Since decomposition of organic material was recently shown to be accelerated by Si, the aim of this study was to examine how Si influences decomposition of carbon and nutrient and toxicant mobilization in peatlands. We selected a fen site in Northern Bavaria with naturally bioavailable Si pore water concentrations of 5 mg/L and conducted a Si addition experiment. At a fourfold higher Si availability, dissolved organic carbon, carbon dioxide, and methane concentrations increased significantly. Furthermore, dissolved nitrogen, phosphorus, iron, manganese, cobalt, zinc, and arsenic concentrations were significantly higher under high Si availability. This enhanced mobilization may result from Si competing for binding sites but also from stronger reducing conditions, caused by accelerated respiration. The stronger reducing conditions also increased reduction of arsenate to arsenite and thus the mobility of this toxicant. Hence, higher Si availability is suggested to decrease carbon storage and increase nutrient and toxicant mobility in peatland ecosystems.Peatlands perform important ecosystem functions, such as carbon storage and nutrient retention, which are affected, among other factors, by vegetation and peat decomposition. The availability of silicon (Si) in peatlands differs strongly, ranging from <1 to >25 mg L Natural peatlands also provide the largest natural source of the potent greenhouse gas (GHG) CH4, yet, they are considered to have an attenuating effect on global warming by a net carbon fixation3, acting as net carbon sinks. However, land use change, such as deforestation, burning, and drainage has turned many peatlands into carbon sources due to e.g. increased peat decomposition4. Besides carbon storage, peatlands perform also other ecosystem functions such as nutrient retention and water quality regulation5. Nutrient retention and water quality regulation are closely linked as peatlands retain atmospherically or agriculturally deposited nitrogen (N) and phosphorus (P), improving the water quality of downstream ecosystems6. Furthermore, peatlands accumulate sulphur7, organic pollutants, toxic metals8, such as arsenic (As), but release dissolved organic carbon9 (DOC) depending on the peat decomposition and hydrology, thus additionally affecting the water quality.Peatlands are important ecosystems with regard to carbon storage. Although covering only a small fraction of the land surface (3%), they store one third of the world’s soil carbon stock (~550Pg)10. Under anaerobic conditions, elements bound to redox labile sorbents such as iron phases are released into the solution as the sorbents are reduced11. This element mobilization due to lowering of the redox potential has been demonstrated also for toxic elements such as As. The As speciation can thereby be used to elucidate dynamics in biogeochemical conditions, because of the shifts in predominant As species depending on changes in redox conditions12. Another controlling factor for peat decomposition is the peat nutrient content, which depends on litter quality, but also on exogenous inputs such as atmospheric deposition and input by waters that are enriched by N and P due to agricultural fertilization. Regarding nutrient content of peat, previous research focused mainly on N and P availability as important factors13. However, for grass dominated peat ecosystems, silicon (Si) was recently shown to be important for stability of litter, because increased Si contents increase decomposition16.Carbon turnover and nutrient retention in peatlands is a result of the budget of inputs and outputs controlled e.g. by the rate of peat accumulation versus peat decomposition. Among other factors, the peat decomposition rate is influenced by temperature, which controls the activity of decomposing microbes and extracellular enzymes, and by the water table level, which predominantly alternates redox conditions. Under waterlogged, anaerobic conditions peat decomposition is slow, whereas dry aerobic conditions accelerate peat decomposition and cause increased carbon losses17. Land use change during the last 250 years has decreased Si availability in soils by increasing export and decreasing Si storage due to higher erosion and a decrease in vegetation potentially accumulating Si, and has led to a twofold to threefold decrease of the base flow delivery of Si19. Due to differences in peat parent material, land use, and peat layer thickness, the Si availability in peatlands is highly variable. Especially in ombrotrophic peatlands, in which mineral weathering and plant growth are mostly decoupled by the peat layer, Si concentrations can be expected to be low in comparison to other ecosystems like forests18. Fen ecosystems connected to local groundwater percolating through mineral matter could be expected to plot in an intermediate range. Most available data for Si, however, is based on bulk concentrations derived from X-ray fluorescence analyses (XRF), which are unable to distinguish between bioavailable Si and refractory forms like quartz20. Only few studies have analyzed the concentrations of bioavailable Si or pore water Si of peatlands, observing varying concentrations between <1 mg/L and >25 mg/L22. In field and laboratory experiments it was found that plants grown under high Si availability showed a higher decomposition rate16. This suggests that Si may influence the ecosystem function of peatlands, especially in grass dominated fens with silicon being beneficial for grasses23, by accelerating the peat decomposition. Higher peat decomposition rates could increase GHG production and decrease the binding capacity of the peat for nutrients and other elements. However, so far, the effect of Si availability carbon turnover in peatlands is not well known.Peatlands and other terrestrial ecosystems represent large reservoirs and filters for Si, controlling the Si transfer to the oceans2 and CH4 as well as DOC release, (ii) mobilizes nutrients, such as N and P, because of competition for binding sites, and (iii) deteriorates water quality by mobilizing potentially toxic trace elements, such as As, due to changes in redox conditions. To this end, a field experiment was conducted at a minerotrophic fen site in Bavaria, Germany. Pore water samples and dissolved gases were analyzed under low and high Si availabilities to identify the effect of Si on CO2 and CH4 production and nutrient and trace element mobilization.Consequently, the aim of this study was to investigate how Si availability influences the biogeochemistry of peatlands. We hypothesize that increased Si availability (i) increases production of COp ≤ 0.001), CH4 (p ≤ 0.001), and CO2 (p = 0.003). With Si addition, DOC concentrations and conductivity exceeded values of the controls by a factor of four one month after Si addition and decreased to about the initial values after three months , the molar CO2:CH4 ratio in both treatments was always larger than one  in pore water pH between both treatments at the end of the experiment. These differences, however, did not exceed previously reported natural variability due to redox dynamics at the study site24, which had only minor effects on element mobilization. Furthermore, no significant correlation between differences in pH and any other investigated parameter was found (data not shown). We therefore deduce that pH is no relevant parameter for explaining the observed differences between high and low Si availability.One month after Si was added, average Si concentrations were threefold higher in the Si+ treatments than in the controls Fig. . The twoths Fig. . In the ion Fig. . In the one Fig. . In Augup ≤ 0.001 each, Fig. Concentrations of total dissolved N, P, Fe, Mn, Co, and Zn in pore water were significantly higher under high compared to low Si availability (t-test)  Fig. . At the st) Fig. , generalst) Fig. , weatherst) Fig. , pH, andst) Fig.  can be f2, CH4, and DOC in pore water under high Si availability clearly confirm the relevance of Si for the carbon cycle and support hypothesis (i) that increased Si availability increases concentrations of CO2 and CH4 in the peat and leads to a release of DOC into porewater  represent products of organic matter decomposition10 Si seems to accelerate decomposition and therefore presumably influences the carbon turnover of such ecosystems. This is in line with the findings of Schaller and Struyf14, who showed a positive correlation of plant litter Si concentration and microbial decomposition rate in a laboratory experiment. The same positive effect of Si on litter decomposition was also proven in a field experiment15. Since in our study plants were not present in the mesocosms, direct plant effects on CO2 formation via rhizosphere respiration could be excluded. Consequently, heterotrophic respiration should be the main source of the surplus of CO225. The increase in CO2, CH4 and DOC concentration in porewaters could thus be explained by Si mobilizing elements like P into pore waters, due to competition of Si and P for binding sites as already suggested for As26  followed by outgassing33. Hence, an increased GHG production is supposed to increase GHG emissions under elevated Si availability.Enhanced decomposition can not only increase CO26, who determined that Si effectively competes with P for binding sites of Fe and Al(hydr)oxides in rice paddy fields and Neu et al. showing a higher P uptake by plants under high Si availability suggesting elevated P mobility23. The fact that Si highly correlated with P in our experiment strongly supports that P is displaced by Si at the binding sites. From previous studies it is known that at this particular fen site, but likely also in other fens, iron oxides are present in notable quantities34. Additional to the competition at binding sites, Si also apparently accelerated the mineralization of organic matter, as obvious from increases in CO2 and CH4, causing additional N and P release from decomposition into the pore water35. The fact that N and P concentrations decreased towards initial concentrations within a few months after the Si application may have partly resulted from increased microbial assimilation of nutrients36. However, leaching might also have been effective due to high runoff rates after intensive precipitation events at the study site9 oxides may have released bound As and P into solution40. Due to the high activity of sulfate reduction and subsequent formation of sulfides at the site the potential of As and P re-adsorption is low and the elements remain in solution41. The shift towards reducing conditions was also reflected in the increase of the reduced As species arsenite under high Si availability oxide surfaces by increased methyl substitution of As. This may explain the increased As release under high Si availability. Nevertheless, high Si availability did not only increase total As concentrations but also released higher concentrations of the more toxic and mobile arsenite into the pore water44. These observations also demonstrate that the analysis of As speciation provided additional insight into the particular biogeochemical conditions, i.e., with Si favoring strongly reducing conditions with concomitant sulfate reduction and methanogenesis.Besides the macronutrients N and P, also the concentrations of micronutrients and potentially toxic elements such as Fe, Mn, Co, Zn, and As increased in pore water after Si addition Fig. , confirm2 and especially CH4 production  in northeastern Bavaria . The study area is characterized by a continental temperate climate with a mean annual precipitation of 1038 mm (1994–2015) and mean annual temperature of 6.5 °C (1994–2015). The minerotrophic peatland has a central open area, which is surrounded by a spruce forest (Picea abies). The open area is covered by grasses and bryophytes45. The thickness of the peat varies from 30 to 120 cm and the organic material is highly decomposed below 5–10 cm depth46. The site has been intensively studied previously with regard to carbon turnover and redox processes , synthetic amorphous silica  was added, which is equal to 23 g of elemental Si, suspended in 1 L deionized water (Si+ treatment) and a control watered with 1 L deionized water. The synthetic amorphous silica is highly dissolvable even at lower pH and is an analogue to nano particulate volcanic ash. The Si was added only once at the beginning of the experiment.The experiment was set up in July 2015. Eight mesocosms  were installed in pairs (each containing one control and one treatment with Si addition about 10 cm apart from each other) randomly selected within homogeneous but sparse vegetation of 50. The gas samples were taken with plastic syringes . Pore water was sampled by polyvinylchloride tubes , which were installed in the top peat layer of each mesocosm. The lowest 15 cm of the tubes, which were placed in the top peat layer just below the surface, were perforated at 5 mm intervals. To eliminate coarse particles, the tubes were covered by nylon prefilters. The pore water samples were taken by syringes to measure pH, temperature , and conductivity . For the analysis of total element and species concentrations, all water samples were filtered after extraction . To prevent precipitation of sulfide or iron minerals, samples for total element analysis 150 µL of H2O2  and 250 µL HNO3  were added per 10 mL sample. Samples used for total N and DOC measurement were frozen after the sampling. Samples for species analysis of As were preserved by immediate flash-freezing in dry ice on site.Pore water and gas samples were obtained from each mesocosm on a monthly basis between July and November, with the first sampling starting directly before Si addition to the Si treatment. To this end, gas samplers  were installed in the top peat layer just below the surface. The polysiloxane tubes were connected to impermeable polyurethane tubes and closed by three-way stopcocks for sampling following a sampling procedure described previously2 and CH4 in the gas samples, which were obtained from the diffusive equilibration samplers, were measured within three hours after the sampling by a gas chromatograph . Concentrations of the dissolved gas in the pore water were recalculated applying the Henry’s law and a temperature correction for in-situ temperatures51. The soil samples were freeze-dried and then ground to fine powder with a zirconium ball mill. Subsequently soil samples were digested in 3 ml of HNO3 and 2 ml H2O2 using a CEM Mars5 microwave digestion system . An ICP-OES  was used to measure total Si concentrations in the pore water samples and in the soil samples after extraction in a 0.1 M Na2CO3 solution at 85 °C for five hours according to Struyf, et al.52. Total dissolved element concentrations in pore water and soil extracts of P, manganese (Mn), iron (Fe), cobalt (Co), zinc (Zn), and As were analyzed by a quadrupole ICP-MS . The elements Mn, Fe, Co, and Zn were measured in KED mode , whereas P and As were measured in oxygen mode (10% oxygen in helium as a reaction gas). Total element concentrations of N and DOC concentrations in pore water were measured with a TOC-Analyzer  with an integrated total N measuring unit. The As speciation analysis was done using an ion chromatography  coupled to a quadrupole ICP-MS . An alkaline eluent with a gradient of 2.5–100 mM NaOH was used at a constant flow rate of 1.2 mL/min as described before53. For quantification, commercially available salts of arsenite, arsenate, monomethylarsenate (MMA), and dimethylarsenate (DMA) were used. Mono-, di- and trithioarsenate were quantified via the arsenate calibration. The validity of this approach has been confirmed before53.Concentrations of COt-test and a multifactorial analysis of variance (ANOVA) of the factors treatment (high and low Si availability) and time (month) were applied using SPSS version 16.01.A Supplementary Information


Our study was aimed to evaluate the risk of a selected non-motor symptom, namely rapid eye movement behavior disorder (RBD) symptoms, among patients with newly diagnosed Parkinson disease compared with health controls.rd, 2016. Eligible studies were those that reported a prevalence of RBD symptoms among newly diagnosed PD and health control. Pooled odds ratios (ORs) with 95% confidence intervals (CIs) were calculated by random-effected models. Heterogeneity across studies was assessed using Cochran Q and I2 statistics.The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines for meta-analysis and Cochrane manual were followed. Studies on RBD symptoms and PD were searched using PubMed, Embase, Web of Science and Cochrane library databases. All studies were published before August 3p = 0.001) with a moderate heterogeneity I2 = 70.5%. After excluding the study of low weight, the overall polled OR was 3.54  and the heterogeneity was completely eliminated (I2 = 0%).We identified eight studies including 2462 PD patients and 3818 health controls. The overall prevalence of RBD symptoms in PD was 582/2462 (23.6%) compared to 131/3818 (3.4%) in control. And the pooled OR was 5.69 (95% CI 3.60 to 9.00; RBD symptoms are common non-motor symptoms of PD, and people with PD are at a higher risk of developing RBD. Further studies are needed to understand the natural history of RBD symptoms in PD and its etiological and clinical implications. Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease with a prevalence of approximately 0.4% in people aged 65 and older, and it increases with age . Apart frd, 2016. We combined medical subject heading (MeSH) terms including “PD, RBD” and text terms including “Idiopathic Parkinson's Disease, Lewy Body Parkinson Disease, Primary Parkinsonism, Parkinsonism, Primary Parkinson Disease, Paralysis Agitans” and “REM Behavior Disorder, Rapid Eye Movement Sleep Behavior Disorder” as our search strategy. Restrictions were made to observational epidemiologic studies involving humans. Titles and abstracts were screened for their suitability. Articles whose abstracts did not report on RBD symptoms and PD were excluded. Full articles were then obtained and reviewed to determine suitability for inclusion or exclusion. Differences of opinion between the researchers were resolved through discussion. The reference lists of all full articles were included. In addition, references from reviews that were identified in the original study, were hand-searched as additional articles, and then subjected to the same filtering process as described above.We conducted a systematic search of original published studies that reported the prevalence of RBD symptoms in PD and health controls. Two researchers (JZ and CYX) independently undertook a search on PubMed, Web of Science, Embase and Cochrane library databases for literature published in English, which were published before August 3Published articles that met the following criteria were included: 1) observational studies with a cohort, case-control or cross-section design; 2) cases where patients were diagnosed with PD according to standard clinical criteria, such as UK Parkinson’s Disease Society Brain Bank Criteria ; 3) contWe excluded studies for the following reasons: 1) studies in the form of case report, review, conference abstracts, or letters that did not report new data; 2) studies that only provided risk estimates such as relative risk without numbers of individuals; 3) duplicate populations; 4) did not provide adequate details of the control group; 5) did not report sufficient data to calculate risk estimates; and 6) non-English publications or non-human studies.Two researchers (JZ and CYX) reviewed all abstracts independently either to determine the eligibility criteria or for examining the appropriateness on the research issue. Data were compared and disagreements were resolved by a consensus. The following information was extracted from each study, and included: name of first author, year of publication, study data, study design, sample size, patient mean age, geographical region, RBD symptoms assessment method and other relevant study characteristics. Finally, studies were assessed for quality using the Newcastle-Ottawa Scale (NOS) .2 test, the percentage of I2 around 25, 50 and 75% mean low, medium and high heterogeneity [Measures of effect were combined using standard meta-analysis methods. We used the odds ratio (OR) with a 95% confidence interval (CI) as the metric of risk, using the random effects model. Heterogeneity across studies was evaluated by the Cochrane’s Q statistic and by Iogeneity . A sensiogeneity . StatistThe literature search yielded 749 records (include three studies from hand searching of references) after duplicates were removed. Of these, 673 records were excluded on the basis of their title and abstract. Review of the remaining 76 full articles led to 68 exclusions based on criteria described above. Finally, eight studies fulfilled our inclusion criteria and were included in our meta-analysis with a total number of 2462 PD patients and 3818 health controls   and 4.14 for “Definite RBD” (95% CI 2.21 to 7.78) with medium heterogeneity . After excluding the study of low weight [2 = 0%; p < 0.00001). Visual inspections of the funnel plot revealed a little asymmetry .The overall prevalence of RBD symptoms in PD was 582/2462 23.6%) compared to 131/3818 (3.4%) in control. And the pooled OR was 5.69 (95% CI 3.60 to 9.01) with a moderate heterogeneity , and the association between PD and the risk of developing RBD symptoms was still statistically significant.The results of our meta-analysis show a definite association between PD and RBD symptoms. Since studies included were of different study design, population bases and methods of exposure ascertainment, the heterogeneity across the studies was high. To identify studies that may have contributed to the pooled heterogeneity, we removed each of the eight studies (one study at a time) and examined the magnitude and direction of the polled relative risk (RR) after the removal of each study. This approach was repeated until the I studies , 19, 21 Our systematic review and meta-analysis offers a confirmation of the association between PD and a subsequent diagnosis of RBD. The pooled prevalence of RBD symptoms in PD and control is 23.6 vs. 3.4%, and the patients who had symptoms of PD carried a 3.60 to 9.00-fold increased risk for developing RBD compared to health controls. It is important to quantify the magnitude of the association between PD and subsequent RBD symptoms, as this may underpin efforts to identify higher risk participants for entry to interventional studies with neuroprotective aims.REM sleep behavior disorder (RBD) is a parasomnia disorder characterized by repeated episodes of dream enactment behavior and REM sleep without atonia (RSWA) during polysomnography (PSG) . The maip = 0.019; Egger test: p = 0.011). This might result in an overestimation of the association between PD and the risk of RBD symptoms. Considering that we only included eight studies which were assessed via means of the NOS, and all studies included in the main analysis had scores ≥ 7/9, we can conclude that the risk estimate that resulted from this analysis may also be viewed as a fairly strict estimate, and a result of the pooling of data from only the highest quality studies.Our study demonstrates that there is potential for publication bias based on the funnel plot and significant publication bias was detected from results of the statistical test (Begg test: Similar to any other meta-analysis of observational studies, our study is subject to several limitations. First, we limited our search to publications in English and therefore might have missed a small number of relevant publications. Second, recall bias may affect the quality of information retrieved from participants in case-control studies and the assessment of RBD symptoms may vary from one study to another. Third, more high quality and large sample sizes observation studies estimating the risk of RBD symptoms in PD should be included in order to avert the publication bias.In conclusion, despite these limitations, we pooled data from 6280 people across eight studies to provide a consolidated risk that estimated relating PD to the development of RBD symptoms. Most of the evidence was of the highest quality and the conclusion was considered robust. Further prospective research is needed to examine the presence of RBD symptoms and its relationship to PD, in order to better understand the etiology and natural history of PD.


Lucilia sericata is the primary causative species. Despite its severity, there has been minimal investigatory work into the disease in rabbits. Here we used text mining to screen electronic health records (EHRs) from a large sentinel network of 389 veterinary practices in GB between March 2014 and April 2017 for confirmed cases of blowfly strike in rabbits.Blowfly strike is a devastating and often rapidly fatal disease in rabbits. In Great Britain (GB), Blowfly strike was identified in 243 of 42,226 rabbit consultations (0.6%), affecting 205 individual rabbits. The anatomical site of recorded blowfly strike lesions was overwhelmingly the perineal area . Less commonly lesions were observed affecting other areas of the body  and head ; in 83 consultations (39.9%), the affected area was not specified. Of the rabbits presenting with blowfly strike, 44.7% were recorded as being euthanized or died.A case control study was used to identify risk factors for blowfly strike in this population. Whilst sex and neuter status in isolation were not significantly associated with blowfly strike, entire female rabbits showed a 3.3 times greater odds of being a case than neutered female rabbits. Rabbits five years of age and over were more than 3.8 times likely to present for blowfly strike. For every 1 °C rise in environmental temperature between 4.67 °C and 17.68 °C, there was a 33% increase risk of blowfly strike, with cases peaking in July or August. Overall blowfly strike cases started earlier and peaked higher in the south of Great Britain. The most northerly latitude studied was at lower risk of blowfly strike than the most southerly . There appeared to be no significant relationship between blowfly strike in rabbits and either the sheep density or rural and urban land coverage types.The results presented here can be used for targeted health messaging to reduce the impact of this deadly disease for rabbits. We propose that real-time temporal and spatial surveillance of the rabbit disease may also help inform sheep control, where the seasonal profile is very similar, and where routine surveillance data is also not available. Our results highlight the value of sentinel databases based on EHRs for research and surveillance. RabbitsThe disease also heavily impacts on sheep, and is associated with a heavy economic burden to the industry, estimated at $280million dollars per annum in Australia . In GB, Lucilia sericata, the Common Green Bottle Fly , which can be collected at large temporal and spatial scales and in real-time from sentinel networks of veterinary practices, have been used to provide new insight into population health for a range of diseases and syndromes , and inc22.1Electronic health records (EHRs) were collected in near real-time through SAVSNET from volunteer veterinary practices across GB; a full description of the data collection protocol has been described elsewhere . BrieflyIn order to explore the seasonality and risk factors for blowfly strike, we defined cases as those animals that were presented for consultation with active blowfly strike lesions, visually confirmed and recorded by the attending veterinary practitioner.https://en.wikipedia.org/wiki/Regular_expression) to detect the word blowfly strike, identified term variants and misspellings. The final regex (Supplementary figure) when applied to the full SAVSNET rabbit database of 42,226 rabbit consultations identified 443 consultations. This data was manually read by two authors (RT and EA), identifying 243 consultations (54.8% of those extracted by the regex) that satisfied the case definition of active blowfly strike disease. Of these 243, any rabbits that had visited the veterinary practitioner more than once in a month were considered to be suffering from the same episode of blowfly strike and thus only the first consultation was kept. Furthermore, rabbits with incomplete data (e.g. age or owner postcode not recorded) were also excluded from analyses. The final dataset consisted of 205 rabbits presenting with blowfly strike; three of these rabbits had two separate blowfly strike episodes longer than one month apart giving a total of 208 episodes of blowfly strike  were randomly chosen from those rabbits that had never presented for an episode of flystrike .2.2For each case and control, the unique consultation ID was used to extract the rest of the information for that animal, including the postcode of the owner, the breed, sex, neuter status and date of consultation. Age was calculated as the difference in years between the date of birth and the date of consultation of each animal, and was categorised into quintiles . Manual The SAVSNET database also collects the breed of the animals. Some breeds have longer coats or more dense fur, which may predispose them to faecal soiling . However2.3http://worldclim.org/version2 , spring (March-May), summer (June-August) and autumn (September to November). The monthly average (night and daytime) temperature (°C) and precipitation (mm) were assessed, to further quantify the influence of climate on the occurrence of blowfly strike . For eacversion2 .Furthermore, the spatial risk of blowfly strike was evaluated based on the coordinates  of owner postcodes. Latitudes were divided into five equal quintiles, each containing 20% of the cases, with Lat1 being the most southerly quintile. For each quintile the number of blowfly strike consultations per one thousand total rabbit consultations was calculated for each month.https://www.ceh.ac.uk/services/land-cover-map-2015. More precisely, the dominant cover of GB is divided into ten classes, i.e. 1 = broadleaf woodland, 2 = coniferous woodland, 3 = arable, 4 = improved grassland, 5 = semi-natural grassland, 6 = mountain, heath, bog, 7 = saltwater, 8 = freshwater, 9 = coastal, 10 = built-up areas and gardens  at: 2.4Descriptive statistics were used to characterise key demographic variables of the blowfly strike-visiting rabbit population. Univariable logistic regression was conducted utilising case control status as a binary outcome variable. Every explanatory variable was explored, with a likelihood ratio test (LRT chi-squared test) being used to assess fit compared to a null model. Explanatory variables with an LRT of p-values ≤0.2 were included in an initial multivariable logistic regression model. Interactions between sex and neuter status were included in the initial multivariable logistic regression model. A backwards explanatory variable selection process was utilised in order to produce a model fit with the lowest Akaike information criterion (AIC) possible, with explanatory variables being removed if their inclusion resulted in an increase in AIC, and by allowing the linear model to be related to the response variable via its ‘logit’ link function. The following assumptions were made: response variable was binary, explanatory variables were linearly related to the log odds, explanatory variables were independent and there was no multicollinearity among the independent variables. All assumptions were checked and verified before analysis. Regression results were considered significant for p-values ≤0.05. All analyses were carried out using R language (version 3.2.0) R Core . Spatial3A total of 42,226 EHRs were collected between 25th March 2014 and 29th April 2017, from a total of 389 SAVSNET-participating veterinary practices throughout GB; the distribution of these practices is shown in The anatomical site of recorded blowfly strike lesions was overwhelmingly the perineal area , including tail base, perineal, anal, vulva, penis and scrotum. Of these animals, 33 recorded either feacal soiling or urine scalding and 10 noted the rabbit suffering from paralysis or lameness of the hind legs. Those consultations that recorded lesions on the head  were always accompanied with recorded ocular and/or nasal discharge. There were nine cases (4.3%) that were on non-perineal areas of the body such as the abdomen (n = 3) and flank (n = 6). In 83 (39.9%) consultations, the affected area was not specified. Additionally, among the affected rabbits, 13 were described as overweight (6.25%).Of the 208 consultations categorised with blowfly strike, 92 recorded that the animal was euthanised and in one, the affected animal was dead on arrival giving a total of 44.7% of blowfly strike consultations leading to the death of the animal.3.13.1.1The age profile of affected rabbits is shown in 3.1.2As shown in The recorded episodes of blowfly strike consultations were in England , Wales  and Scotland  A.The seasonality by latitude is shown in Compared to latitude 1, the risk of blowfly strike was only significantly different (lower) for latitude 5  . For eveUrbanisation and sheep density had no significant association with blowfly strike risk .3.2Multivariable logistic regression showed the significant predictors of blowfly strike in this population were the rabbit’s age, the neuter status of female rabbits and the average monthly temperature . This increased to 4.78 times for rabbits aged over seven years . Compared to female neutered rabbits, female entire rabbits showed a 3.30 greater risk ; male entire and male neutered rabbits were not associated with altered risk to neutered does.Analysis of temperature showed that, on average, each 1 °C increase in average monthly environmental temperature (4.67 °C–17.68 °C) resulted in a 33% increase in odds of blowfly strike . Only 11 consultations from the 208 were reported at times when the average monthly environmental temperature was below 8.5 °C, including one in March, two in April, and seven in November and December.4Blowfly strike is recognised as an important welfare concern in both rabbits and sheep. However, the historical absence of routinely collected population health data for companion animal species, has led to an overall lack of understanding of important risk factors for the disease in rabbits. Here we have taken a novel approach to fill this gap using clinical narratives from EHRs available at large scale, confirming blowfly strike to be important in this population (0.6% of consultations of the SAVSNET network in GB), and identifying key risk factors that could inform improved targeted health messaging and control strategies.Among the animals identified with confirmed blowfly strike in this population, 44.7% of cases ended in euthanasia or the death of the animal, much higher than the 2.7% reported for sheep . BlowflyThe age of a rabbit was shown to have a significant association with the risk of blowfly strike in this population, with those aged five years and over being greater than 3.8 times more likely to present for blowfly strike than rabbits less than one year old. Older rabbits are likely to suffer more from lumbar spondylosis , dental problems, decreased caecotrophy and less activity, and as a consequence, are less able to efficiently groom themselves . It is aSex and neuter status were not independently associated with blowfly strike risk. However, when combined, entire female rabbits were 3.30 times more likely to suffer blowfly strike than neutered females. Currently we can only speculate on the causes underlying this observation. The rabbit breeding season occurs simultaneously with the main blowfly strike season in GB and female rabbits (does) can have multiple litters in one season . HoweverUnsurprisingly, average monthly environmental temperature showed a stronger association with blowfly strike than any other environmental or geographical variable examined, explaining the pronounced seasonality of blowfly strike in the study population, with the majority of cases occurring in summer when the average monthly temperature is highest. Warmer climate in southerly latitudes of GB likely explains the earliest cases being recorded in the most southerly quintile in May. The more southerly regions also appeared to have a longer recorded blowfly strike season, and a higher proportion of cases during their peak months.The geographical and seasonal patterns observed here in rabbits are consistent with those observed for sheep. A retrospective farmer survey showed the proportion of farms with at least one case of blowfly strike was highest in south-west England and lowest in Scotland . The firLucilia cuprina (a relative of L. sericata) showed that flies can cause strike in sheep independent of temperature and high fly abundance, suggesting the availability of organic matter is a more important driver of blowfly strike development than environmental temperature for breach strike in some sheep (Some cases of rabbit blowfly strike were identified in May in northerly latitudes and in November and December, when average environmental temperatures were below the predicted threshold for oviposition of 8.5 °C . These cme sheep . It is pUnlike temperature, precipitation was not deemed significant in the multivariable model, suggesting perhaps not surprisingly that all months have sufficient rainfall in GB to create the local humidity necessary for blowfly strike to occur . Lack ofAlthough EHRs can provide new insight into disease, their use has certain limitations. Despite having a highly efficient system for extracting cases of blowfly strike from the SAVSNET database, some true cases of blowfly strike were likely missed either because they were not recorded, or because they were recorded in a way not picked up by the regex used. As such the level of disease reported here is likely to be an underestimate of the true incidence of blowfly strike. A large proportion of consultations in the study population lacked recorded breed information. This level of missing data is much higher than seen for cats and dogs , and sugIn conclusion, this is the first study to confirm a countrywide seasonality of blowfly strike in rabbits. We have found that the main predictive risk factors for blowfly strike are increased age, being an entire female rabbit and increased average monthly temperature. Our use of the SAVSNET data illustrates the utility of large databases of EHRs to investigate disease burden, especially in companion animals, which otherwise lack coordinated disease surveillance. The results presented here can be used to generate targeted health messages for risks of blowfly strike aimed at rabbit owners and veterinary practitioners.RT designed and planned the study, performed all preliminary data analyses and drafted the manuscript. EA, PJ and DS finalised the statistical and epidemiological analyses. AR conceived and helped design the study, and recruit practices, and wrote the final manuscript. AR, BB, PJ and PJN helped fund the project, collected the data and participated in the epidemiological analyses. All authors read, edited and approved the final manuscript.The authors declare that they have no competing interestsThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.


Chlamydia trachomatis infects the urogenital tract (UGT) and eyes. Anatomical tropism is correlated with variation in the major outer membrane protein encoded by ompA. Strains possessing the ocular ompA variants A, B, Ba and C are typically found within the phylogenetically coherent “classical ocular lineage”. However, variants B, Ba and C have also been found within three distinct strains in Australia, all associated with ocular disease in children and outside the classical ocular lineage. CtGEM genotyping is a method for detecting and discriminating ocular strains and also the major phylogenetic lineages. The rationale was facilitation of surveillance to inform responses to C. trachomatis detection in UGT specimens from young children. CtGEM typing is based on high resolution melting analysis (HRMA) of two PCR amplified fragments with high combinatorial resolving power, as defined by computerised comparison of 65 whole genomes. One fragment is from the hypothetical gene defined by Jali-1891 in the C. trachomatis B_Jali20 genome, while the other is from ompA. Twenty combinatorial CtGEM types have been shown to exist, and these encompass unique genotypes for all known ocular strains, and also delineate the TI and T2 major phylogenetic lineages, identify LGV strains and provide additional resolution beyond this. CtGEM typing and Sanger sequencing were compared with 42 C. trachomatis positive clinical specimens, and there were no disjunctions. CtGEM typing is a highly efficient method designed and tested using large scale comparative genomics. It divides C. trachomatis into clinically and biologically meaningful groups, and may have broad application in surveillance. Chlamydia trachomatis encompasses four major evolutionary lineages [Whole genome-based studies have shown that lineages –4. The eC. trachomatis in accordance with variation in ompA [OmpA confers variation in immunoreactivity, is associated with tropism towards the urogenital tract (UGT) or ocular sites, and pathogenic properties. Specifically, the “LGV” ompA genotypes L1, L2, and L3 are LGV associated, the UGT genotypes D, E, F, G, H, Ia, J, Ja and K are associated with the non-invasive C. trachomatis STIs, and the “ocular” genotypes A, B, Ba and C are associated with trachoma and similar ocular disease exhibiting characteristic “face to face” transmission amongst children. There is significant ompA homoplasy with respect to evolutionary lineages defined by genome wide SNPs [ompA genotypes B, Ba and C are not only found in the classical ocular lineage, but also in other lineages isolates from northern Australia, from children with ocular disease, and strongly associated with the ocular site [ompA genotypes Ba and C (termed AusBa and AusC). On the basis of genome wide orthologous SNPs, these are closely related to each other within the T1 lineage and also closely related to UGT strains of ompA genotypes E and F. The third Australian strain is ompA genotype B (termed AusB). AusB is unrelated to AusBa and AusC and is within the T2 lineage, closely related to UGT strains of ompA genotypes G, H, K and D. A very recent large-scale study of global C. trachomatis genome diversity has shown that while the AusBa and AusC clones have as yet been found only in Australia, isolates essentially identical to AusB have been found in Finland and The Netherlands [It has long been common practice to define sub-groups within the species  in ompA –8 which ide SNPs , 9. Of pC. trachomatis genotyping method that i) discriminated ocular lineages from UGT lineages, ii) discriminated the classical ocular lineage and the three Australian ocular lineages from each other, and iii) discriminated the major evolutionary lineages of the entire species. The proximate rationale was the enhancement of the evidence base for interpreting instances of C. trachomatis detection in young children in remote northern Australia. The issue of child sexual abuse in remote northern Australia has a high public profile and is socio-politically contentious [C. trachomatis in UGT specimens in the adult/post pubescent population will provide a comparator for genotypes found in young children, when instances of possible abuse are being investigated [C. trachomatis from a paediatric UGT specimen does not provide evidence for contact with adult sexual networks and may be suggestive of autoinoculation, or of similar processes that drive trachoma transmission. Conversely, if nominally ocular strains are prevalent in sexual networks, then the genotype of the strain in a paediatric specimen is less informative of origin [The objective of this study was to develop an efficient tentious . Reportstentious . Howevertentious . While ttentious –14, it istigated , 16. Of stigated , 17, 18.f origin .The technology platform chosen was high resolution melting analysis (HRMA) –21, becaC. trachomatis genomes was mined for SNP combinations with high Simpsons Index of Diversity using the software Minimum SNPs [C. trachomatis B_Jali20 genome [ompA. This led us to conjecture that a method encompassing both these loci would identify ocular strains and discriminate major phylogenetic groups. The SNP at position 222834 is within a hypothetical gene  that is annotated as encoding a hypothetical protein that is likely membrane located [The conceptual and technical basis of the method resembles “Minim typing’ which makes use of HRMA of fragments within multilocus sequence typing (MLST) loci that are nucleated by SNPs selected for high combinatorial resolving power –25. Howemum SNPs , 27. It 0 genome  , and refer to the genotypes from this method as CtGEM types 1–32. Detailed information regarding the CtGEM types, and their relationship to the phylogeny and ompA allelic status are provided in We elected to number the rg1-ofr combinatorial genotypes from 1–32, in accordance with all possible combinations of the four HRMA alleles of rg1 and eight HRMA alleles of the ofr, even though all 32 genotypes may not exist in nature. We named the procedure “in silico the performance of CtGEM typing across known diversity of the species. First, we determined how the genotyping assay would be predicted to resolve the 65 genomes used to design the CtGEM typing. It can be seen in CtGEM types defined by these genomes encompass four CtGEM types containing only “classical ocular” genomes, and three CtGEM types containing only Australian ocular B, Australian ocular Ba, and Australian ocular C genomes. In intuitive terms, rg1 resolves C. trachomatis into the T2 lineage (haplotype GA), the T1 lineage , and the classical ocular lineage plus the L1 and L2 lineages (haplotype GC). It is fortuitous that the combinatorial resolving power of the two fragments is what is required for the method to meet the performance requirements. In particular, the Australian ocular genomes are resolved from their close UGT-associated relatives on the basis of ompA HRMA types. Also fortuitous are the distinctive ompA HRMA curves we obtained from ompA genotypes E, A and L1/L2. Upon careful analysis of the sequence data we noted that the LGV associated L1-440 has an aberrant ofr sequence that is predicted to yield an HRMA curve indistinguishable from the ofr Ba+D curve. Because L1-440 possesses the rg1 GC haplotype, this strain is predicted to be CtGEM type 25, as for ompA genotype Ba strains in the classical ocular lineage, and accordingly should be regarded a false positive for an ocular strain.The availability of many whole genome data sets enabled us to assess in silico experiments, we extended the analyses to a much larger recently published set of whole genome information of C. trachomatis isolates, comprising the 65 genomes we had already considered and 494 additional genomes , as does AusB. OmpA genotype D isolates NL4, NL5, NL6, NL8, NL19, Fin163 and S2761 have the rg1 haplotype GT (CtGEM type 9), as does AusBa. OmpA genotype J strains J_NL55 and J_Sou106 possess the rg1GT haplotype (CtGEM type 11), as does AusC. No novel SNPs were identified in region 1. Novel SNPs in the HRMA fragments were extremely rare and in only one instance was predicted on the basis of G+C content to impact the HRMA curve. This was strain S4641, which possesses a novel ompA sequence for which it is unclear whether to classify as genotype E or genotype G. The ofr sequence is predicted to yield a curve indistinguishable from ompA genotype G. The rg1 haplotype is AT, so the predicted CtGEM result is type 24. Strain S4641 is the only known or putative representative of CtGEM type 24.For the second stage of the  genomes . Two queng ARIBA . The isoCtGEM false positives for ocular strains is 12; one from the 65 genome set and a further 11 from the additional 494 genomes, as indicated above. Of the 559 genomes in the total set, 89 have ocular ompA genotypes, so there are 470 true negatives for ocular strains. Specificity = true negatives/, so this equates to a specificity of 0.975 if CtGEM typing were used to screen the 559 genomes for ocular strains. Six of the false positives are “NL” strains from the Netherlands. The equivalent specificity for “NL’ designated isolates alone is 0.902, showing that there is potential for regional variation.The number of predicted The inferred sensitivity for ocular strains is 1.0, primarily because of absolute linkage between the classical ocular and L lineages, and the rg1 GC haplotype.CtGEM typing enables screening for ocular strains with high sensitivity and specificity, and also provides a low resolution but robust indication of evolutionary position as defined by genome wide orthologous SNP-based phylogenetic inference.It was concluded that CtGEM typing was tested using 113 C. trachomatis positive UGT clinical specimens, obtained from public pathology service providers in the Australian state of Queensland. To generate a gold standard comparator, the regions subjected to HRMA were first analysed by Sanger sequencing of PCR fragments, in a different laboratory to where the CtGEM typing was performed. For both targets, specimens were tested either once or twice. For rg1, a subset of specimens where genotyping failed was re-tested, but this did not increase the success rate. For ofr, a subset of specimens that initially failed to be genotyped was re-tested using a reaction that incorporated a small reduction in the primer annealing temperature . This resulted in six initially “fail” specimens being successfully genotyped.The robustness of CtGEM types obtained and their relative abundances were similar to that shown by the in silico analysis of genome sequences. The predominant mode of failure was complete amplification failure, and there was considerable consistency between failure of HRMA and failure of sequence determination  ompA genotypes.Not only does CtGEM typing has limitations. First, given that the method is based on known genetic variation, it is inherently vulnerable to being compromised by undiscovered variation. However, the recently publication of hundreds of C. trachomatis genome sequences from all over the world provided an extremely valuable resource [CtGEM typing and population structure defined by the smaller set of genomes used to design the method remains valid with the larger set of genomes. Also, while there is concordance between CtGEM type relative abundances in the global set of genomes, and clinical specimens from Queensland, the global set of genomes contained no strains of ompA genotype Ja, whereas these are quite common in the Queensland set. Further, one Queensland ompA genotype Ja strain possessed a rg1 GT haplotype (as opposed to the expected T2 associated GA haplotype that was found in the other Queensland ompA genotype Ja strains). The combination of rg1 haplotype GT and the ofr CHIaJJaK allele is CtGEM type 11, the same as the AusC strain. Therefore this strain constitutes a single false positive for the ocular AusC isolates, that is absent from the global set of genomes [It is inevitable that such a streamlined method as resource . We demo genomes . As discC. trachomatis strains.The global set of genomes defined only a small number of additional SNPs in the regions subject to HRMA, and these were in very rare strains, The predicted effects on HRMA are to produce novel curves rather than to result in HRMA allele mis-calling. It may be that in the future the CtGEM allele designations and genotype numbering system will be expanded. However, the current protocol is predicted to be valid for the vast majority of extant CtGEM data analysis protocol specifies that ompA genotypes C, H, Ia, J, Ja, and K are not discriminated from each other. We regard this as acceptable because these ompA variants are closely related [ompA genotypes in this group (i.e. not C) are associated with the T2 lineage and appear to have similar distributions and ecology [ompA genotypes C, K, Ja, and H+Ia+J . However, we have taken a conservative approach and not incorporated this into our protocol for data analysis, primarily because this still fails to provide 100% specificity for ompA genotype C. We therefore suggest sequence analysis of the ofr if more discrimination is needed, in particular when confirming the identification of an ompA genotype C strain strain.The  related –43 and t ecology , 3. Sequd H+Ia+J . OmpA geompA genotype Ia, which has an identical ofr sequence to genotypes H and J). The ofr from ompA genotype A has an identical sequence to ompA genotypes H, Ia, and J except for a T→C substitution at ofr position 140, and although there is evidence for an early melting domain from ompA genotype A, it is much less conspicuous a than in HRMA curves from C, H, Ia, J, Ja, and K. This suggests that the existence of the conspicuous early melting domain is largely dependent on a T at position 140. The association between the allelic state of the position 140 T-C SNP and the nature of the early melting domain is only seen with the closely related ompA genotypes A, C, H, Ia, J, Ja, and K derived ofr sequences. Apart from the ofr derived from ompA genotype L3, all other known ofr variants possess a T at position 140, and yet do not have the ofr early melting domain. This is explicable as there are many differences between ofr sequences from ompA H, Ia, J, Ja, K, and the other variants of ompA, which could easily change the melting domain structure. Further, there was complete concordance between our observations of early melt domain magnitude and presence/absence, and in silico prediction using the software uMELT [A critical aid to the interpretation of the ofr HRMA is a conspicuous early melting domain in the ofr’s from genotypes C, H, Ia, J, Ja and K  were obtained from a study of mother and child pairs conducted in the Northern Territory, Australia between 1985 and 1993 [3 culture flasks at 80–100% infectivity stored at -80°C using a QIAamp DNA Mini Kit .and 1993 . DNA wasC. trachomatis reference strains A2497 [Chlamydia Biobank, University of Southampton, UK. The strains were cultured in HeLa cell monolayers which were propagated in DMEM supplemented with 10% foetal calf serum. The inocula were centrifuged onto a confluent monolayer at 750xg for 30 minutes at room temperature. Infected cultures were grown in DMEM with 10% foetal calf serum plus 1μg/mL cyclohexamide and 20μg/mL gentamicin to 80–100% infectivity. Elementary bodies were harvested from three 25cm3 culture flasks by scraping the cells from the surface of the flask and centrifuging at 3000xg. The cell pellet was resuspended in cold PBS containing glass beads and vortexed at high speed for 1 minute to lyse the cells. Cell debris was removed by centrifugation at 250xg for 5 minutes and DNA was extracted from the elementary bodies in the supernatant using a GenElute Bacterial Genomic DNA Kit .Genome-sequenced ns A2497  and UCH1ns A2497  were obtC. trachomatis-positive diagnostic urogenital swab specimen, collected as part of a prospective study of C. trachomatis genotypes in the Northern Territory, Australia [PS297 is a ustralia . Royal DC. trachomatis DNA control .DNA from reference strain ATCC VR-902B was purchased as Amplirun® C. trachomatis-positive clinical samples from Queensland, Australia were collected as part of a study to understand current serovar and genetic differences in ocular and urine samples. Specimens were urine remnant DNA extracts from the Roche 4800 CT/NG test .C. trachomatis isolates [in silico, to determine a maximally efficient means of meeting the assay specifications. This process included manual investigations of the feasibility of PCR primer design, and the potential effects of other nearby SNPs which have the potential to add resolving power, or conversely, confound an HRMA assay if the SNPs are linked and have opposing effects on amplimer G+C content.We used a previously described genome-wide orthologous SNPs matrix inferred from whole genome sequencing data from 65 diverse isolates . The ortisolates , 27. ThiompA, and the other within a universally present putative membrane protein-encoding gene that is annotated as Jali_1891 in the genome of isolate B_Jali20, which we designated “region 1”. The format was a nested (region 1) or semi-nested (ompA) PCR in which the products of the first round PCR were subjected to a second round of PCR, followed by HRMA. For the region 1 fragment, an unlabelled probe and second round asymmetric PCR were used to facilitate discrimination of known variants.The optimised method encompasses the HRMA analysis of two DNA fragments, one within ompA fragment (ofr), the first round PCR contained 0.4μM of each primer and 1μl of DNA extraction. The second round PCR and HRMA reaction contained 1μl of the first round PCR product and 0.4μM of each primer. For the region 1 fragment (Rg1), the first round PCR contained 0.5μM of each primer and 1μl of DNA extraction. The second round PCR and HRMA reaction contained 1μl of the first round PCR product, 0.04μM of the forward primer, 0.4μM of the reverse primer, and 1μM of the probe. A detailed account of the assay design and method development is given in The primer and probe sequences, and PCR and HRMA temperature conditions are shown in Each region was initially amplified by PCR using the primers described in C. trachomatis phylogeny [in silico to determine their CtGEM type. A reference set of known genotypes from the 65 genomes [ompA fragment HRM amplicon. For rg1, the reference genotypes were 400bp which encompassed the 74bp of the region 1 HRM amplicon. Short read genome sequence data for individual isolates was aligned against the reference genotypes for ofr and rg1 using ARIBA [CtGEM fragments , yielding complete CtGEM predictions for 559 isolates.The original 65 genome dataset  and addihylogeny  were ana genomes  used in ng ARIBA . Of the ng ARIBA , 63 wereThis study was approved by the Human Research Ethics Committee of the Northern Territory Department of Health and Menzies School of Health Research (permit number 10–1337) and the Human Research Ethics Committee of the University of Queensland (permit number 2015000301).S1 FileCtGEM method.Additional information regarding development of the (DOCX)Click here for additional data file.S2 FileA detailed protocol for HRMA curve interpretation.(DOCX)Click here for additional data file.S3 FileRg1 sequences derived from the Queensland specimens.(TXT)Click here for additional data file.S4 FileompA sequences derived from the Queensland specimens.Partial (TXT)Click here for additional data file.S5 FileGenotypes inferred from HRMA, and HRMA raw data.(XLSX)Click here for additional data file.


Bacillus amyloliquefaciens TOA5001 improved the above mentioned characteristics of the male broiler breeders. Thus, B. amyloliquefaciens TOA5001 might improve the reproductive performance of male broiler breeders.This study aimed to investigate the effect of probiotics on the intestinal morphology, intestinal microflora, oxidative activity , and semen quality of male broiler breeders. For this, 180 Cobb male broiler breeders (60 weeks of age) were randomly distributed into two groups. The control group was fed a basal diet, and the probiotics group was fed basal diet supplemented with probiotics for 6 weeks. Probiotics containing  Antibiotics are used to improve the health and productive performance of animals used for human consumption; however, they also result in the emergence of drug-resistant microorganisms. The European Union has banned the use of antibiotics as growth-promoting agents in the poultry industry, and many countries are increasingly restricting the prophylactic use of antibiotics in animals raised for food. Therefore, the poultry industry is actively searching for alternatives to antibiotics, and many different functional preparations such as herbs, essential oils, organic acids, and probiotics have been tested.The sub-therapeutic use of antibiotics in animal nutrition has been found to be beneficial for the improvement of growth performance traits such as weight gain, feed efficiency, and mortality rate2. Supplementation of probiotics in the feed of chickens has been found to improve (i) growth and productive performances such as body weight, daily weight gain, carcass percentage, absolute organ weight and organ weight/body weight ratio13; (ii) nutrient digestibility16; (iii) intestinal microflora modulation19; (iv) pathogen growth inhibition16; (v) immunomodulation and gut mucosal immunity19; and (vi) antioxidant status20. Various microorganisms have been used as probiotics, including species of Bacillus, Bifidobacterium, Enterococcus, Lactobacillus, Lactococcus, Streptococcus, and numerous yeast strains. Although many studies have investigated the beneficial effects of probiotics in chickens, few have determined their effects on male broiler breeders. This study aimed to determine the effect of probiotics on the semen traits, intestinal morphology, intestinal microflora, and antioxidant status of male broiler breeders.Probiotics are living microorganisms that improve animal health when included in the diet. They act by balancing the intestinal flora, influencing intestinal villi, and improving nutrient digestion and absorptionThis study was conducted at a commercial poultry farm in Kagoshima Prefecture, Japan, and performed under the fundamental guidelines for the proper conduct of animal experiments and related activities at academic research institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology. This study was approved by the Ethics Committee of the Inatomi Animal Clinic .−1. The birds in group 1 were fed a basal diet, and those in group 2 were fed basal diet containing probiotics for 6 weeks. The probiotics  containing Bacillus amyloliquefaciens TOA5001 at 1 × 108 colony-forming units·g−1 in rice bran was supplemented to the feed at 0.2% (w/w). The basal diet consisted of commercially available antibiotic-free male broiler breeder feed . The basal diet was used in a mashed form and formulated to meet the nutritional requirements of 60–66-week-old male broiler breeders as per the guidelines of the Kagoshima Agricultural Economic Federation. The composition of the basal diet and nutrient content are shown in Table ad libitum.In this study, 180 Cobb male broiler breeders that were 60 weeks old were used. The birds were randomly divided into 2 treatment groups (groups 1 and 2) of 90 birds each. Before starting this study, semen of all birds were analysed and there were no differences in semen trait in each group. The birds were individually placed for 6 weeks in cages having the following dimensions: 30 cm × 37.5 cm × 52.5 cm × 52.5 cm. Nipple drinkers and trough feeders were included in the cages. All birds were housed in a windowless and environmentally controlled room, with the room temperature maintained at 22 °C to 24 °C. The illumination period was 16 h·d21. A total of 10 intact, well-oriented crypt-villus units were selected in triplicate for each intestinal cross-section. The villus height, measured from the tip to the crypt junction, and crypt depth, defined as the depth of the invagination between adjacent villi, were measured using Image-Pro Plus  as described in detail by Touchette et al.22. The villus height:crypt depth ratio was also calculated.After the semen, serum, and plasma samples were collected, all birds were killed at 66 weeks of age to collect intestinal segments. The intestinal segment samples were collected from the jejunum and ileum and, after their contents were flushed with physiological saline, the samples were submerged in a fixative solution  containing 3% glutaraldehyde, 2% paraformaldehyde, and 1.5% acrolein. They were then brought to the laboratory to determine the morphological changes. Three cross-sections for each intestinal sample were prepared after staining with azure A and eosin by using standard paraffin embedding procedures23. Gut contents (10 g) were aseptically collected from the dissected jejunum and ileum of each bird and placed in 90 mL sterilised physiological saline , and the pH was determined using a pH meter .The pH of the different parts of the gastrointestinal tract was measured for all birds as described previously24. Approximately 1 g of jejunal and ileal digesta was obtained from all birds and serially diluted 10-fold (from 10−1 to 10−7) with sterile physiological saline solution (0.9% NaCl) and subsequently homogenised for 3 min by using an ultra-turrax. Dilutions were then plated onto selective agar medium for enumeration of the target bacterial groups. Escherichia coli were grown on MacConkey agar . Lactobacilli were cultivated using de Man–Rogosa–Sharpe agar . Lactobacillus plates were incubated anaerobically, whereas E. coli plates were incubated aerobically at 37 °C for 24 h. Bacteria were enumerated by visual counting of colonies by using the best replicate set from dilutions that resulted in 30 to 300 colonies per plate. The microbial enumerations of jejunum and ileum were expressed as base-10 logarithm colony-forming units per gram of jejunum and ileum digesta.The microbial counts of jejunum and ileum were obtained as previously describedg for 15 min at 4 °C; the top yellow serum layer was pipetted into two 1-mL conical tubes and held at −80 °C. The α-tocopherol level in the serum was determined using high-performance liquid chromatography25.For the analysis of serum α-tocopherol concentrations, blood samples of all birds were collected from the wing vein and allowed to clot for 30 min. Blood clots were centrifuged at 3,000 × N,N-diethylparaphenylendiamine in acidic buffer. Such oxidant capacity is mainly attributed to hydroperoxides, with the contribution of other minor oxidant factors. The biological antioxidant potential (BAP) test evaluates the capacity of the plasma sample to reduce ferric ions to ferrous ions. BAP varies primarily as a function of the titres of the major oxidative barriers in the plasma . For the analysis d-ROMs and BAP, blood samples of all birds were collected from the wing vein in EDTA-containing blood collection tubes and centrifuged . The plasma supernatants were stored at −80 °C until assayed. The d-ROMs and BAP were determined using commercial kits  and FRAS4 , respectively26.The reactive oxygen metabolite-derived compound (d-ROM) test provides a measure of the whole oxidant capacity of plasma against 27. The spermatozoa density or sperm count in the semen was estimated using a colorimetric method28. The live and dead spermatozoa were differentiated by staining with eosin and nigrosine by using the method described by Swanson and Bearden29.The semen from all birds from each group was collected using the massage method as per the procedure of Burrows and QuinnU tests were performed using EZR software ; EZR is a graphical user interface for R . The significance level was set at p < 0.05.Mann–Whitney The villus height, crypt depth, and villus height:crypt depth ratio from each group are shown in Table Lactobacillus were significantly higher in group 2 than in group 1, whereas those of E. coli were significantly lower in group 2 than in group 1  such that the addition of 2 g·kg−1 of diet would yield 108 colony-forming units per kg diet. Probiotics are known to be efficacious in animals at the daily intake level of 107–109 microorganisms38.The major microbes used as probiotics in poultry production include B. amyloliquefaciens TOA5001 did not show any adverse effects on male broiler breeders. The structure and integrity of the intestinal epithelium are important factors contributing to gut health and subsequent digestive capacity. Villus height is generally recognised as a good indicator of the function and activation of intestinal villi39. Better villus height and villus height to crypt depth ratio suggest an improvement in nutrient digestibility and absorption capacity of the small intestine40. The present study showed that B. amyloliquefaciens TOA5001 improved the gut structure and resulted in a greater absorption surface, as indicated by the improved villus height and villus height to crypt depth ratio. The effects of probiotics on gut structure and integrity have also been reported in the literature. Jayaraman et al.41. and Xinjian et al.32, respectively, reported that the inclusion of B. subtilis and B. amyloliquefaciens in broiler diets led to better villus height and villus height to crypt depth ratio. These two factors have been shown to be related to the epithelial cell turnover42. Inflammatory responses induced by pathogens or their toxins might cause the rapid epithelial cell turnover43. Thus, the suppression of pathogenic bacteria by B. amyloliquefaciens TOA5001 might have resulted in the better villus height and villus height to crypt depth ratio.In this study, dietary supplementation with 44. Various studies have shown that probiotics can positively modulate the composition of the intestinal microflora of chickens via the stimulation of potentially beneficial bacterial populations and/or the reduction of potentially pathogenic bacteria45. In the present study, jejunum and ileum samples of birds fed a diet containing B. amyloliquefaciens TOA5001 had lower pH, higher Lactobacillus concentration, and reduced E. coli counts. The B. amyloliquefaciens TOA5001-mediated reduction in intestinal pH might be favourable for the colonisation of lactobacilli and the suppression of E. coli46.In the gastrointestinal tract, numerous microorganisms co-exist and constitute a symbiotic ecosystem in equilibrium47 indicated that irregular intestinal microflora can cause malabsorption of vitamins. In mouse, rotavirus infection was shown to cause acute diarrhoea and vitamin deficiency48. In the present study, serum vitamin E (α-tocopherol) concentrations were significantly higher in group 2 than in group 1. Probiotics have been shown to increase serum vitamin E concentration in cattle by improving the intestinal environment49. Thus, the probiotics containing B. amyloliquefaciens TOA5001 might improve the digestive health of male broiler breeders.Mitsuoka51. It also increased plasma BAP in sheep exposed to heat stress52. However, few studies have linked BAP and probiotic supplements in chickens. In the present study, although d-ROM was not markedly different between groups 1 and 2, BAP was significantly higher in group 2 than in group 1. These data indicate that male broiler breeders in groups 1 and 2 were under the same oxidative stress conditions, but probiotic supplements increased the antioxidative activity of the male broiler breeders. B. amyloliquefaciens TOA5001 supplementation was suggested to increase serum vitamin E concentrations and improve the antioxidative activity of male broiler breeders by increasing antioxidant absorption in the intestine.Reactive oxygen metabolites are produced as a by-product of oxidative metabolism or exposure to oxidants in food or the environment. Oxidant exposure leads to the production of toxic reactive oxygen species such as free radicals, which in turn modify biological macromolecules. Vitamin E is known as an excellent biological chain-breaking antioxidant that protects cells and tissues from lipid peroxidation induced by free radicals53. The results of the proportion of live sperms was in agreement with that of a previous study in which broiler breeders after zinc-induced moulting were fed diets supplemented with probiotics54. The observed improvement in sperm concentration in the B. amyloliquefaciens TOA5001-fed males might have been due to the enhanced availability of nutrients facilitated by more efficient nutrient absorption by the gastrointestinal tract. Furthermore, several studies have indicated higher antioxidant activity in chickens fed probiotic-supplemented diets55, and B. amyloliquefaciens TOA5001 improved the antioxidative activity of male broiler breeders in this study. Thus, the relationship between the improvement in the activity of antioxidants such as glutathione peroxidase (GSH-Px) and superoxide dismutase in B. amyloliquefaciens TOA5001-fed birds and spermatozoa production and maturation needs to be considered. High levels of GSH-Px are found in the testes, and they act as powerful antioxidants in developing spermatids and spermatozoa56. Spermatozoa are adversely affected by high concentrations of peroxides in the testes, semen, and uterovaginal sperm host glands58. In organs such as testes that have high metabolic rates, levels of antioxidants required to ensure the survival of spermatozoa in aerobic environments are high. Thus, the high density of spermatozoa and high proportion of live sperms recorded in B. amyloliquefaciens TOA5001-fed males in this study might be attributed to the influence on the antioxidant activity.In the present study, the sperm density and proportion of live sperms were significantly higher in group 2 than in group 1. The results of sperm density are in agreement with those of a previous study in which broiler breeders were fed diets supplemented with yeast cultureB. amyloliquefaciens TOA5001 improve intestinal morphology, intestinal microflora, oxidative activity , and semen quality (sperm count and liver sperm) of male broiler breeders. Therefore, their use would be advantageous to the poultry industry.Our results indicate that probiotics containing


P = 0.044). Our findings suggest that VDAC3 genetic variants may be associated with human sperm count.Voltage-dependent anion channel (VDAC) is a multifunctional channel protein across the outer mitochondrial membrane of somatic cells and participates in many physiological and pathophysiological processes. Up to now, only a few studies, including our previous studies, showed that VDAC exists in mammalian spermatozoa and is involved in spermatogenesis and sperm functions. There is no report about VDAC genetic variants in germinal tissues or cells. To investigate the possible association between VDAC genetic variants and human sperm quality, we performed semen analysis and variant Genotyping of VDAC3 subtype  of 523 Han-Chinese males with idiopathic infertility respectively by computer assisted semen analysis (CASA) and single nucleotide polymorphism (SNP) Genotyping assay. No significant association was found between rs7004637 and rs6773 genotypes and semen quality. However, the AG genotype of rs16891278 showed a significantly lower sperm concentration compared with the AA genotype ( Voltage-dependent anion channels (VDAC), also known as mitochondrial porins, is first identified in the outer mitochondrial membrane of Paramecium aurelia . Then itAlthough VDAC has been extensively studied, only a few studies focus on the localization and functions of VDAC in male germinal tissues and cells. A few studies now available about VDAC in mammalian reproductive tissues and cells have suggested that VDAC might play important roles in spermatogenesis, motility, capacitation and acrosome reaction , 14. TheIn three isoforms, VDAC3 is the most widely distributed in germinal tissues and cells, and different in pore-forming ability comparing with others. Furthermore, male mice lacking VDAC3 show markedly reduced sperm motility and are infertile despite having normal testes and sperm numbers . Taken tVDAC3 is located on chromosome 8p11.2, spanning 13.3 kbp and is made up of 9 exons . It is cThis study contained 523 ethnic Han-Chinese males with definite idiopathic infertility at 31.75 ± 5.47 years of age. The mean duration of sexual abstinence before semen collection was 4.88 ± 2.66 days. The sperm parameters between the stratification of selected characteristics are listed in Table P = 0.092). For the rs16891278, the AG genotype carriers had significantly lower sperm concentration compared with the AA genotype (P = 0.044), but no statistical significance was found regarding those with the GG genotype, as well as the combination of AG and GG genotype. For the rs6773, the semen volume, concentration, motility, VCL, VSL and VAP were not significantly different between the genotypes of the variant.Table This study represents the first attempt to investigate the impact of VDAC3 genetic variants on semen quality. Three tagSNPs  in the VDAC3 gene were selected using the tagSNP method. 523 Han-Chinese idiopathic infertility males were genotyped for these SNPs using TaqMan-based genotyping. Significant associations were detected between the genetic variant rs16891278 and sperm concentration. The other two SNPs do not affect semen quality. Since all the three SNP do not affect amino acid: rs16891278 (chr8:42401679–42401679) and rs7004637 (chr8:42392929–42392929) are both transcript variants occurring within the intron, while rs6773 locates at chr8:42405639–42405639 and is a UTR variant of the 3′ UTR. We suspect that rs16891278 may play a role at the transcription level. The characteristics of sperm number are one of the most informative parameters in semen quality analysis . As the This study found that the rs16891278 AG genotype carriers had significantly lower sperm concentration compared with the AA genotype. A possible explanation for this finding could be the participation of VDAC3 in the process of spermatogenesis. Outer mitochondrial membrane permeability is a central event in apoptotic cell death. VDAC interacted with Bcl-2 family members or with other proteins probably acts as a convergence point for a variety of life-or-death signals. When outer mitochondrial membrane permeability is increased, it releases several apoptogenic factors such as cytochrome c into the cytoplasm, which can activate the downstream destructive processes , thus VDAlthough, in the study of bovine sperm, VDAC3 protein is observed to be localized abundantly in bovine outer dense fiber (ODF) of the flagella , which iIn conclusion, our present study showed that VDAC3 genetic variant, rs16891278, is associated with human sperm concentration. To our knowledge, it focuses on the relationship between VDAC genetic variants and human semen quality for the first time. Our findings would be beneficial to find more functions of VDAC in male reproductive system, to search for the cause and mechanism of some clinical idiopathic male infertility, and to explore new diagnostic and therapeutic methods for patients with poor semen quality.From March 2014 to June 2015, a total of 523 ethnic Han-Chinese men visiting the Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University were consecutively recruited. They were all married and failed to conceive for at least 12 months. All patients were well-developed men with normal secondary sexual characteristics and testicular volume. The serum testosterone, LH, and FSH levels were within the normal range: testosterone = (3∼5.7) ng/mL, LH = (0.80∼5.10) mIU/mL and FSH = (0.80∼6.30) mIU/mL. Patients with orchitis, obstruction, cryptorchidism, varicocele, congenital bilateral absence of vas deferens, cytogenetic abnormalities, and Y chromosome microdeletions were excluded from the study after a complete historical and physical examination , 34. TheEach patient provided informed consent and completed a questionnaire including information about age, smoking, drinking habits and other lifestyle factors. Besides, each subject donated 5 ml of peripheral blood for genomic DNA extraction and an ejaculate for semen analysis. This study protocol was approved by the ethics review board of Nanjing Medical University.All participants gave written informed consent, and the study was approved by the Institutional Ethics Committee of Nanjing Medical University (Process n.2012/3).According to World Health Organization guidelines , the computer-assisted semen analysis system  was applied to perform the semen analysis. For the statistical analysis for an association with VDAC3 genetic variants, six parameters of semen quality were chosen: semen volume (ml) and sperm concentration (106/ml) (these two parameters represent sperm number); motility, curvilinear velocity , straight line velocity  and average path velocity  (these four parameters indicate the sperm motility characteristics). The sperm numbers and sperm motility characteristics provided a reliable estimation of the fertilizing ability of human spermatozoa . Strict http://hapmap.ncbi.nlm.nih.gov), the SNPs were selected within the 17023 bp human VDAC3 gene as well as 1500 bp upstream and 1500 bp downstream, which had a minor allele frequency > 0.05 in Han Chinese in Beijing and was pinpointed to chromosome 8 from 42367047 to 42384070, thirteen SNPs were captured in this region. A linkage disequilibrium (LD) plot of this region was made based on the r2 values, which indicate the ability of a certain SNP to predict another SNP [Using genotype data obtained from unrelated Han Chinese in Beijing individuals in the HapMap . TaqMan SNP Genotyping Assays were performed for genotyping using the Taq amplification method in a ABI Prism 7900 HT Fast Real-Time PCR system . PCR amplification was performed at 95°C for 10 minutes followed by 40 cycles at 95°C for 15 seconds, 56°C for 10 seconds and 60°C for 1 minute, with one additional cycle at 60°C for 10 minutes. Ten percent of study participants were randomly chosen and genotyped in duplicate to confirm the concordance of the genotyping results. In our study, all the call rates for these SNP genotyping were 100% and the concordance of duplicates was 100%.χ2 test was used to evaluate differences in selected demographic variables, smoking and alcohol status. Continuous variables, including age and BMI, were evaluated by the analysis of variance (ANOVA). All results were reported as the mean ± SD, and P < 0.05 was considered to be significant.Computations were carried out using the Stata statistical package . Sperm concentration was undertaken logarithmic transformation to achieve homogeneity of variance and normal distribution of residuals. No transformation was performed for the remaining parameters. For statistical analysis, a multiple linear regression analysis was applied on the comparison of semen parameters that was considered for the genotypes of each SNP. Age, smoking, drinking, body mass index (BMI) and the duration of sexual abstinence (Abs) were initially considered as potential confounders. The


Listeria monocytogenes serotype 4b variant (4bV) strains. Three of these incidents can be linked to a defined geographical region, while the fourth is likely to be linked. In this study, whole genome sequencing (WGS) of strains from these incidents was used for genomic comparisons using two approached. The first was JSpecies tetramer, which analyzed tetranucleotide frequency to assess relatedness. The second, the CFSAN SNP Pipeline, was used to perform WGS SNP analyses against three different reference genomes to evaluate relatedness by SNP distances. In each case, unrelated strains were included as controls. The analyses showed that strains from these incidents form a highly related clade with SNP differences of ≤101 within the clade and >9000 against other strains. Multi-Virulence-Locus Sequence Typing, a third standardized approach for evaluation relatedness, was used to assess the genetic drift in six conserved, known virulence loci and showed a different clustering pattern indicating possible differences in selection pressure experienced by these genes. These data suggest a high degree of relatedness among these 4bV strains linked to a defined geographic region and also highlight the possibility of alterations related to adaptation and virulence.Four listeriosis incidences/outbreaks, spanning 19 months, have been linked to  Listeria monocytogenes is the causative agent of listeriosis, an illness usually linked to the consumption of contaminated ready-to-eat (RTE) foods and afflicting the elderly, immune compromised, pregnant women and neonates. Historically, foods associated with cases of listeriosis have been deli meats and soft cheeses; however, a recent shift has been observed with an increasing number of outbreaks being linked to fresh produce and other atypical products [L. monocytogenes strains can be grouped into 13 different serotypes, over 95% of illnesses and food contaminations are linked to just four serotypes, 1/2a, 1/2b, 1/2c and 4b [L. monocytogenes isolated from food and food processing environments are typically serotype 1/2a [products –5. Whilec and 4b . Among tc and 4b . Conversype 1/2a –9. Recenype 1/2a . A uniquype 1/2a  and modiype 1/2a . This vaype 1/2a . By tradype 1/2a . For easype 1/2a . Data frype 1/2a suggests During 2014 through 2016, 4bV did appear to have a higher than anticipated frequency (two of nine outbreaks reported via CDC) compared to the low frequency observed in LeClercq et al’s study , 16. ExaEvaluation of the region flanking the 6.3 kb DNA segment identified an internalin protein in all strains examined that is significantly divergent in 1/2b and 4b strains . Additioin vivo, with the genes up-regulated within the mouse intestine, though lmo0737, the molecular target used in Doumith et al’s assay [Examination of the genes encoded by the 6.3kb segment identified the presence of a transcriptional regulator and genes putatively involved in ribose metabolism. Knockout mutants of this region in EGD-e, a 1/2a strain, found no effect on invasion, intracellular growth or growth in nutrient limitation, including ribose limiting conditions . However’s assay  that ide’s assay , 21.Until recently, such variants of serotype 4b have been reported from different parts of the world although never associated with any major foodborne outbreak –15. The In July 2014, there was a large recall of stone fruits (SF), linked to a company in the San Joaquin Valley, California, USA. During the investigation of this outbreak, four clinical cases were identified with matching PFGE patterns to the 4bV strain isolated from the fruit, suggesting a high probability of relatedness to the strains isolates from the fruits. WGS analysis identified two of these cases as highly related, though only one was linked via traditional epidemiology . For theListeria monocytogenes (Lm). Additionally, three of the four cases had Lm strains with PFGE patterns matching that of the 4bV strains suggesting the possibility of a bias for these strains to cause atypical infections.Later in 2014, an outbreak was linked to caramel covered apples (App) that were produced using apples grown in the San Joaquin Valley. The apples were found to be contaminated by two strains, one a 4b and the other a 4bV strain with the same PFGE pattern as the stone fruit incident. This outbreak had several clinical cases including three cases affecting young males, age 5–15, that were otherwise deemed healthy. A fourth case, affecting a male, age 18–25, was also identified though his overall health status was unclear. These cases didn’t fit the typical at risk population for The third cluster (Cl3) occurred in September 2015 and involved a cluster of nine cases of listeriosis that had WGS matches to FDA historical isolates from two firms in the Salinas Valley region  of California. One strain was from a spinach sample and the others were from a cheese firm. Ultimately, the cheese company was considered as the suspected source of one of the cases; however, the other cases were unable to be linked by WGS and traditional epidemiology as an outbreak and remain a suspect incident.Cl3 was followed by an outbreak in January 2016 (Cl4) that implicated leafy greens, including bagged lettuce salads, and involved 33 clinical cases . The proL. monocytogenes. Our study showed that these isolates are very closely related to each other although the extent of such association can be varied depending on the tool used for such analysis. The relative geographical proximity of the products associated with these outbreaks/incidents and the genetic relatedness of the L. monocytogenes strains associated with these outbreaks/incidents indicates the possibility of an endemic clade of strains in this geographical area.In this study we examined the genetic relatedness of the isolates associated with the four above mentioned outbreaks/incidents by WGS followed by SNP analysis of the core genomes and also by MVLST analysis of six virulence related genes of lmo0737 marker, the indication of serotype 4bV. Additionally, strains collected during and/or from the same or close geographic region as the outbreaks were examined via BLAST to identify other 4bV strains. The metadata available on the identified isolates is provided in Strains linked to the four listeriosis 4bV incidents/outbreaks were identified from FDA internal investigation reports. Additionally, a search of the NCBI SRA (Sequence Read Archive) was done to identify other 4bV strains that may be associated with the outbreaks, by searching for isolates with the same PFGE pattern (GX6A16.0135/GX6A12.0349) that were not reported in the four incidents. The initial search was done without consideration for whether the candidate isolates contained the 6.3kb region. These candidate strains were verified as serotype 4b or 4bV by BLAST analyses looking for the presence of the targets used in Doumith et al’s assay , specifiSequencing data for all strains were obtained either from NCBI or from within our lab. Paired end sequencing reads for each strain were trimmed and assembled using CLC Genomics Workbench, version 7.0.4. Reference genomes were assembled in prior studies and in many cases, lack the publically available data to perform our own assemblies due to the sequencing technology available for those studies. All newly assembled sequences were submitted to NCBI and their accession numbers are available in The genome assemblies were analyzed using the JSpecies tetranucleotide frequency tool as previously described , 26. AddThe CFSAN SNP pipeline generated SNP alignment files that were used to generate Maximum likelihood trees with node confidence assessed by 1,000 bootstrap replicates using the Tamura-Nei model in MEGA6 , 32.Strains were selected based on prior identification with epidemic clones (ECs) or clonal complexes (CCs) to serve as reference strains , 34. AddIn addition to identifying sequences linked to the four incidents using FDA internal investigation reports, NCBI was queried to identify other possible matches from the same timeframe, geographical region, or same PFGE profile. A total of 107 strains were included in this study, though 11 were not used in the CFSAN Pipeline analysis due to file incompatibility . These f2 value that, as it approaches 1, indicates increasing relatedness, or clonality [2 values >0.99992 when compared with each other . These were still labeled SF since they overlapped with the SF timeframe and matched by PFGE. SFc2 showed high relatedness to SF isolates by WGS but was not linked by traditional epidemiology [The pipeline analysis, using the 4bV strain CFSAN023463 as the reference genome, suggested a slight genomic SNP divergence between apple 4b strains and the associated 4b clinical isolates Figs . Given tclp, dal, inlB, inlC lisR and prfA, that are present in nearly all Lm isolates to evaluate relatedness of Lm strains using a SNP-based comparison to identify allelic differences. These loci are distributed throughout the genome with only prfA linked to a pathogenicity island (LIPI-1)[As two of these outbreaks , 23 had  LIPI-1). This to (LIPI-1), 41.Whil (LIPI-1), 41. Str (LIPI-1), 42–44 ( (LIPI-1), 42–44.  (LIPI-1), 42, 45. (LIPI-1). All of  (LIPI-1). However (LIPI-1) is phylo. This to (LIPI-1).L. monocytogenes. This report demonstrates that isolates from these four incidents were highly related (<125 SNPs between the strains) versus various 4b and 4bV isolates unlinked by epidemiology (>9000 SNPs). This supports the assessment that this group may represent an emerging risk, having been responsible for four independent listeriosis events within less than 2 years, making further evaluation of this group as well as other 4bV strains critical. Furthermore, the association of this group of strains with atypical clinical cases, observed in otherwise healthy children [With the recent occurrence of four incidents of listeriosis linked to 4bV strains, concerns were raised about a possible emerging pathogenic strain of children , 47, givAs noted above, this cluster with the associated short time span between outbreaks raises concerns of an emerging trend in listeriosis and highlights the need for better understanding of how these genetic variations impact environmental survival and pathogenicity. Furthermore, the four incidents are linked to foods produced in both the Salinas Valley or San Joaquin Valley and this added detail suggests the possibility of either endemic contamination in the area or cross-contamination between facilities, not necessarily limited to those linked to the outbreaks, as there are several, possible sporadic clinical cases, either identified during the Cl3 investigation or during data mining (SRR1805362)  without Evaluation of these strains by MVLST showed clustering of these strains with an unrelated 4bV strain. This is surprising as previous work has shown agreement between the results obtained via MLST and MVLST . Indeed,S1 TableStrains were renamed according to incident association  and source . Cl3a and Cl3b strains were associated with the Cl3 event but showed notable divergence and poor epidemiological evidence. n/a, not applicable. ND, analysis not performed.(XLSX)Click here for additional data file.S2 TableValues >0.99998 are highlighted red and values of 0.99992 through 0.99998 are highlighted yellow.(XLSX)Click here for additional data file.S3 TableSNP differences less than 70 are highlighted green. Clusters deemed related and highly related are highlighted by black and red borders, respectively. The highly related group generally corresponds to incident.(XLSX)Click here for additional data file.S4 TableSNP distances of the apple 4b isolates are highlighted by a black border.(XLSX)Click here for additional data file.