurology Clinic  for 5 years up to August 2011, with a history of hospitalization in Vali-e-Asr Hospital's NICU, Tehran, Iran. Overall, 325 neonates were enrolled based on the inclusion criteria. Infants clinically and para clinically proved hearing loss assumed as case group and the control group was those who had no hearing impairment. An auditory evoked potential system  was used to record ABR. A click-type stimulus was used twice with rarefaction polarity, with Intensity of 35-80 dB NHL, under 21/1 pulse set based on 1500 trials and analysis time of 10-15 ms. All of the neonates had been sedated by oral chlorate hydrate, 50 mg/kg half an hour before the test. Four electrodes were applied as follows: two active ones were placed on mastoid bones, the reference electrode on vertex, and the ground electrode on the forehead. Recorded waves were analyzed and interpreted by an expert audiologist. Measurable and comparable variables consisted of mean latency of V, III and I waves, interpeak interval of I-III, III-V, I-V waves and no waves. Neonates were divided into two groups according to the normal and abnormal results of the ABR. After assessment of hearing status, 12 neonates showed impairment (case group) and 4 times more neonates enrolled as a control group (n=48). Both of groups matched in gestational age (± 2 wk) and birth weight (± 200 gr). Hearing loss detected using ABR test. Recorded variables collected in 3 categories:1. Pregnancy variables: A) fetal infections e.g. CMV, rubella, hepatitis B, HIV, syphilis, and chickenpox; B) mothers drug abuse during pregnancy; C) mothers diseases including preeclampsia, gestational diabetes mellitus (GDM), chronic diabetes mellitus, chronic hypertension and heart diseases.2. Neonatal variables: mechanical ventilation more than 2 d, low Apgar score (<7), RDS, metabolic diseases  and hyper/hypothyroidism), sepsis, icterus requiring phototherapy or exchange transfusion.3. Independent variables: hearing loss of mothers, fathers or both of them.Informed consent was received verbally from parents. The name and personal information of the patients remained confidential Statistical AnalysisA P-value of less than 0.05 considered as significant and for significant correlations, Odd ration with confidence interval of 95% calculated for significant correlations.Data were analyzed using SPSS software 17.2 . Bijective dependent variables analyzed via logistic regression; student t-test used for assessment of correlations among quantitative variables including Apgar score, mechanical ventilation days and qualitative variables including history of icterus, RDS, PPROM sepsis, preeclampsia, thyroid dysfunction and Delivery type analyzed by Chi-square test. Among 325 neonates, 12 infants (3.6%) showed hearing loss according to ABR results as mild (threshold of hearing 15-30 db) and moderate (30-50 db) hearing loss. There was 3 females (25%) and 9 males (75%) in these 12 cases with male to female ratio as 3 to 1. Bilateral hearing loss was shown in 3 neonates (0.92%).History of icterus, acute RDS and low Apgar score showed significant with impaired hearing screening results .There were no histories of meconium-stained liquor, maternal heart diseases and diabetes in both groups. In the history of any of the members of the two groups, there was no evidence of TORCH. The family history of hearing impairment was negative for members of both groups. Number and percent of not significant variables are listed in No significant differences were noticed between case and control groups in Delivery type, Sepsis, PROM, preeclampsia, thyroid dysfunction and ventilation; NVD: normal vaginal delivery, CS: cesarean section, PROM: preterm rupture of membrane.In the present study, 3.6% of infants showed hearing loss according to ABR result with male to female ratio as 3 to 1. All of infants in case group showed impaired OAE test and 75% of these infants had a history of icterus while only 16.7% of infants in control group had this history. A history of acute RDS showed in 25% of neonates in case group and there was no case of RDS in control group. Low Apgar score was recorded for 33.3% of neonates in case group and 6.3% of cases in control group. The total cost of education for not screened hard-hearing children imposes high costs on governments . The preth d after birth using OAE test and the prevalence of hearing impairment showed in 10.8% cases in this stage; ABR test was done for these neonates in next stage and hearing loss confirmed for 6.2% by ABR . Nowadays, the importance of icterus, especially severe degrees, is well marked in auditory neuropathy , 23. TheThe present study showed that there was no correlation between maternal preeclampsia and neonatal hearing loss, reported already . NeonateThe present findings showed correlation between neonatal RDS and hearing loss; however few studies investigate this relationship. RDS was not a risk factor for hearing loss ; while aIn conclusion, the newborn hearing screening program is possible, beneficial and justified in terms of scientific and economic principles that provide early treatment interventions that can significantly reduce further costs for patients. Moreover, because the incidence of congenital bilateral hearing loss is higher than the prevalence of other disorder screening, newborn hearing screening is done as soon as possible-preferably before three months of age and early intervention programs and the hearing strengthening tools should be available in health centers. Given the prevalence of hearing impairment 3.6% and risk factors such as phototherapy treated icterus, RDS and low birth Apgar score and also the cost of hard-hearing patients training, it seems necessary considering of these risk factors in hearing screening. Nowadays, neonatal hearing status screening performs routinely using OAE in Iran; however, ABR is a more accurate test to replace with OAE in the first step of screening.


Lawsonia intracellularis, an obligate intracellular bacterium, is an important enteric pathogen in pig herds and horse farms worldwide. The hallmark feature of L. intracellularis infection is the proliferation of epithelial cells in intestinal crypts. A major limitation to the study of L. intracellularis infection is the lack of an in vitro model that reproduces the changes observed in proliferative enteropathy. Here we investigated the suitability of mouse enteroids as a model to study L. intracellularis infection. Mouse enteroids were microinjected with L. intracellularis, filter-sterilized L. intracellularis culture supernatant, or sterile cell culture media (DMEM). L. intracellularis antigen was detected in mouse enteroids by immunohistochemistry and was located mostly in the basal region of the epithelium. There was no differential growth of enteroids among treatment groups, and cellular proliferation was not increased in L. intracellularis-infected enteroids in relation to non-infected enteroids based on immunofluorescence staining. L. intracellularis infection did not induce changes in gene expression of Ki-67 (proliferation marker), Sox9 (marker for transit amplifying cells) and Muc2 (marker for goblet cells). These results indicate that although L. intracellularis antigen is detectable in mouse enteroids, indicating susceptibility to infection, mouse enteroids fail to replicate the cellular proliferation and gene expression changes observed in proliferative enteropathy. Nevertheless, we have successfully demonstrated that mouse enteroids can be used to model days-long intracellular pathogen infection, serving as potential models for the study of other pathogens of interest in veterinary medicine. Lawsonia intracellularis and is characterized by the thickening of the small intestinal mucosa due to proliferation of intestinal epithelial cells. PE causes diarrhea and compromised weight gain in pigs [Pig herds and horse farms worldwide are regularly challenged by proliferative enteropathy (PE). This disease is caused by  in pigs , 2. In w in pigs . Other s in pigs .Lawsonia intracellularis is a Gram-negative bacterium that requires an intracellular culture system and a specific atmosphere to be propagated in vitro [L. intracellularis propagation, but they have failed to reproduce the increased cellular proliferation observed in PE-affected animals [L. intracellularis. Hence, identification of new alternatives for control and prevention of PE has been difficult, and pig producers and horse owners have been relying on available commercial vaccines (whole cell vaccines) and antimicrobials to prevent and control PE [in vitro . Traditi animals . The lacntrol PE –10. The ntrol PE . Therefontrol PE –14.L. intracellularis infects intestinal epithelial cells, especially in the crypt compartment, leading to cellular proliferation and decreased differentiation in goblet cells along with increased apoptotic events [L. intracellularis and intestinal epithelial cells, determined using a controlled environment, could generate a better understanding of how L. intracellularis induces cellular proliferation.The currently held hypothesis is that c events , 15, 16.A promising alternative to single cell cultures are tridimensional multi-cell type cultures, also known as organoids. One of the most relevant advantages of organoids in relation to the traditional in vitro single cell culture is the similarity of the organoids to their respective tissue of origin , 18. EntL. intracellularis infection. Infected enteroids were monitored over time by size along with changes in expression of genes that have been reported to change during PE. We found evidence of infection with L. intracellularis in enteroids followed for up to 7 days post-infection (dpi). The genetic profile of infected mouse enteroids, however, diverged from the gene expression changes observed in PE.The objective of this study was to evaluate mouse enteroids as an in vitro model for Mouse enteroid preparation and maintenance were performed as described elsewhere   at low (≤ 20) passage [® CRL-1696™). McCoy cells were cultured as described elsewhere [7L. intracellularis and incubated at 37 °C under a controlled microaerophilic atmosphere created with a mixture of gas containing 10% hydrogen, 10% carbon dioxide, and 80% nitrogen [g to separate cell debris from L. intracellularis organisms. The pellets were discarded and the remaining supernatant was then filtered through 0.80 µm sterile filters  to remove any remaining McCoy cells and nuclei. After a final centrifugation at 8000 × g at 4 °C the bacterial pellet was either used to re-infect McCoy cells, or to prepare the inoculum suspended in approximately 500 µL of its own supernatant and used as inoculum.A  passage  was proplsewhere . Cells wnitrogen . Seven dL. intracellularis growth requires special atmospheric conditions [L. intracellularis growth would impact growth and development of mouse enteroids. One plate with enteroids was maintained under atmospheric conditions specific for L. intracellularis i.e., 10% hydrogen, 10% carbon dioxide, and 80% nitrogen [2 as a control. Both plates were incubated at 37 °C. Since L. intracellularis manipulation in vitro requires an antibiotic-free sterile system, antibiotics (Penicillin/Streptomycin) were excluded from both plates to assess sterility of the cultures. The plates were observed every 48 h for 3 weeks, with media replacement and enteroid passage as described previously.As nditions , we firsnitrogen  while anL. intracellularis, we adopted the “incubation and seed” method [L. intracellularis suspended in 100 µL of enteroid culture medium (resulting in an inoculum with ~109L. intracellularis organisms/mL), with occasional gentle agitation, and incubated at 37 °C under special atmospheric conditions generated with a gas tank of 10% hydrogen, 10% carbon dioxide, and 80% nitrogen for 30 min. The infected enteroids were briefly centrifuged at 200 × g for 1 min, mixed with 50–100 µL ice-chilled Matrigel , and then seeded into culture plates. Enteroids were harvested 15 days post-infection (dpi) by removing culture media and suspending the Matrigel in phosphate buffered saline (PBS). After centrifugation for 1 min at 200 × g, the pellet was suspended in 4% formaldehyde and incubated for 2 h at room temperature. Enteroids were then washed in PBS, centrifuged at 200 × g for 2 min, suspended in Histogel  and placed into a biopsy mold. Histogel blocks were inserted into a cassette and maintained in 70% alcohol until they were processed for histological evaluation. Four-micrometer sections were placed onto glass slides and used for hematoxylin and eosin (H&E) staining and immunohistochemistry [L. intracellularis antigen was detected by IHC in the infected enteroids by this method  operated by a trained technician. Experiments were performed in four independent replicates.Mouse enteroids were cultured and passaged until a sufficient number of enteroids  with 100 µm diameter were obtained for the trial. Wnt3a protein, which regulates the signaling pathways related to the cellular proliferation in the intestinal crypt compartment , was remMicroinjected enteroids were monitored for 1 week, from the day prior to infection until 7 dpi. Images were captured at 200× magnification using an inverted microscope coupled with a digital camera immediately after injection (0 dpi), and at 1, 3 and 7 dpi. The enteroid area was measured using an image software . The relative enteroid area (enteroid growth) was measured by subtracting the enteroid area from the area recorded at its previous measurement.g for 4 min. The supernatant was discarded, and the pellet suspended in 1 mL of PBS. This step was repeated two times to remove Matrigel. The resulting enteroid pellet was suspended and fixed in 4% paraformaldehyde for 1 h at room temperature. After fixation, enteroids were washed three times in PBS and centrifuged at 200 × g for 4 min.Enteroids from each treatment group were harvested at 1, 3 and 7 dpi and prepared for immunofluorescence. Matrigel was disrupted with a sterile pipet tip and the contents in each well were transferred to a 1.5 mL tube and centrifuged at 200 × Fixed enteroids were embedded in Histogel  and then either placed in optimum cutting temperature  for preparing cryosections or dehydrated in 70% ethanol and embedded in paraffin.L. intracellularis using specific rabbit polyclonal antibodies and a previously described method [Paraffin-embedded sections (4 μm) on charged slides were used for IHC for detection of d method . StainedOCT-embedded blocks were sectioned (4 µm), placed on charged glass slides and stored at −20 °C until immunofluorescence staining. Anti-Ki-67 immunofluorescence was performed as described elsewhere [Enteroids were harvested at 0, 1, 3 and 7 dpi, released from Matrigel as described above, lysed with 1.5 mL of TRIzol  and stored at −80 °C until RNA extraction. RNA extraction was performed using chloroform and isopropanol precipitation following TRIzol protocols and then cleaning with the RNeasy mini kit  according to the manufacturer’s instructions. Total RNA was quantified using a Nanodrop 2000  and 400 ng of RNA were transcribed to cDNA using the High Capacity cDNA Reverse Transcription kit  with random hexamer primers.GAPDH was monitored as reference. Relative gene expressions were normalized to GAPDH using the primer efficiencies. The expression levels of Ki-67 (marker for cellular proliferation), Sox9 (marker for transit amplifying cells) and Muc2 (marker for goblet cells) in enteroids of all treatment groups were measured at 1, 3 and 7 dpi. Fold change in each case was calculated in relation to expression in enteroids from day 0 (control). All primers used are listed in Table Quantitative PCR reactions were performed using SYBR Green PCR Master Mix (Applied Biosystems) according to the manufacturer’s instructions. Amplification was performed with the following conditions: initial activation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing at 60 °C for 60 s. Expression of the housekeeping gene p <0.05 considered statistically significant. GraphPad Prism 8.1 software for Windows  was used to perform the analysis.Results are presented as a mean ± standard deviation of the mean (SEM) for each group. Two-way ANOVA followed by Geisser-Greenhouse correction was used to verify statistical differences with 2 incubator (data not shown), indicating that growth conditions required for L. intracellularis do not impact enteroid viability or growth.There was no difference in growth or morphology between enteroids maintained for 1 week in the microaerophilic atmosphere relative to those maintained under regular conditions, i.e., 5% COL. intracellularis by microinjection. Enteroids were harvested and analyzed by IHC for L. intracellularis antigen at 1, 3, and 7 dpi. L. intracellularis antigen presence was observed at all time points, indicating successful infection with L. intracellularis. Unexpectedly, although L. intracellularis antigen was observed in the cytoplasm of enteroid cells, most of the antigen, especially at 7 dpi, was found in the basal region of epithelial cells , the antigen was mostly observed localized in the extracellular space proximal to the basal region of the epithelium of the mouse enteroids. In vivo, L. intracellularis organisms are consistently observed on the apical side of the cytoplasm at similar infection time points to those used in this study [L. intracellularis to move towards the nutrients to support their growth. It is also possible that conditions of the enteroid culture environment could result in less permissible conditions for L. intracellularis closer to the lumen. Another possibility is that the fibroblasts and immune cells in the basal region of the intestinal epithelium in vivo may have a regulatory effect on L. intracellularis traffic in the intestine, and the lack of those cells in the mouse enteroid culture would modify L. intracellularis traffic in vitro.One of the most intriguing findings in the present experiment was the unexpected location of is study , 36. A pL. intracellularis is considered the gold standard method for diagnosis of proliferative enteropathy [L. intracellularis suspension (~106L. intracellularis organisms/mL), were infected and to visually demonstrate the level of infection in these microinjected enteroids. Our results were not only surprising in regards to the localization of L. intracellularis antigen, as discussed before, but also in regards to the amount and morphology of the stained antigen. Generally, L. intracellularis antigen stained by immunohistochemistry is observed as small bacilli in the cytoplasm of cell cultures [L. intracellularis antigen, we observed mostly the antigen as a “bundle” accumulated on the basal region of the enteroid cells. Nevertheless, occasional well-shaped L. intracellularis antigens were observed in the cytoplasm of some enteroid cells (Figure L. intracellularis organisms gained entrance into the cell cytoplasm. The events involving the changes in the morphology of the antigen and its final location in the basal side of enteroid cells remains unclear and may deserve more investigation. The homeostasis of the intestinal epithelium involves various signaling pathways of which canonical Wnt signaling is responsible for maintaining a balance between the pool of undifferentiated cells with proliferation capacity, named transit amplifying cells, and differentiated cells [L. intracellularis on cell proliferation, therefore we removed Wnt3a from the culture media 3 days before microinjection and during the experiment to allow the epithelial cells to differentiate, and better reproduce the proportion of proliferative cells of the normal intestinal epithelium. However, we failed to detect increased proliferation in L. intracellularis-infected enteroids by RT-qPCR or immunofluorescence. It is possible that the removal of Wnt3a 3 days before microinjection was not sufficient to reduce proliferation to a level where we could detect changes induced by L. intracellularis. Another possible explanation for this lack of difference in proliferation between treatment groups was the small amount of L. intracellularis delivered to the enteroid lumen, thus requiring a longer time for the bacteria to replicate in numbers to produce a detectable change in proliferation markers. The low amount of antigen detected in the microinjected enteroids indicate that L. intracellularis did not encounter an ideal environment for propagation as it does in McCoy cells and in the intestines of mice and other rodents [L. intracellularis susceptible species, such as pigs and horses, would help to confirm this hypothesis.Immunohistochemistry for eropathy , 46. In cultures , 47, 48 s Figure D, which ed cells , 49. In ed cells , resulti rodents , 43, 50.Changes in the area of enteroids can indicate an expansion on the number of cells or an increase in secretory activity. Secretory activity of enteroids has been demonstrated by swollen enteroids as a result of accumulation of luminal secretions after stimulation with forskolin . In the L. intracellularis is a unique bacterium, requiring intracellular conditions and special atmospheric conditions for propagation in vitro. Little is known about L. intracelllaris survival in the cellular cytosol, or the host-cellular pathways that are disrupted during L. intracellularis infection. By using mouse enteroids as an in vitro model for L. intracellularis infectioin, our aim was to develop a tool to further define several aspects of L. intracellularis pathogenesis. The localization of L. intracellularis more abundantly in outside of the basal side of the enteroid epithelial cells may indicate nutrients or a microenvironment preferred by L. intracellularis and merits further investigation. Likewise, little is known about mouse enteroids maintained with a pathogenic enteric bacterium for a period longer than 24 h [L. intracellularis infection that have been noted in vivo in more susceptible species. Testing L. intracellularis infection in enteroids from other more susceptible species will provide further insight to species susceptibility to pathogenesis.han 24 h , 21. Mor


Lawsonia intracellularis is one of the world’s most important infectious diseases in pork production with regard to economic losses. So far, studies are missing that describe the effects of a natural infection of piglets on the digestibility of nutrients, possible effects on performance and the morphometrics of the intestine depending on whether piglets are vaccinated, clinically healthy or clinically affected with regard to Lawsonia intracellularis induced diarrhoea.Lawsonia intracellularis infection in a trial with three repetitions. Nine out of 27 animals were vaccinated as suckling pigs with a commercial Lawsonia intracellularis vaccine . Half of the remaining 18 animals were without clinical signs of infection (non-vac/cs-), half showed moderate clinical signs of Lawsonia intracellularis induced diarrhoea (non-vac/cs+). All three groups were fed one identical complete diet ad libitum. Faecal shedding of Lawsonia intracellularis was found in all groups . Numerically, the mean excretion in the group non-vac/cs + (7.69 ± 1.65 log10 copies/ g faeces) was higher in comparison to the group non-vac/cs- (5.83 ± 2.35 log10 copies/ g faeces) and vaccinated animals (vac: 6.00 ± 2.89log10 copies/ g faeces). The average daily weight gain  differed significantly . The apparent total tract digestibility (ATTD) of nitrogen was significantly lower in clinically affected animals .The total length of the small intestine in clinically affected animals increased significantly . The relative body weight depending on the length of the small intestine was lower for clinically affected animals .Digestibility studies were performed on a total of 27 eight-week-old piglets with naturally occurring L. intracellularis infections lead to significantly lower ADGs in comparison to vaccinated animals. The disease is also found in altered intestinal morphometry and reduced total N digestibility if clinical signs occur.These studies show that clinically moderate  Lawsonia intracellularis (L. intracellularis) in pigs lead to economic losses both in the case of clinical manifestations and subclinical infections [L. intracellularis infection in practice [Infections with fections –4. Lowerfections –8. The ofections , 9–11. Tpractice . Clinicapractice . These mpractice .L. intracellularis [In order to convert the absorbed nutrients at their highest rate into weight gain, a good state of health of the growing animals is essential. This excludes any avoidable activation of an inflammatory immune response, which might also be common for gastrointestinal infections . High collularis .L. intracellularis infection. With regard to the pathomorphological changes in the intestines of infected pigs, it has to be assumed that due to the reduced permeability of the intestinal wall there is a reduced absorption of certain nutrients in cases of an L. intracellularis infection [In general, an immediate reduction in average daily feed intake (ADFI) for pigs affected by digestive bacterial infections can be estimated at 15% . Nonethenfection , 21. Fornfection . Howevernfection . This isnfection . In genenfection . Mechaninfection . There anfection . These anfection . A leadinfection , 26. Entnfection .L. intracellularis might affect the digestibility of nutrients, performance, IGF-1 status and gastrointestinal tract morphometry as a result of natural exposure when non-vaccinated and vaccinated animals are compared.To date, no investigation has been carried out on how an infection with Animal experiments were carried out according to German regulations. It was not an animal experiment requiring a notification or an approval according to the Animal Protection Act . Interventions before dissection were not carried out. The animals were killed according to § 4, paragraph 3 of the Animal Protection Act, exclusively to use their organs or tissues for scientific purposes.Erysipelothrix rhusiopathiae, Escherichia coli + Clostridium perfringens dam vaccine) as were the piglets . In the context of regular and long-term screening programmes, the herd showed no signs of Salmonella and Brachyspira infections. Clinical symptoms of an L. intracellularis infection (confirmed by pathogen detection in faeces) were, on the other hand, a common finding in piglets at an age of seven to nine weeks. Before the starting the experimental trials, additional results of serological analyses of piglets were obtained from the veterinarian of the herd. From these random tests in the rearing phase of piglets, it was found that there were no serological positive animals in the middle of this phase whereas at the end of the rearing phase, only 10% were still serological negative (data not shown).All animals used in these trials came from one farm with 420 sows of Danish genetics (DK: Danish Landrace 50% x Yorkshire 50%). In total, 27 piglets (DK x Pietrain) were used in a trial with three repetitions. The sows in the herd were regularly vaccinated  on the 21st day of life. The vaccination was done via oral drenching. Subsequently, the piglets were marked individually.The production system on the farm was organised in a two-week rhythm. During the trial period every two weeks approximately 50 piglets from a total of four to five complete litters were vaccinated with a commercial L. intracellularis infection time point, samples from the individual stables were tested for their L. intracellularis status by real-time PCR using established methods [At weaning all 450 to 500 piglets in a group were placed in a piglet rearing compartment in groups of 12 to about 30 animals on fully slatted floors . Visual contact to other animals was possible at all times. The boxes had a concrete floor. All three repetitions of the trial were performed identically.®, HS-Kraftfutterwerk, Ochtrup, Germany; Table The pigs continued to receive the diet they were accustomed to on the farm. The diet contained 41.0% wheat, 35.0% barley, 18.0% soybean meal, 2.00% soybean oil and 4.00% of a mineral and vitamin supplement (MOVIKALIN Speed 10L. intracellularis infection. Chromium oxide (0.5%) was added to the diet as a marker for calculating the precaecal digestibility. The diet was offered ad libitum. The feed was changed once a day. Feed refusals were removed and the amount determined after drying at 103 °C. The collected faeces were weighed by means of electronic scales  and then frozen. At the end of the collection period, an aliquot of the total amount of faeces was collected after thawing and homogenisation for further analysis.The nutrient composition was in accordance with the official recommendation for piglets in Germany. The complete diet for all trial repetitions derived from one batch. The digestibility trial consisted of two phases starting with a 3-day adaptation period followed by a five-day collection period. The adaptation period was shortened to only three days due to the use of the identical diet already on the farm. In this way, the experiment still focused on the clinically conspicuous phase of the natural ® 10%, Serumwerke Bernburg, Bernburg, Germany; active ingredient: ketamine hydrochloride, dosage: 15 mg/kg body weight intramuscularly) and azaperone . In the course of the dissection a blood sample was taken intracardially from the pigs. After that, pigs were euthanised with T61®intracardially .At day of the dissection the pigs were fed in a timely manner. There was an interval of nine hours between offering the fresh diet and death of each animal. In each repetition 3 out of 9 animals were dissected on the day of finishing the digestibility trial, the remaining animals on the following day. Prior to the dissection the animals were anaesthetised with neuroleptanalgesia by means of a combination of ketamine , which operates according to the principle of a catalytic tube combustion (DUMAS combustion method). The molecular nitrogen formed by reduction from nitric oxide was detected by a thermal conductivity detector and the nitrogen content was calculated by the device software. The crude protein content of the sample was calculated by multiplication with a constant factor of 6.25. The crude fat content was determined after acid hydrolysis in the Soxhlet apparatus. The content of crude fibre was determined after washing in diluted acids and alkalis by established methods. The determination of starch contents was carried out polarimetrically . The sugar content was analysed according to Luff-Schoorl by titration with sodium thiosulphate. The mineral content was determined in accordance with the official methods [Diets were analysed by standard procedures in accordance with the official methods of the VDLUFA .The anal methods  by atomiL. intracellularis genome equivalents (GE) was done from the aliquot of the homogenised faeces via quantitative PCR according to established methods [The determination of mean  methods . The numInsulin-like growth factor I (IGF-1) concentration was determined using a commercial radioimmunoassay acoording to the standard operation manual . The assay was validated for porcine plasma by determining the recovery and an intra-assay coefficient of variation by measuring 20 times one sample within one test routine. The recovery was 94,5% and the intra-assay was 6,3%.Normal distribution of the model residuals was confirmed by Kolmogorov-Smirnov-Test and visual assessment of q-q - plots. All data were included into a descriptive analysis with calculation of the arithmetic mean, standard deviation, minimum and maximum.The influence of the factor group  on the quantitative variables  was carried out by a one way variance analysis of variance (ANOVA) with REGWQ (Ryan-Einot-Gabriel-Welsch multiple Range-Test) post hoc test for multiple pairwise comparisons, concerning the experimentwise errorrate.® Enterprise Guide 5.1. Differences were taken to be statistically significant when P < 0.05.Analyses were carried out with the statistical software SAS, version 9.3 , using SASL. intracellularis).The investigations were carried out on a total of 27 piglets. In all three repetitions of the trial, the general health status of each animal was checked at least twice a day. The 27 animals showed an undisturbed general state. Moderate clinical diarrhoea in all animals of the non-vaccinated, clinically affected group (non-vac/cs+) previously found on the farm was included in the study as a selection criteria . There were no statistically significant differences in the absolute level of pathogen excretion  in proportion to the nose-rump length. Therefore, per unit body length more gain in body mass is concentrated. The vaccinated and also the clinically inconspicuous animals each had the highest ADG in relation to the length of the small intestine (+ 18.2% per m and + 21.5% per m).L. intracellularis infection (+ 18.2%) relative to vaccinated animals. Vaccinated animals had the highest ADG (+ 13.0%) in relation to the mass of the stomach. There was a significantly reduced amount of stomach content  in clinically conspicuous animals.Total mass of the caecum tissue . There were also no significant differences between the mass of the small intestine and the mass of the stomach. Numerically, the mass of the small intestine was lowest in relation to the mass of the stomach in the clinically healthy animals .L. intracellularis infection result from lower average daily gains and a feed conversion ratio which is worse [L. intracellularis in pigs are the main issue for the pig industry [Performance losses in the course of an is worse –8. The eindustry –4.L. intracellularis infection, a reduced feed intake was observed in animals aged 28 to 49 days. When groups with different inoculation doses (counts per pig) were compared (7.2 × 107 to 3.8 × 105 and 7.2 × 107 to 2.2 × 106), the reduction in feed intake was 1.27% and 7.85%, respectively [10L. intracellularis organisms in the inoculum) in comparison with animals treated with 50 ppm tylvalosin for 14 further days after experimental infection [E. coli in piglets) the reduction was 15% [L. intracellularis in the acute phase seems to have a somewhat lower effect on the feed intake than viral or classical bacterial intestinal infection in piglet rearing. In the present study, the effects of an L. intracellularis infection on the feed intake were higher in percentage terms than in the literature [L. intracellularis are freshly harvested from McCoy cells [L. intracellularis infection may simulate the conditions in the field better. This is because interactions between the natural composition of the microbiome in pigs and nutrient digestibility are proven [A high feed intake is the basic prerequisite for exploiting the performance potential of pigs , 31. Theectively . In anotnfection . For virnfection . In a re was 15% . In concterature . This caoy cells –37. On te proven . This isThe Insulin-like growth factor I is an important anabolic hormone that mediates growth and development of individuals . IGF-1 iNumerically, the vaccinated animals had the highest mean values (285 ng / mL). Non-vaccinated, clinically healthy animals (− 4.72%) and pigs with clinical signs of infection (− 20.9%) had lower IGF-1 concentrations in the serum at the time of dissection.P < 0.01) [From literature it is known that pigs with higher post-weaning average daily gains have significantly increased odds of elevated IGF-1 expression in the liver . For exa < 0.01) .p < 0.001; [p < 0.01; [In a five-week feeding experiment with piglets on the effects of antibiotics  the levels of IGF-1 in untreated animals were significant lower . This o < 0.01; ). From s < 0.01; . In geneL. intracellularis in pigs is a downregulation of various genes in the enterocytes. These play an important role in the mechanisms of nutrient absorption [L. intracellularis can have an influence on the digestibility of nutrients. No significant differences were found in the study with regard to the precaecal digestibility of the nutrients, although numerically the digestibility rate of nitrogen decreased by 2.3% percentage points in the presence of clinical manifestations of the disease (non-vac/cs + vs. non-vac/cs-). Until now, there is no comparative study dealing with the influence of this relevant infectious agent of the pig on digestibility of nutrients [One of the characteristics of an infection with the pathogen sorption . Therefoutrients . For porutrients . This isThe response of the gastrointestinal tract to ingested nutrients is a complex and closely controlled process . A leadiIn the present study, it was seen that average total tract digestibility of nitrogen was significantly reduced in clinically affected animals. This was also demonstrated in the above-mentioned study for the combined infection of PRRSV and PED . The totBrachyspira infection in swine [L. intracellularis is also a prevalent cause of naturally acquired colitis in pigs. About 25% of endogenous nitrogen originates from the large intestine while 59.9% originates from the small intestine [It can be presumed that mucosal alterations might lead to increased presence of nitrogen compounds in the intestine as hypothesised for in swine . This mintestine . The reantestine  for an nntestine  and 16.1ntestine  with reaL. intracellularis infection. The lower average weight gain is primarily caused by a lower feed intake. The precaecal digestibility of the nutrients was not reduced significantly. Adaptation mechanisms lead to increased length and relative mass of the small intestine in diseased animals. Therefore, the relative efficiency of nutrient utilisation might be adversely affected. In the case of vaccinated animals, the lowest effects of an L. intracellularis infection were observed concerning the performance of the animals.This study clearly shows that the performance of pigs is adversely affected by an