Heterodera attack cereals, particularly wheat, causing costly financial losses due to impact on yield. The soil borne pathogens program at the International Maize and Wheat Improvement Center (CIMMYT) in Turkey has focused on screening wheat germplasm to identify sources of Heterodera resistant varieties for almost 20 years. The aim of this current study was to validate the finding that resistant lines demonstrate resistant reactions under controlled conditions and to test whether they present tolerant reactions when challenged with cyst nematodes under two different locations in field conditions. The results of this study, including the check lines, indicated that 27 and 28 lines maintained their reactions to H. filipjevi in Eskisehir and Yozgat field, respectively, and 23 lines were the same in both locations. In terms of tolerance, 3 and 13 lines proven to be tolerant and moderately tolerant to H. filipjevi in Yozgat field. In Eskisehir field, 13 and 14 lines were tolerant and moderately tolerant. In both locations, L7 showed tolerance reaction, although it was susceptible. The majority of the resistant germplasm  of screened lines from the Turkey CIMMYT–ICARDA (TCI) nursery were found to be resistant to both H. filipjevi populations including L1, L3, L6, L15, L21, L26, and L34, whereas 17% (four lines) from the USA had the same reaction. L32 showed a high level of resistance and tolerance in both locations and could prove to be promising lines in the breeding programs. The International Winter Wheat Improvement Program (IWWIP) formerly used these resistant lines in the crossing block and subsequently distributed them to more than 150 international collaborators. Regression analysis revealed a negative correlation between yield and RF of H. filipjevi in both nematode populations, which describes the negative impact of this pest on winter wheat. The results of this study are very important for breeding programs especially for the IWWIP, a joint program between the Turkish Ministry of Agriculture and Forestry, CIMMYT, and the International Centre for Agricultural Research in the Dry Areas (ICARDA).Nematodes attack cereal crops resulting in significant yield losses, estimated at 10%. The plant parasitic nematodes of the genus  Triticum aestivum L.) is a vital staple food crop. An estimated 750 million tons (MT) of the grain was grown on more than 220 million hectares (Mha) worldwide in 2017 (Wheat ( in 2017 . Turkey  in 2017 . The wor in 2017 .The average area (in Mha) of production (in MT) and yield (tonnes/ha) of wheat from 2008 to 2012 by country in the Central West Asia and North Africa (CWANA) region is depicted as reviewed by Heterodera avenae complex; CCN) is found globally and leads to significant economic yield losses, particularly in areas where dryland and cereal monoculture systems are practiced (H. avenae) is calculated by Cereal cyst nematode , other cyst-forming nematodes  and two Fusarium species (F. culmorum and F. pseudograminearum). The most resistant lines are then distributed to international collaborators for use in their breeding programs.The soil-borne pathogen program at CIMMYT Turkey annually receives about 1,000 accessions of wheat from the CIMMYT Mexico spring wheat program and IWWIP. They are screened under growth room, greenhouse and field conditions at various locations in Turkey. Cultivars are also screened for multiple disease resistances, such as resistance to different species of root lesion nematodes  to screen winter wheat germplasm provided by IWWIP against the cereal cyst nematode H. filipjevi, (ii) validate the resistance of those lines under the infested fields with H. filipjevi, and (iii) study and compare the tolerance reaction of lines under field conditions with the proven resistant ones under controlled conditions.However, the habits and nature of CCN is complex due to the existence of pathotypes, which require long-term investment strategies. This is vital to ensure that a selected resistant wheat line can target as many different pathotypes as possible in addition to other H. filipjevi under growth room conditions at the Transitional Zone Agricultural Research Institute (TZARI) in Eskisehir, Turkey and were selected for this study. Four standard check lines: Katya (MR), Sonmez (MR), Kutluk (S), and Bezostaya (S), being well-recognized for their resistant/susceptible reactions to H. filipjevi, were also included as known controls  in Yozgat, Turkey  and one at TZARI in Eskisehir . The trials in each of these two locations were conducted during the 2014 to 2015 season and repeated in 2015 to 2016. The ICAE site is identified as historically infested with substantial populations of fication , while tfication . In 2015fication . The meafication . GeneralTrials were planted in October for both locations and growing seasons, with each entry sown in a plot (1 m wide × 2 m long) consisting of four rows with 20 cm between rows in the TZARI field, whereas plots in ICEA were 5 m long × 1.2 m wide consisting of six rows with 20 cm between rows. Seeds were sown at a rate of 550 to 600 seeds per square meter. A randomized complete block design was used in both locations with three replicates. Trials were fertilized at sowing time with 10–10–10–5 NPKS at 200 kg/ha. Bronate® herbicide (MCPA + bromoxynil) was applied at 470 ml/ha to control broadleaf weeds. Trials were terminated in July. At harvest, spikes from each plot were manually harvested using a sickle and threshed with a small harvester at TZARI and by experimental harvesting machine in ICEA. Grain weight per plot was weighed and recorded.3 each were taken per plot in a zigzag pattern at the beginning of the growing seasons to assess nematode initial populations (Pi). Samples were taken from around seed rows at 20 cm depth using a soil auger (2.5 cm diameter). Extraction of the cysts from the soil was performed as per Pf) per plot by taking soil samples from the root niche. The nematode Pi and Pf were used to calculate the nematode reproduction factor (RF) based on equation: RF = Pf/Pi.Six soil subsamples of 250 cmAn accession’s resistant reaction was classified into one of five distinctive groups based on the reproduction factor (RF): resistant (R) = RF equal or less than 1; moderately resistant (MR) = RF between 1 and 2, a few more cysts than in a resistant check; moderately susceptible (MS) = RF between 2 and 3, distinctly more cysts than in a resistant check, but less than in the susceptible check; susceptible (S) = RF between 3 and 4, more cysts than in the susceptible check; and highly susceptible (HS) = RF more than 4, cyst number higher than in the susceptible check  and takiP < 0.001 using SPSS statistical software V 17.0 . Linear regression analyses were conducted to describe the relationship between the H. filipjevi reproduction factor (RF) and the grain yield for each line in the two experimental areas. Principal component analysis was used to determine population structure using R® 3.4.3 software to distinguish principal groups of wheat lines based on their tolerance to H. filipjevi. All other analyses  were described using XLSTAT software 2016.02.28451 .The CCN data were transformed and then analyzed using analysis of variance (ANOVA). To meet assumptions of normality, H. filipjevi population in Yozgat, while 9 and 13 lines were tolerant and moderately tolerant in Eskisehir region, respectively. Around 60% (14 lines) of the resistant lines from TCI were found resistant to both H. filipjevi populations including L1, L3, L6, L15, L21, L26, and L34, whereas 17% (four lines) from the USA had the same reaction including L9, L29, L32, and L33. Line 32 gave tolerance and resistance reactions to both H. filipjevi populations in both regions. The two susceptible check cultivars “Bezostaya” and “Kutluk” confirmed their susceptibility and intolerance reactions in both locations. Additionally, the two resistant cvs., ‘Katya’ and ‘Sonmez’ conserved their resistance in both locations. Analysis of population structure based on grain (resistance reaction) displayed two distinct groups among the evaluated 35 lines (including checks) in Eskisehir and three distinct groups in Yozgat experiments  showed susceptibility reaction under both locations . Three leriments . The sameriments .In the Eskisehir (TZARI) experiment, the first group comprised 27 resistant lines (R), including L12, L13, L14, L22, and L32. Group 2 described eight susceptible line (S), including L12, L16, and L31 . With thIn Yozgat (ICAE), we obtained 28 resistant lines (R), including L1, L5, L14, L15, and L32, alongside six susceptible lines (S), including L4, L7, L16, and L20 and one moderately susceptible line (MS), L10 , whereasH. filipjevi. Those lines could be accepted as tolerant lines in the present study. For lines L6, L23, L24, L26, and L29 in ICEA experiment, grain yield was low even at low initial densities of nematodes (Pi), indicating intolerance and susceptibility of those lines to H. filipjevi. Line L7 was the only line that combined susceptibility and tolerance to this nematode in both locations, providing a high yield despite high RF.As shown in P < 0.001) in the host status of the 31 IWWIP lines tested to H. filipjevi based on their yield and reproduction factor (RF) in field conditions in TZARI (RF) (H. filipjevi (L27) and significantly lower RF compared to the other IWWIP lines evaluated in this study. The lowest RF was achieved by L3 (0.4), while L12 gave the highest RF in the Eskisehir field experiment (2.2) (We detected significant differences (ARI (RF) . The hignt (2.2) .P < 0.001) in the host status of the IWWIP lines to H. filipjevi, ranging from resistant to susceptible in terms of the reproduction factor, which fluctuated from 0.2 to 1.4 in ICEA field (H. filipjevi (L18 and L27) and significantly lower RF compared to the other IWWIP lines evaluated in this study. The lowest RF was achieved by L1 (0.2), while both L20 and L35 gave the highest RF values . A total of 27 lines were shown to maintain resistance in Yozgat region  with a resistance capacity and intolerant reaction even with a RF value of 0.9 , we found a negative relationship between yield and RF of ilipjevi , which dilipjevi . Referenperiment . Our resectively .H. filipjevi infested fields in the provinces of Yozgat and Eskisehir in Turkey. The results of this study indicate that H. filipjevi is considered an important factor which causes severe damage to different wheat cultivars and has potential to decrease growth and yield of wheat in Turkey , while 13 lines were tolerant, and 14 lines were moderately tolerant in Eskisehir region. This location indicated four intolerant lines, fewer in comparison to Yozgat region, as it comprised nine intolerant lines, which can explain the reduction of tolerance can be caused by high nematodes densities.Resistant or tolerant wheat cultivars are the most successful and preferable method to manage nematodes in cereals . Toleranct yield . Therefoct yield . As a re regions . FindingHeterodera species is currently available. Therefore, there is an urgent need to identify new resistance sources and to pyramid and incorporate them into high-yielding cultivars. Many studies have attempted to seek new genotypes of cyst nematode resistance , MACKELLER, CPI 133842, CPI 133814, and CROC_1/AE.SQUARROSA (224) // OPATA*1. H. avenae in Saudi Arabia and their results showed that the 10 studied wheat genotypes were significantly different in terms of their resistance and tolerance to nematode. On the other hand, CIMMYT spring wheat genotypes  plus the cvs. AUS-30851 and Yecora Rojo were found to be the most susceptible genotypes to the tested Saudi population of H. avenae. H. filipjevi as well as other growth parameters in all Back–cross Rowshan, Pishtaz, and Parsi cultivars in the province of Isfahan in Iran.Nowadays, wheat landraces and domesticated wheat have been used to identify many important agronomical traits . Althougsistance . Nematodsistance . HajihasHeterodera resistance for decades due to high infection pressure. H. filipjevi under in vitro conditions and found that 5 international  and 10 national wheat genotypes  were the most resistant wheat variety and pedigrees found against H. filipjevi Haymana population. H. avenae under in vitro conditions. They reported that 4 national varieties (including Adana 99 (PFAU/SERI82//BOG“S”), 23 international germplasm, and 17 wild genotypes were found moderately resistant to nematode. H. filipjevi, respectively. H. avenae under field conditions. They found no varieties with complete resistance to H. avenae. However, they reported Adana 99, Ceyhan 99, and Silverstar were moderately resistant and that Karatopak, Osmaniyem, and Seri 82 were moderately susceptible against H. avenae. H. filipjevi, one germplasm [PI628144 (syn. AUS28321)] was resistant and five other accessions were moderately resistant under greenhouse conditions. P. thornei of which 56 lines were pre-identified as resistant under controlled growth room conditions. These lines were further evaluated for their resistance and tolerance reactions under field conditions, where 14 accessions maintained their resistance, and 16 were moderately resistant against P. thornei. Four lines gave excellent resistant and tolerance reactions to P. thornei. The relationship between the nematode reproduction factor (Pf/Pi) and wheat grain yield in field experiments fit a linear regression model. In this study, the reaction of the 31 lines plus the four checks showed that around 10 lines were resistant and tolerant in Eskisehir, whereas just two lines gave the same reaction in Yozgat. The line ‘L32’ provided resistant reaction and tolerance capacity to both H. filipjevi populations.In Turkey, breeders have selected wheat varieties for H. filipjevi as they were resistant against it. The RF normally goes to a value that exceeds six in nurseries screened for the first time. Our results indicated significant negative relationship between RF and wheat yields. The reduction in grain yield increased with both increasing Pi and Pf. Similar results reported by H. avenae and grain yield of three cultivars Seri 82, Osmaniyem, and Karatopak in the Eastern Mediterranean region of Turkey. The negative relationship between nematode Pi densities and grain yields for the tested germplasm in Eskisehir are similar to those of microplot trials conducted in Iran on Sardari cultivar by The low reproduction factor of the tested lines is normal, as those lines were screened many times under the controlled conditions for H. filipjevi and identifying a new resistant wheat germplasm will allow breeders to develop new crosses with their local varieties, improving their genetic resistance to the targeted nematode species. So far, hundreds of CCN-resistant genotypes have been identified from the IWWIP materials, but the genetic nature of the resistance and genetic diversity of these germplasms are not yet known. Therefore, studying the genetic background of those lines is important to understand the resistance novelty of those lines and use those lines with different genetic backgrounds in crosses and for pyramiding a new resistance and high yield that increases grain yield to support food security.Wheat germplasm with acceptable levels of resistance and tolerance are subsequently crossed with high-yielding susceptible cultivars. This is an essential step as most of the locally adapted wheat varieties are susceptible to


There is increasing evidence that intrauterine environment and, consequently, growth in utero have both immediate and far-reaching consequences for health. Neonatal body composition might be a more sensitive marker of intrauterine environment and neonatal adiposity than birth weight and could serve as a predictor for non-communicable diseases later in life.To perform a systematic literature review on neonatal body composition determined by air displacement plethysmography in healthy infants. The systematic review was performed using the search terms “air displacement plethysmography”, “infant” and “newborn” in Pubmed. Data are displayed as mean (Standard deviation).n = 6231 infants) using air displacement plethysmography fulfilled inclusion criteria for meta-analysis. In these, weighted mean body fat percentage was 10.0 (4.1) % and weighted mean fat free mass was 2883 (356) g in healthy term infants. Female infants had a higher body fat percentage (11.1 (4.1) % vs. 9.6 (4.0) %) and lower fat free mass (2827 (316) g vs. 2979 (344) g). In the Caucasian subpopulation (n = 2202 infants) mean body fat percentage was 10.8 (4.1), whereas data for reference values of other ethnic groups are still sparse.Fourteen studies (including Body composition varies depending on gender and ethnicity. These aggregated data may serve as reference for body composition in healthy, term, singletons at least for the Caucasian subpopulation. There is strong evidence that fetal and early postnatal environment play an important role in fetal programming and in determining the risk for disease in adulthood such as obesity, diabetes and cardiovascular disease , 2. PropFetal growth and body composition at birth are influenced by numerous factors, some non-modifiable such as gender, gestational age and ethnic/genetic background, others modifiable such as maternal diet as well as weight gain and (metabolic) health during pregnancy. These modifiable prenatal factors may affect the health of the offspring throughout his/her entire life , 6.In a recently published meta-analysis including 477,620 children aged 2 to 13 years in Europe, the pooled prevalence of overweight and obesity ranged from 13 to 23% in various regions in the period between 2011 and 2016 , indicatNeonatal body composition parameters such as fat mass (FM), fat free mass (FFM) and the proportion of FM divided by total body mass (BF%), might be more sensitive markers of the in-utero environment and neonatal adiposity than birth weight and length alone, because variability in FM and FFM has been reported in newborns of similar weight and length , 14, andNeonatal body composition can be determined by skinfold thickness, isotope dilution, dual energy x-ray absorptiometry (DXA), magnetic resonance imaging (MRI) and air displacement plethysmography (ADP), where the latter was shown to produce highly reproducible and accurate measurements and may be suitable for large epidemiological studies , 15. FurTo inform future studies, we performed a systematic review of the literature and meta-analysis for available measurements of body composition at birth in healthy, term, singleton infants using ADP. Our aim was to establish reference values for different ethnic groups and to investigate factors potentially influencing body composition.https://www.cosmed.com/images/pdf/bibliography/PEA_POD_Bibliography.pdf).An all-language literature search was carried out on September 14, 2018 in Pubmed using the search strategy ((“air displacement plethysmography”[All Fields]) AND  OR “newborn infant”[All Fields] OR (“infant”[All Fields] AND “newborn”[All Fields]) OR “infant, newborn”[All Fields]). This was complemented by an online list of references provided by the manufacturer of the ADP device  during the first 96 postnatal hours selected. If investigators published more than one report on the same study cohort or several reports with overlapping populations, only the most recent publication or that with the most representative population was included. As we aimed for reference data, reports on less than 100 infants were excluded because such small populations were deemed potentially non-representative. Data on body composition , anthropometry and birth details were extracted from relevant full-text articles. This process war carried out by two of the authors .p < 0.05.Observational data was pooled calculating weighted mean and standard deviation assuming that the summarized populations are samples of a single overall population . Between group comparisons were done based on means, standard deviations and sample size using two-sided t-test or ANOVA and post hoc Tukey’s multiple comparison test. The degree of variability of body composition of healthy singletons was assessed by calculating the coefficient of variation (coefficient of variation = (standard deviation / mean) * 100). Analyses were performed with GraphPad Prism® 8.1.0  and the level of significance was Search results are detailed in Fig. The remaining 15 publications reported on 13 distinct populations. Two populations were reported twice, but with different aspects relevant to this review.n = 6231 infants studied in Europe, Australia, Asia, North and South America, India and Africa. Characteristics of studies and participants as well as countries of origin are shown in Table Additionally, data from a German Caucasian cohort recently studied at Tuebingen University Hospital was included as 14th population , resultiThe (weighted) mean (SD) gestational age at birth was 39.6 (1.2) weeks and mean birth weight 3382 (456) g; mean age at assessment was 40.8 (23.1) h. The overall male/female ratio was 0.96.In the healthy term singleton infants included in the 14 study populations (n = 6231), mean BF% was 10.0 (4.1) % and mean FFM 2883 (356) g . Meta-analysis showed that females had a higher mean BF% (11.1%) than males ) , we classified ethnic background into five different groups . Meta-analysis showed that Hispanics recruited in the USA had the highest BF% with 14.3%, followed by African-American infants with 11.2% ; mean postnatal age in these studies was 46.9 (6.8) h [Body composition according to postnatal age (in days after birth) at measurement was reported in four studies ( (6.8) h , 22, 25. (6.8) h , almost p < 0.0001 each), BF% and birth weights were similar in all four postnatal age groups   Table .Table 4Aim of this meta-analysis was to summarize and compare the currently available data on neonatal body composition in healthy term infants determined by ADP to inform future studies. In the studies selected for meta-analysis, median BF% was 10.0% SD 4.1%) and mean FFM 2883 (356) g. German infants showed values for BF% (10.6%)  similar .1% and mMeta-analysis of eight studies enabled comparisons of different ethnic backgrounds and showed that Hispanic infants recruited in the USA had the highest BF% with 14.3%, followed by African-American infants with 11.2%. The lowest BF% was reported for East-African newborns (7.8%). FFM was highest in Caucasian infants with 2903 (363) g, followed by East-Africans with 2840 (310) g and lowest in African-Americans (2674 (N/A) g).Differences in total body fat between populations of different ethnic background have already been reported in adults and children , 32, butIn a cross-sectional Australian study including 599 term infants, gender showed the strongest association with neonatal BF%, followed by maternal ethnicity . ConsistBesides ethnic factors and gender, the quality of maternal diet and intake of macro- and micronutrients during pregnancy as modifiable factors have demonstrated significant impact on birth outcomes including body composition , 39. TheSparkes et al. hypothesized that fetal FFM is primarily influenced by genetic factors, whereas fetal FM is influenced by the maternal metabolic and nutritional environment . This isHealthy newborn infants typically loose about 6–7% of their initial birth weight in the first days after birth , 46, andLimitations of our analysis are the limited number of studies and their heterogeneous design. Data on various influencing factors (e.g. age at measurement) were not published for all study populations. Nonetheless, body composition was measured in an objective and reproducible fashion using the same technique in healthy term (and predominantly singleton) neonates. The relatively homogeneous results found for body composition in our meta-analysis suggest good generalizability to other industrialized countries.Our systematic review revealed different body composition results for infants from different ethnic or socioeconomic backgrounds. Therefore, reference data for individual populations may be needed. Gender seems to affect not only body weight, but also body composition and thus also needs to be considered. Increasing postnatal age during the first 96 postnatal hours did not seem to affect BF% but was associated with decreased body weight at assessment and FFM.


Aspergillus section Flavi frequently contaminate staple crops with aflatoxins. Aflatoxins negatively impact health, income, trade, food security, and development sectors. Aspergillus flavus is the most common causal agent of contamination. However, certain A. flavus genotypes do not produce aflatoxins . An aflatoxin biocontrol technology employing atoxigenic genotypes to limit crop contamination was developed in the United States. The technology was adapted and improved for use in maize and groundnut in SSA under the trademark Aflasafe. Nigeria was the first African nation for which an aflatoxin biocontrol product was developed. The current study includes tests to assess biocontrol performance across Nigeria over the past decade. The presented data on efficacy spans years in which a relatively small number of maize and groundnut fields (8–51 per year) were treated through use on circa 36,000 ha in commercially-produced maize in 2018. During the testing phase (2009–2012), fields treated during one year were not treated in the other years while during commercial usage (2013–2019), many fields were treated in multiple years. This is the first report of a large-scale, long-term efficacy study of any biocontrol product developed to date for a field crop. Most (>95%) of 213,406 tons of maize grains harvested from treated fields contained <20 ppb total aflatoxins, and a significant proportion (>90%) contained <4 ppb total aflatoxins. Grains from treated plots had preponderantly >80% less aflatoxin content than untreated crops. The frequency of the biocontrol active ingredient atoxigenic genotypes in grains from treated fields was significantly higher than in grains from control fields. A higher proportion of grains from treated fields met various aflatoxin standards compared to grains from untreated fields. Results indicate that efficacy of the biocontrol product in limiting aflatoxin contamination is stable regardless of environment and cropping system. In summary, the biocontrol technology allows farmers across Nigeria to produce safer crops for consumption and increases potential for access to premium markets that require aflatoxin-compliant crops.In sub-Saharan Africa (SSA), diverse fungi belonging to  Aspergillus species frequently contaminate with aflatoxins several staple crops, including maize and groundnut (Throughout sub-Saharan Africa (SSA), certain roundnut . In SSA,roundnut . Consumproundnut . Chronicroundnut . Trade sroundnut . Becauseroundnut .Aspergillus species of agricultural importance belong to section Flavi  with aflatoxin B1 both the most toxic and prevalent and classified as a Group 1 carcinogen by the International Agency for Research on Cancer  for use in cotton  with superiority in adaptation, fitness, and competitiveness . In 2014, Nigeria’s National Agency for Food and Drug Administration and Control (NAFDAC) approved the full registration of the biocontrol product for unrestricted use in both maize and groundnut across Nigeria  started a collaboration with USDA-ARS to adapt and improve the biocontrol technology for use in Nigeria and subsequently in other SSA nations . Initialtiveness . In parativeness . Moreovetiveness , and in tiveness . Four su Nigeria .Several technologies aiming to improve crop productivity and/or quality—including technologies to reduce crop aflatoxin content—are often evaluated in experimental stations, or on farmer fields managed by researchers, and typically in a few fields . EvaluatThe current study reports the longest-term efficacy study of any aflatoxin biocontrol product developed to date. We present results of biocontrol product efficacy across Nigeria at first in (i) field trials in maize and groundnut farmers’ fields (2009–2012), and then in (ii) commercially-produced maize (2013–2018). Post-harvest benefits of using pre-harvest biocontrol are discussed as well. The results obtained during these 10 years demonstrate that biocontrol provides stable aflatoxin reductions and is a valuable aflatoxin mitigation tool used by maize and groundnut farmers across Nigeria regardless of their farming practices, crop varieties, or environmental challenges. Biocontrol allows farmers in Nigeria to produce maize and groundnut with aflatoxin concentrations safe for consumption and trade.A. flavus L morphotype isolates  that belong to atoxigenic AAVs native and relatively common in the major maize and groundnut producing areas of Nigeria  and in commercial maize  fields (2013–2018) . All fieProject’ .® 80. Suspensions were adjusted to a concentration of 106 spores ml–1 using a hemocytometer. White sorghum grains were soaked in water for 2 h, drained, and autoclaved for 45 min in polyethylene bags (45 cm × 20 cm). Batches of 1-kg of grain were seeded independently with 100 ml of spore suspensions of each atoxigenic active ingredient isolate, mixed to spread the suspension uniformly, and incubated for 18 h at 31°C. Then, grains were dried at 55°C for 4 days to stop fungal growth. Several batches were prepared for each active ingredient isolate. The final product was formulated by combining batches of 2.5 kg of each isolate into a polyethylene bag and mixed by hand shaking. Product in the polyethylene bags were placed within 10-kg-capacity plastic containers, sealed, and stored at room temperature until use.For the field trials (2009–2012), the biocontrol product was produced using a laboratory-scale method described earlier . Briefly® 80 was added to each bottle to harvest spores. Bottles were placed on a Roto-Shake Genie reciprocal shaker  at 200 rpm for 20 min. The Teflon balls helped to dislodge spores from sorghum grains. For each atoxigenic isolate, a suspension was adjusted to 4 × 107 spores ml–1 using an Orbeco-Helling digital direct reading turbidimeter  and a nephelometric turbidity unit (NTU) vs. CFU (colony-forming units) standard curve . To prepare 100 kg of the product, a spore suspension  of the constituent atoxigenic isolates were individually combined with 150 ml of a polymer  and 200 ml of a blue non-toxic dye , and coated on roasted, sterile sorghum grain with a seed treater  was produced in the Aflasafe Manufacturing Plant in IITA-Ibadan . Briefly treater .–1, 20 g l–1glucose), and Violet Red Bile Agar . After incubation  plates were examined to count numbers of grains colonized by A. flavus and to detect presence or absence of any other microorganism, including fecal coliforms on VRBA. Spore yield was evaluated by placing 24 grains from each batch in individual wells of 24-well cell culture plates, in triplicate, and incubated as above. After incubation, three replicates of two randomly selected grains were rinsed three times with 10 ml 100% ethanol. The resulting wash from each replicate was mixed with 10 ml distilled water and transferred into a turbidimeter vial. Spore yield was quantified by turbidity as above.Samples from biocontrol product batches produced either in the laboratory or the manufacturing plant were examined. For each sample, 100 sorghum grains were plated onto two plates each of 5-2 agar, Nutrient Agar  were generated and saved as previously described   . Compleme enzyme . Complem–1. Prior to application, areas of fields were measured using Garmin eTrex GPS units  to determine the quantity of product required to treat individual fields. Extension agents, trainers, and/or IITA staff supervised biocontrol product application.During 2009–2012, efficacy of the biocontrol product was tested by smallholder farmers who voluntarily consented to participate in the experiments in Enugu North, Isi-Uzo, Oji River, and Uzo-Uwani (Enugu state); Birnin-Gwari, Lere, Maigana, Giwa, Soba, and Ikara (Kaduna state); Gwarzo, Tudun Wada, Tsanyanwa, Danbata, Rinin Gado, Rano, Dawakin, Tofa, Albasu, Doguwa, and Warawa (Kano state); and Ogbomosho (Oyo state) . All parDuring 2013–2018, biocontrol was used only in commercial maize fields by farmers and implementers of the AgResults Project . BrieflyIn both 2009 and 2010, soils of all treated and control fields were collected prior to biocontrol application. Soil from the top 2-cm layer were collected by sub-sampling (∼50 times) from three random locations within each field . ComposiDuring 2009–2012, 25 maize ears were randomly harvested by hand from treated and control fields. The ears were placed in paper bags and transported to the laboratory. Aflatoxins were quantified on two sets of grain: (i) grain immediately after harvest (15 ears) and (ii) grain subjected to simulated poor storage where 10 ears with husks were wetted for 4 h and allowed to dry slowly over a 10-day period, followed by drying at 48°C for 48 h. Ears from each set were husked and shelled for aflatoxin analysis . Sub-samFor maize from commercial fields (2013–2018), farmers harvested the ears and the implementers aggregated the maize in their stores. Some implementers assisted farmers with threshing, bagging, labeling, and transporting the maize to aggregation stores. At the aggregation point, maize bags (containing around 100 kg maize) were tagged with farmers’ details and arranged according to the quantity supplied by each farmer. Since it was not possible to quantify aflatoxin concentration in individual fields of the >90,000 farmers, grain samples for each 30 tons of treated maize were examined, except for 2013 when samples were collected from individual farmers’ fields. Field officers of the AgResults Project collected a 5-kg sample for each 30 tons of aggregated maize by randomly sampling 50 g of maize from each of 100 bags. The samples were transported to IITA-Ibadan, where they were homogenized, milled, and stored as above.Aspergillus section Flavi fungi were obtained from soil and grain by dilution plate technique on Modified Rose Bengal Agar (MRBA) . Aspergillus isolates were assigned to their corresponding species based on colony morphology, spore ornamentation, and aflatoxin production profile  . Briefly profile . Isolatenit) were generated as previously described , aflatoxins were extracted from sub-samples (20 g ground sample) by adding 100 ml 70% methanol  for maiz® Q+ for Aflatoxin kit . Briefly, a 20-g sub-sample was transferred into a 500 ml media bottle and 100 ml 65% ethanol was added. The mixture was shaken for 3 min using an orbital shaker at 200 rpm, allowed to settle for 3 min, and then filtered through Whatman No. 1 filter paper  into a Tri-Pour® beaker. Thereafter, 500 μl of sample diluent was transferred to a sample cup and 100 μl of sample filtrate was added. A 100-μl aliquot of diluted sample was transferred into a new cup and mixed thoroughly and mixed by aspiration. A new test lateral flow strip was placed in the sample cup for 6 min ensuring that the strip touched the mixture. Then the strip was removed and read either on a Neogene AccuScan® Pro or Gold Reader.For maize from commercial fields (2013–2018), aflatoxins were quantified using GIPSA-approved Neogen Reveal–1, Aspergillus species distribution, incidence of atoxigenic AAVs, and aflatoxin concentration  were transformed using the equation y = log10(1 + x) to stabilize the variance prior to analysis. Depending on the comparisons, means were separated using paired t-tests  or protected Fisher’s least significant difference test  using SAS software . Untransformed data are presented in summary tables and graphs in this paper.Data on CFU gA. flavus. Other microorganisms were not found in any of the examined carrier grains of any of the 382 batches. VCA revealed that the recovered fungi belonged solely to the four atoxigenic AAV active ingredients of the biocontrol product. In general, each of the four atoxigenic AAVs was found on 25 ± 8% of the carrier grains. On an average, there were 3,500 ± 500 CFU g–1 of product.During the 10-year study, 382 batches of the biocontrol product were produced and examined for quality control parameters. Batches include the biocontrol formulation produced using both the laboratory- and industrial-scale processes. The quantity of the biocontrol product produced per year varied (range = 1.54 tons in 2009 to 779.42 tons in 2017). In all examined batches, 100% of carrier grains were colonized by Aspergillus section Flavi densities ranged from 304 CFU g–1 to 1,050 CFU g–1 of soil (P > 0.05) in treated and control fields. At harvest, maize and groundnut grains from treated fields contained Aspergillus section Flavi densities ranging from 185 CFU g–1 to 4,117 CFU g–1 compared to 72 CFU g–1 to 4,839 CFU g–1 in grains from control fields (P > 0.05) differences in CFU g–1 between grains from treated and control fields.Across treated and control fields,  of soil . In all l fields . There wAspergillus fungi found in soil were potentially high aflatoxin producers in all farmers’ fields where efficacy trials were conducted. In 2009, groundnut fields had high levels of SBG strains while in 2010, maize fields had high levels of both SBG strains and A. parasiticus before treatment  except for both the L morphotype and SBG strains in treated groundnut soil during 2009 (P > 0.05) higher frequencies than the other types  different between fields to be treated (range = 1.1–6.5%) and their respective control fields (range = 0.7–7.8%)  . The obsP < 0.05) in treated crops (range = 68.8–83.8%) than in control crops (range = 2.5–11.6%). In treated fields, the frequencies of atoxigenic AAVs in soil before inoculation were always lower (P < 0.05) than in grains at harvest (P > 0.05) in soils before inoculation and grains at harvest  among atoxigenic AAV frequencies in maize soil to be treated during both 2009 and 2010 and groundnut soil to be treated during 2010 (Frequencies did not differ significantly among the biocontrol product AAVs in soils before treatment in control plots . There wing 2010 . Ka16127ing 2010 . In graiing 2010 .P < 0.05) less aflatoxins (82–95% less) than untreated crops  generally contained low aflatoxin content and significantly  and after simulated poor storage (2009 and 2010) compared to controls . In 2009After simulated poor storage, frequencies of biocontrol-treated crops containing <4 ppb total aflatoxins ranged from 7.9 to 31.4% while for untreated crops the range was 0–5.5% . The proFor commercially produced maize treated with the biocontrol product during the 2013 to 2018 seasons, the proportion of samples containing <4 ppb total aflatoxins ranged from 65.8 to 98.5%  with onl® in maize and groundnut cropped in Nigeria. Aflasafe® is the first aflatoxin biocontrol product registered for use in any African nation and the third worldwide. The 10-year record provides substantial evidence of the stability of the biocontrol product efficacy in limiting aflatoxin content in farmers’ fields across the diverse cropping systems of Nigeria within fluctuating and sometimes challenging environmental conditions. Biocontrol-treated crops became contaminated with significantly less aflatoxins than untreated crops. Most of the treated crops met the stringent tolerance thresholds of local and international food and feed premium markets. A significant proportion (94%) of the 7,681 biocontrol-treated commercial crop samples that were examined had less than the World Food Program maximum limit of 10 ppb total aflatoxins. Initially (2009–2012), biocontrol efficacy was evaluated in a relatively modest number of maize and groundnut fields . However, the number of fields and the size of each field (∼1 ha) during the 2009–2012 efficacy evaluations were considerably larger than other reported biocontrol efficacy studies that used fewer than five replicates per year with some plots less than 0.01 ha  other than farmer training in application method and provision of the biocontrol product for a single application allowed testing under real-life situations that smallholder Nigerian farmers face. A natural-setting strategy, although in large-scale commercial agriculture, was conducted during efficacy trials to register the biocontrol products AF36  and Afla®  in the U® , 2004. T® , (ii) si® , (iii) p® , 5, and ® , 7. Beca pending .A. flavus L morphotype predominated, and most of the L morphotype isolates belonged to the atoxigenic active ingredient AAVs of the biocontrol product  in Nigeria and elsewhere in SSA would benefit if the technology is adapted and registered for its use in those crops.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.RB and PC designed the overall projects from which data are derived. JA, AA, AO-B, PC, and RB contributed to the conception and design of the experiments. JA, AA, AO-B, and TF conducted the experiments and field studies, and collected and analyzed the data. AA, PC, and RB provided guidance. AO-B drafted the original manuscript. JA, TF, PC, and RB edited the manuscript. All authors read, reviewed, and approved the final manuscript. RB secured funds for the study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Moschus berezovskii; FMD) is an artiodactyl mammal and is both economically valuable and highly endangered. To investigate the genetic mechanisms of musk secretion and adaptive immunity in FMD, we compared its genome to nine other artiodactyl genomes. Comparative genomics demonstrated that eight positively selected genes (PSGs) in FMD were annotated in three KEGG pathways that were related to metabolic and synthetic activity of musk, similar to previous transcriptome studies. Functional enrichment analysis indicated that many PSGs were involved in the regulation of immune system processes, implying important reorganization of the immune system in FMD. FMD-specific missense mutations were found in two PSGs (MHC class II antigen DRA and ADA) that were classified as deleterious by PolyPhen-2, possibly contributing to immune adaptation to infectious diseases. Functional assessment showed that the FMD-specific mutation enhanced the ADA activity, which was likely to strengthen the immune defense against pathogenic invasion. Single nucleotide polymorphism-based inference showed the recent demographic trajectory for FMD. Our data and findings provide valuable genomic resources not only for studying the genetic mechanisms of musk secretion and adaptive immunity, but also for facilitating more effective management of the captive breeding programs for this endangered species.The Chinese forest musk deer ( Moschus berezovskii; FMD) is primarily found in Southern Asia and is well known for the secretion of musk  class II proteins were associated with the resistance or susceptibility of FMD to infectious diseases , wild yak , water buffalo , goat , white-tailed deer , sheep , Tibetan antelope , wild boar , and wild Bactrian camel .The full genome sequence of one male FMD was sequenced and annotated by our laboratory . In ordeCap. hircus/Ov. aries; 2.85–3.89 Ma for Bo. mutus/Bi. bison; 6.85–7.50 Ma for Bo. mutus/Bu. bubalis; 11.5–19.7 Ma for the emergence of Bovinae tribes) (We used orthoMCL  to defin tribes) . Analyse tribes) . GO enri tribes) .ω) by ML with the codeml program within PAML under the branch-site model  using TBlastN. Lastly, we performed PCR amplification and sequencing with eight FMD muscle samples to verify FMD-specific missense mutation sites in gene MHC class II antigen DRA. None of the FMD muscle samples was used for the genome sequencing, as they were collected from individuals from Miyaluo Farm . Primers /ADA construction was performed by Tsingke Biotechnology Company . The ADA gene and the vector pET28a (+) plasmid were subjected to digestion with Nco I and EcoR I, and ligated using T4 DNA ligase. The recombinant pET28a(+)/ADA was transformed into Escherichiacoli DH (5α) competent cells for amplification. Positive colonies resistant to ampicillin on a Luria–Bertani (LB) plate were selected and the plasmid pET28a(+)/ADA was confirmed by restriction enzyme mapping and DNA sequencing.The ADA-Ser-F [forward]: CCTGCCATTGC GGGC TGCCGGGAGG and ADA-Ser-R [reverse]: CATGTAG TAGTTGAACTTGGTCAGG, product size: 6,383 bp) (in which Pro located at position 70 was substituted by Ser by changing the TCT codon to CCT codon), with the recombinant plasmid containing ADA gene template. The amplification was carried out in 25 ml total volume containing 12.5 ml of I-5 2×High-Fidelity Master Mix , 1 μl of each primer (10 mM), and ADA gene template. The PCR reactions were performed through the following profile: An initial predenaturation for 5 min at 95 °C followed by 35 cycles at 95 °C—30s, 55 °C—30s, 72 °C—5 min, and a final extension at 72 °C for 10 min. Mutated PCR products were digested with dpnI and then we used the BKL Kit (Takara) to generate annular pET28a(+)/ADA-Pro70Ser plasmid. The recombinant pET28a(+)/ADA-Pro70Ser was transformed into E. coli DH (5α) competent cells for amplification. Positive colonies resistant to ampicillin on a Luria–Bertani (LB) plate were selected and the plasmid pET28a(+)/ADA-Pro70Ser was confirmed by restriction enzyme mapping and DNA sequencing.Mutations were generated via PCR using Taq polymerase with mutagenic primers (ADA and pET28a(+)/ADA-Pro70Ser were transformed into E. coli BL21(DE3) (TransGen) competent cells for expression. The expression of the proteins was induced by the addition of 0.1 mM isopropyl-β-d-thiogalactoside (IPTG) once the optical density of 600 nm (OD600) of the culture had reached 0.6–0.8. After 4 h of induction, 1 ml culture was centrifuged at 8,000 × g for 5 min. The cell pellet was resuspended in 100 µl phosphate-buffered saline (PBS) and analyzed by SDS-PAGE. The expression level of recombinant ADA was determined by protein bands.The recombinant plasmids were extracted using a plasmid extraction kit , and then the recombinant plasmids pET28a(+)/Following induction, 1 l culture was centrifuged at 8,000 × g for 5 min. The pellet was resuspended in 45 ml PBS and placed in an ice bath for ultrasonic lysis . The supernatant was further purified by 80% ammonium sulphate fractionation, dialysis and desalt, filtered using a 0.22 µm filtration membrane and loaded onto a Ni-NTA agarose column . The column was washed with buffer (equilibration buffer) and the protein was eluted with elution buffer . The eluate was concentrated through a 10 kDa cutoff Centriprep filter  for SDS-PAGE.ADA activity using a commercial kit .We assayed the We used SAMtools to detect SNPs between diploid chromosomes for the sequenced FMD . We usedBo. mutus (Bovidae), Od. virginianus (Cervidae), S.scrofa (Suidae), Cam. ferus (Camelidae), and FMD (Moschidae), and 315 gene families were specific to FMD  annotation classified the PSGs into three categories: Molecular functions, cellular components, and biological processes  online. a, supplementary fig. S2b, We identified the biochemical pathways on the basis of the PSGs detected in FMD. The KEGG annotation of the PSGs suggested that they were distributed in 43 pathways related to metabolism (108 genes), genetic information processing (111 genes), environmental information processing (143 genes), cellular processes (126 genes), organismal systems (171 genes), and human diseases (179 genes) , Cytokine-cytokine receptor interaction , Measles , Influenza A , Malaria , and Toll-like receptor signaling pathway   .Table 1Actinobacillus pleuropneumoniae showed a significant representation of genes belonging to this GO term  distributed in phototransduction pathway and retinol metabolism pathways, possibly enhancing adaptation to low-light and night-time environments. In addition, males and females of FMD were visibly very similar, but male individuals were armed with two freakishly long canine teeth in the upper jaw used for fighting with rivals. We also found evidence of strong positive selection in four candidate genes  associated with odontogenesis and amelogenesis.FMD was a nocturnal mammal and foraged during night or crepuscule . CurrentAKR1D1) involved in steroid metabolism was annotated in steroid hormone biosynthesis (map00140)   online. eostasis .Table 2MVD, FNTA, and DHDDS) were annotated in terpenoid backbone biosynthesis (map00900)  related to aldosterone metabolism, which were annotated in aldosterone-regulated sodium reabsorption (map04960)   online,  acetate . This pa acetate , and som acetate . DHDDS cynthesis . FNTA isted gene . Additioap04960)  online.Vibrio cholerae infection, Epithelial cell signaling in Helicobacter pylori infection, pathogenic E. coli infection, Salmonella infection, and Shigellosis)  . Previouisorders . Infectid nausea .Table 3Staphylococcus aureus infection pathway. Staphylococcus aureus was reported to be the most frequent cause of human skin or soft tissue abscesses  of ADA.After the examination of the PSGs identified in FMD. We found two PSGs containing FMD-specific missense mutations. The first was at position 123 (Thr) of MHC class II antigen DRA (p.Glu123Thr)  of mutations were predicted to be functionally damaging (The mutation at u123Thr) a was clau123Thr) , and prou123Thr) b–d. The nfection . MHC clan humans a. We assdamaging b and c.ADA gene (p.Pro70Ser)  a that watabolism , and the pathway . Primarie system .ADA activity, we conducted the E. coli expression of wild-type ADA (Ser) and mutated ADA (Pro). We found that the activity of mutated ADA was significantly lower than that of the wild type  could affect the ild type b–d, whicWe identified 7,015,181 heterozygous SNPs in the FMD genome. The distribution in genome-wide SNP density for FMD is shown in Od. virginianus) and Bovidae  were more closely grouped, and FMD appeared to be their sister group. This genome-wide phylogenetic tree indicated that Moschidae, Cervidae, and Bovidae were three monophyletic groups. This phylogeny was consistent with other studies that placed Moschidae as an independent family (Complete genome sequences provided considerable resolution to the phylogenetic relationship and demography of FMD. The genome-wide phylogenetic tree showed that Cervidae (t family . Cervidat family .AKR1D1 was annotated in steroid hormone biosynthesis (map00140). AKR1D1 was found to inactivate the major classes of steroid hormones in steroid hormone metabolism and catalyze key steps of the biosynthetic pathway of bile acids, regulating lipid emulsification, and cholesterol homoeostasis  were annotated in terpenoid backbone biosynthesis (map00900) and participated in terpenoid metabolism. MVD catalyzed the final step in the mevalonate pathway, which was responsible for the biosynthesis of isoprenoids from acetate , which had FMD-specific mutations, have been demonstrated to be associated with the immune response  distributed in phototransduction (map04744) and retinol metabolism pathways (map00830)   . Previous (CSNB) . SAG was cascade . Mutatiolindness . RDH12 eystrophy . Therefoystrophy . CurrentKLK4, ACP4, CSF3R, and SSUH2) that were associated with odontogenesis and amelogenesis. KLK4  is a glycosylated chymotrypsin-like serine protease and has been identified as essential participants in amelogenesis (KLK4 can lead to autosomal recessive, nonsyndromic enamel malformations in humans and mice (ACPT localization has been detected in secretory ameloblasts, odontoblasts, and osteoblasts, and its biallelic mutations could cause nonsyndromic, generalized hypoplastic autosomal-recessive amelogenesis imperfecta in humans (SSUH2 disrupts dental formation and was involved in tooth development (We found evidence of strong positive selection in four candidate genes (ogenesis . Mutatioand mice . ACPT lon humans . After tn humans . SSUH2 delopment . Males aIn summary, this is the first report conducting the comparative genomics of FMD to other Artiodactyla species. Comparative genomics confirmed the PSGs annotated in functional pathways related to steroid compounds metabolism, such as steroid hormone biosynthesis, were present in FMD. The obtained PSGs data provide comprehensive gene information at the genomic level, which can contribute to a better understanding of the genetic mechanisms of musk formation and secretion in FMD. Our identification of the PSGs related to the common susceptible diseases in FMD provide a framework for future studies of these diseases in this species. Furthermore, our work can help enhance the understanding of the pathogenesis and immune system function of FMD-susceptible diseases. The PSGs found in FMD in this study will provide a strong foundation for other functional and comparative genomic studies.Genome Biology and Evolution online.Supplementary DataClick here for additional data file.


To assess the value of contrast-enhanced ultrasonography (CEUS) for monitoring disease activity of Takayasu arteritis (TA).TA patients were recruited in a Chinese TA clinical center from January 2016 to September 2017. The physician global assessment was used as the referential standard for disease activity. Clinical data, acute phase reactants, and CEUS scans were simultaneously recorded at baseline and after a 3-month therapy.p = 0.001). Severe neovascularization (grade 2) was observed in 29 active cases (61.70%) and in 9 inactive cases (24.32%) (p = 0.001). Receiver operating characteristic analysis showed that the combination of CEUS parameters (cutoff of thickness was 1.75 mm or neovascularization grade 2) and erythrocyte sedimentation rate (ESR) (cutoff of 20 mm/H) could help differentiate between active and inactive TA patients with a sensitivity and specificity of 81.1% and 81.5%, respectively. Youdon’s index was 0.626. Furthermore, our study found that patients with decreased ESR and C-reactive protein (CRP) still had a progression of vascular wall inflammation at 3 months of follow-up.A total of 84 TA patients were enrolled, and 47 (55.95%) cases were active at baseline. Macaroni sign and entire artery involvement were characteristic findings of CEUS in TA. The average vascular full thickness of the carotid artery in active TA patients was significantly higher than that in inactive patients (2.36 ± 0.86 vs. 1.79 ± 0.49 mm; The evaluation of vascular inflammation by CEUS is more sensitive than acute phase reactants. Neovascularization can still be observed in the vascular lesion sites of those who have reached clinical remission after treatment. Thus, CEUS can be used as an alternative method to assess disease activity for TA patients. Takayasu arteritis (TA) is a type of chronic inflammatory vasculitis that develops mainly in Asian women of childbearing age. The condition primarily involves the aorta and its branches, as well as the ascending aorta, abdominal aorta, renal artery, and pulmonary artery. Pathological data suggest that inflammation progresses from the adventitia to the intima and eventually encompasses the whole layer, leading to stenosis, occlusion, or expansion and consequently to organ ischemia and infarction. Once TA is diagnosed, comprehensive disease assessments, including disease activity, vascular stenosis, organ function, and drug-related adverse events, should be conducted to guide the therapeutic strategy. However, up to date, the biggest challenge is the lack of quantitative, reliable, and effective measures to monitor disease activity in TA.Commonly used parameters, such as erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), lack specificity and may not reflect the local inflammation of the vascular wall. Imaging techniques play an important role in the diagnosis and surveillance of TA. Previous studies have shown that color Doppler ultrasonography (CDUS) and computed tomography angiography (CTA) can be used for diagnosing TA, although these methods are not significantly associated with disease activity. Magnetic resonance angiography (MRA) yields unclear results when assessing the carotid artery, possibly because of considerable interference. Positron emission tomography/computed tomography (PET/CT) cannot be widely used for monitoring disease activity because of the high cost and risk of nuclear radiation , 2. HencContrast-enhanced ultrasonography (CEUS) has been widely used in atherosclerotic disease as the supplementary imaging technique for CDUS. The use of ultrasound contrast agents facilitates the identification and quantification of intraplane neovascularization in the carotid artery. Retrospective studies indicated that the enhancement of neovascularization in carotid plaques on CEUS is associated with clinical symptoms, cardiovascular events, and plaque types –5. Some According to the Numano classification , TA can We enrolled 84 patients diagnosed with TA (based on the 1990 American College of Rheumatology diagnostic criteria ) in the Demographic parameters, clinical symptoms, and physical examination findings of TA patients were electronically recorded. Serological tests, such as assessments for complete blood count, ESR, CRP, serum amyloid A (SAA), immunoglobulin, and cytokines, were performed within 3 days before CEUS examination. Physician’s global assessment (PGA)  was usedAll patients in both the TA group and the atherosclerosis control group accepted carotid CDUS, which aimed to identify the specific characteristics of TA. CDUS was performed on a Philips Elite ultrasound instrument , with a L9-3 linear array probe. The extracranial carotid arteries  were assessed by the same experienced physician. In the supine position, all patients turned their heads laterally and fully exposed their necks. For each patient, the bilateral carotid arteries were examined and the wall thickness of the normal carotid artery bifurcation was measured as a standard image of intima-media thickness (IMT). The diameter of the lesion wall was also measured as the wall thickness. The mechanical index was 0.07, and gain was 70%. The instrument parameters were kept consistent for all patients.CEUS was performed at the thickest site of the common carotid artery found by CDUS in TA patients. The US contrast agent  was injected through the middle elbow vein and immediately followed by 5.0 mL of saline. The instrument’s built-in timer was initiated simultaneously to prepare for image acquisition. The probe was fixed for 2 min, and images were continuously and automatically acquired; dynamic images were then stored in the instrument hard disk. A specific medical digital image transmission format  was used to store images for offline analysis.The degree of neovascularization at the thickening wall on CEUS was defined as follows : grade 0χ2 test. All continuous variables were described as mean ± SD and compared using Student’s t test. The clinical value of the vascularization grade of carotid lesions as a disease activity parameter was evaluated using the logistic regression model. Odds ratios (ORs) and 95% confidence intervals (CIs) were examined. Sensitivity and specificity of a selection of the following screening measures were tested using the receiver operating characteristic (ROC) analysis for individual and combined tests: CEUS parameters, ESR, and CRP. Significance was defined at p < 0.05. Statistical analysis was performed using SPSS for Windows (version 22).The cohort of the TA clinical center was prospectively studied. According to the optimal cutoff value for PGA concentration, TA patients were divided into an active and inactive group. Categorical variables were compared between the two groups using the n = 37), 9.5% (n = 8), 2.4% (n = 2), and 44% (n = 37) cases in the study, respectively. At baseline, 49 patients had received treatment previously. All of these patients received glucocorticoid  combined with immunosuppressors, including 5 azathioprine (50 mg/day), 7 methotrexate (10–15 mg/week), 5 cyclophosphamide (15 mg/kg/month), 15 mycophenolate mofetil (30 mg/kg/day), 16 leflunomide (20 mg/day), and 2 tocilizumab (8 mg/kg/month). The other 35 patients had not received any treatments previously.Both newly treated patients and those undergoing long-term treatment were included in the study. The average patient age was 31.88 ± 10.01 years . The female-to-male ratio was 6.64:1. The disease duration ranged from 1 month to 27 years . The longest follow-up duration was 1.5 years . Imaging tests were done in all the patients including CTA (4 cases), vascular ultrasound (20 cases), or MRA (78 cases). According to the 1996 Numano classification, types I, IIa, IIb, and V were observed in 44%  patients complained of new symptoms such as neck pain, dizziness, pulseless, fatigue, fever, or asymmetrical blood pressure. Furthermore, only one inactive patient complained of dizziness. In the present study, 20 (23.81%) patients had comorbidities including hypertension, whereas 7 (8.33%) had cerebral infarction.p = 0.001). With regard to serological tests, there was a significant difference in ESR, CRP, SAA, and platelet count between the two groups. Compared with the inactive group, both Kerr scores  and ITAS2010 index  were higher in the active group , and most (86.90%) were female. On CDUS, local vascular lesions were more common in cases with carotid atherosclerosis . Carotid thickening in TA mainly occurred in the initial segment, followed by the entire carotid artery. On contrast imaging of TA, carotid plaques were found to be more common at the bifurcation of the carotid artery, and the echo of the plaques was not uniform. A significantly narrower lumen  and thicker artery wall  were observed in cases with TA. The macaroni sign was a characteristic manifestation of TA and was not found in any atherosclerosis patient  than that in the inactive group. We observed a wider range of diffuse lesions in the active group, although the difference was not significant. Both the wall thickness and diffuse lesions indicate the presence of vascular damage. Furthermore, in the inactive group, the proportion of luminal stenosis and luminal occlusion was higher , which indicated that vascular inflammation could be observed in clinical inactive patients (Table Carotid CEUS showed severe vascularization (grade 2) in 29 61.70%) active cases and 9 (24.32%) inactive cases , proportions of luminal stenosis and luminal occlusion, and vascularization grade of the carotid artery were evaluated for their relationship with disease activity biomarkers. The results suggested that ESR, CRP, and SAA levels had a moderate relationship with wall thickness (p = 0.001) by reference to grade 0. After adjusting for ESR and CRP, the OR was still significant . The OR of developing disease activity was 4.40  when the wall thickness increased every 1 cm. After adjusting for ESR and CRP, the OR was 3.32 .To further clarify the risk of disease activity in different vascularization grades, we conducted logistic regression analysis. The OR of vascularization grade 2 was 4.72  and disease duration  of the two groups were not significantly different. After 3 months of treatment, the proportion of disease activity, according to PGA, decreased from 65.8 to 18.4%. The ESR and CRP levels decreased from 37.84 ± 34.81 to 21.89 ± 20.26 mm/H (p = 0.019) and 31.11 ± 49.52 to 15.37 ± 26.25 mg/L, respectively . The proportion of severe vascularization (grade 2) decreased from 52.6 to 34.3% (p = 0.036). The lumen diameter increased, though the improvement was not statistically significant. The proportion of stenosis and occlusion did not change significantly after 3 months’ treatment, suggesting that the two parameters would be changed in the chronic phase of vascular disease. In addition, peak flow rate and RI did not change significantly during follow-up.At the end of 3 months’ follow-up, the thickness of the lesion wall was significantly improved compared to baseline  In contrast to carotid plaque, TA has the macaroni sign, which is characterized by a uniform annular thickening of the transverse section of the affected arterial wall. (2) In this study, we enrolled patients with TA alone. Compared with previous studies, our sample size was the largest. (3) The thickness and severe vascularization of the vascular wall were specific to active TA patients. These two indices were reduced after 3 months of treatment. (4) During the follow-up period, the ESR decreased, but a small amount of neovascularization signal was still visible in the vascular wall, suggesting that CEUS was more sensitive for detecting vascular inflammation.With advancements in imaging technology, the sensitivity of detecting blood vessel inflammation and vascular lesions has been increased. Previous studies reported that common vascular involvement in Caucasian patients primarily encompasses the abdominal aorta, superior mesenteric artery, subclavian artery, and common carotid artery. In our previous TA cohort study, it was found that the left subclavian artery, left common carotid artery, brachiocephalic artery, thoracic aorta, abdominal aorta, and renal artery were commonly involved in the Chinese population, which suggested the existence of an ethnic difference. Our previous study found that type V, followed by type IIb and type I, was common among TA patients. In the present study, we found 44% of patients with types V and I, followed by 9.5% with type IIa and 2.4% with type IIb. These studies suggest that carotid artery involvement is common in patients with TA in the Chinese population. MRA is not optimal for carotid artery assessment, but ultrasound has a higher resolution both in the diagnosis and assessment of inflammatory activity in TA. Thus, ultrasound is worth being promoted or should be preferred for application in patients with carotid artery involvement.In TA patients, although systemic inflammation is significantly associated with local inflammation of the vessel wall, they are also independent in some cases . PreviouCarotid CEUS is an effective diagnostic method for assessing the risk of cardiovascular disease. Previous studies have indicated that, on carotid CEUS, plaque neovascularization is positively correlated with inflammation activity . Thus, CIn the present study, there were differences between the active and inactive groups in terms of the CDUS and CEUS parameters. The wall thickness of the carotid lesion was significantly greater in the active group. Carotid wall vascularization grade 2 was observed more often in the active cases. After regular treatment, these two indicators can be significantly improved, and some patients return to completely normal carotid. However, the proportions of vascular stenosis and occlusion were significantly higher in the inactive group. Notably, Fan et al.  reportedROC analysis showed that the combination of CEUS parameters and ESR yielded better results than separate indicators. The optimal cutoff points for ESR, carotid artery wall thickness, and vascularization grade were 20 mm/H, 1.75 mm, and vascularization grade 2, respectively. The combined indicators could differentiate between active and inactive TA patients, with a sensitivity of 81.1% and specificity of 81.5%. Giuseppe et al. observed a significant relationship between carotid CEUS vascularization grade and PET/CT uptake. Moreover, CEUS had a sensitivity of 100% and specificity of 92% for identifying the active disease of large-vessel vasculitis using PET/CT as a reference standard, which was much higher than our findings . Howeverp = 0.041). Makita et al. [Carotid ultrasound examination can reflect vascular structural and morphological changes under the route of disease development, which help to assess the patient’s outcomes after treatment. We found the change in carotid wall thickness was the most sensitive parameter. At 3 months’ follow-up, it was observed that the thickness of the lesion wall was significantly improved than the baseline (1.85 ± 0.61 vs 2.24 ± 0.89 mm, a et al.  observeda et al.  also conThus, CEUS appears to have some potential for assessing disease activity in TA and during follow-up.The present study has certain limitations. First, the 84 cases included in this study contained newly treated patients as well as those undergoing long-term treatment. The average treatment duration was 47.49 ± 66.03 months. In our previous study, we found that carotid ultrasound contrast microbubble development was affected by artery wall fibrosis and collateral circulation in some patients, which may have overestimated contrast activity at the artery wall. Second, CEUS used a semi-quantitative assessment in the present study; hence, it may have underestimated wall inflammation.In conclusion, CEUS is a convenient and rapid noninvasive technique for assessing the inflammation of the TA carotid artery wall. CEUS can also analyze the neovascularization of the TA carotid artery wall. The moderate relationship between the grade of neovascularization in CEUS and the levels of inflammatory markers suggests CEUS can be used as an effective method to assess disease activity in patients with TA. The sensitivity and specificity of combined CEUS parameters and ESR were 81.1% and 81.5%, respectively, for predicting TA activity. Ultrasound can more sensitively reflect changes in blood vessels and inflammation compared with acute phase reactants.


Invasive meningococcal disease surveillance in Europe combines isolate characterisation and epidemiological data to support public health intervention. A representative European Meningococcal Strain Collection (EMSC) of IMD isolates was obtained, and whole genome sequenced to characterise 799 EMSC isolates from the epidemiological year July 2011–June 2012. To establish a genome library (GL), the isolate information was deposited in the pubMLST.org/neisseria database. Genomes were curated and annotated at 2,429 meningococcal loci, including those defining clonal complex, capsule, antigens, and antimicrobial resistance. Most genomes contained genes encoding B  or C  capsules; isolates were genetically highly diverse, with >20 genomic lineages, five of which comprising 60.7% (n = 485) of isolates. There were >350 antigenic fine-types: 307 were present once, the most frequent  comprised 8% (n = 64) of isolates. Each genome was characterised for Bexsero Antigen Sequence Typing (BAST): 25.5% (n = 204) of isolates contained alleles encoding the fHbp and/or the PorA VR1 vaccine component, but most genomes  did not contain the NadA component. EMSC-GL will support an integrated surveillance of disease-associated genotypes in Europe, enabling the monitoring of hyperinvasive lineages, outbreak identification, and supporting vaccine programme implementation. Neisseria meningitidis, the meningococcus, develop severe disease, usually meningitis and/or septicaemia, with survivors often suffering permanent sequela [Invasive meningococcal disease (IMD) is a contagious, occasionally epidemic, infectious disease that causes significant morbidity and mortality with varying incidence globally . Only a  sequela . While v sequela ,4. To ci sequela ,6.Understanding the impact of meningococcal vaccines requires surveillance that includes knowledge of disease-associated meningococci and the antigens and capsule types that they possess. However, comprehensive and timely surveillance of IMD is complicated by the high genetic and antigenic diversity of meningococcal populations . While m, where the analysis tools of the BIGSdb platform can be used [The establishment of a European meningococcal genome library has potential to enhance IMD surveillance continent-wide, providing comprehensive and comparable long-term typing data. To do this, all national reference laboratories for meningococci in Europe were asked to make available in 2014 isolates from one epidemiological year 2011–2012) to contribute to the European Meningococcal Strain Collection (EMSC) in order to provide a bank of isolate genomes representative of those causing invasive disease in Europe at that time. The EMSC Genome Library (EMSC-GL) was created to exploit whole genome sequence (WGS) data for surveillance, characterisation, and vaccine intervention studies to analyse longitudinal data using both historical, current and future datasets. The WGS data are hosted on the pubMLST.org/neisseria database to contrThe EMSC was established in 2002 at the Norwegian Institute of Public Health, Oslo, Norway with the support of the European Centre for Disease Prevention and Control (ECDC), to assure preservation of meningococcal isolates causing disease in Europe. Additional funding was allocated by the ECDC in 2011 for the implementation of the EMSC-GL. Participation of each country in the creation of the EMSC-GL was voluntary. All 799 invasive meningococcal isolates received from the epidemiological year July 2011–June 2012, from 16 participating national reference laboratories, were included in the EMSC-GL.2 and DNA extracted using an epMotion 5075 pipetting robot (Eppendorf) and NucleoSpin 8 Tissue kit (Macherey-Nagel) according to the manufacturer’s instructions. DNA was visualised with the FlashGel DNA system (Lonza), its quantity assessed using Qubit , and sent frozen in 96-well plates to the Oxford Genomics Centre (OGC), University of Oxford, United Kingdom (UK).Isolates were grown on blood agar plates overnight at 37 °C in an atmosphere of 5% COThe DNA samples were normalised and sequenced at the OGC. Quantification and integrity of DNA extracts were assessed using the Invitrogen PicoGreen assay on a 1% E-GelEX. DNA was fragmented using acoustic shearing and the fragment distribution determined using the Agilent Tapestation D1200 system. NEBNext DNA Sample Prep Master (New England BioLabs) was used with minor modifications to construct the libraries. Ligation of the adapters was completed using Illumina Adapters and PCR enriched. Libraries were pooled and quantified using with the MX3005PTM instrument (Agilent) and the Agilent qPCR Library Quantification Kit. Paired-end 100 base reads were sequenced using the Illumina HiSeq 2000 sequencing platform.de novo as described previously [Isolate records were created in the pubMLST.org/neisseria database and included provenance and phenotypic typing information . Short-reviously , and, upNeisseria (NEIS) locus identifier and an allele number assigned if an identical reference sequence was present in the database. Novel alleles with ≥ 98% sequence identity to a reference sequence in the database, were automatically assigned new reference numbers. This process was repeated until all NEIS loci had been curated and as many alleles as possible had been defined and assigned in each genome assembly. The new alleles with < 98% sequence identity were manually checked and curated before assigning a new reference number. The annotated loci included those defining antigens, genes known to effect antibiotic sensitivity, the capsule operon genes, and the 1,605 loci belonging to the N. meningitidis core genome MLST (cgMLST) scheme (version 1.0) [Each genome assembly was queried against the pubMLST.org/neisseria sequence definition database to identify the loci within individual genomes. As identified, loci were automatically annotated with a ion 1.0) ,14. Alleion 1.0) -17.Using the ‘sequence bin breakdown’ tool, the assembly statistics and genome coverage were assessed for each genome assembly. The BIGSdb Genome Comparator Tool was used to generate allele-based pairwise comparison distance matrices that were visualised as Neighbour-Net phylogenies. Cramer’s V coefficient was used to measure the association of clonal complex (cc) with Bexsero Antigen Sequence Typing (BAST) and individual vaccine antigens, using the ‘cramersV’ function in the ‘lsr’ package in R 3.3.0 . The coeThe data confidentiality and security: no personal identification is recorded in the database and isolates are identified by an anonymised sample name and an automatically assigned isolate accession number. Each reference laboratory is provided immediate access to their submitted data once it has been received and assessed by a curator. The pubMLST database and its curators are currently supported by The Wellcome Trust (grant 104992) and the UK Department of Health (grant H2R00080) respectively.N. meningitidis genome, based on finished genomes , was 1,967 genes.The isolates (n = 799) represented IMD cases from 16 EU countries during the epidemiological year July 2011–June 2012. The genome assemblies contained an average of 466 contigs, with an average length of 5,211 nt per contig, and an average genome length of 2,143,632 nt. The larger number of contigs and unassembled genes compared to other studies was due to fragmentation of the DNA during extraction process ,19. The Isolates had been tested for capsule phenotype by the submitting laboratory: 65.8% were serogroup B (n = 526); 20.8% were serogroup C (n = 166); 7.5% were serogroup Y (n = 60); 4.0% were serogroup A, W, or W/Y ; and 1.9% were non-groupable (NG) or had no serogroup data recorded . Analysis of capsule operon regions A and C from the WGS data: (i) genotypically confirmed the phenotypic serogrouping of 767/799 isolates (96.0%); (ii) defined the genogroup of 14 NG isolates and of one isolate without serological data; (iii) corrected the capsule designation of 14 (1.8%) isolates; and (iv) partially confirmed one B and one C capsule and found one mosaic capsule operon .The isolate without serological typing information contained capsule type B genes. Two genomes had a poorly assembled region A and the gene that determines capsule type was incompletely assembled; however, the assembled sequence indicated that genetically one isolate was B and one isolate was C. cnl); and one typed as serogroup W was genetically a capsule type Y. Of the serogroup records that did not match capsule genotype: three isolates assigned to serogroup C were capsule type B (n = 2) and capsule type Y (n = 1); three serogroup Y were capsule type B; seven isolates assigned to serogroup B were capsule type C (n = 5), capsule type E (n = 1), and one isolate did not contain the capsular regions A or C and was confirmed to be a capsule null  MLST data obtained by conventional means were also available. The WGS data confirmed the sequence type (ST) and cc for 377/402 (93.8%) of these isolates and determined the ST and cc for 395 of the remaining 397 (99.5%) isolates. gdh MLST locus was incomplete but the remaining six MLST alleles were identified: one isolate had a profile consistent with the ST-269cc, but was nonetheless not assigned to any specific ST, while the other was unique and did not match any currently defined ST; (ii) and for 23 isolates, the WGS-derived MLST differed from that previously reported. Most (17/23) differed at a single MLST locus , three differed at two loci , and three isolates differed at all seven loci. The isolates where the ST was one or two loci different were still assigned to the same cc, the three isolates with seven loci differences were each reassigned to a different cc. These results were consistent with the downstream effects of splitting samples in seven-locus PCR-based MLST and mislabelling of samples [Concerning the 25 isolates among the 402, where the conventional MLST and the WGS data did not concur: (i) for two the  samples . In total, 795 isolates had an assigned ST, among which 108 with no cc association. Two-hundred-ninety STs were defined  and twenty-nine cc were identified . The three most common cc, cc41/44 (n = 177 isolates), cc32 (n = 132), and cc11 (n = 92), represented 50.2% of the 799 isolates. Eight additional cc had at least 10 representatives each and comprised 29.0% (232) of the isolates . The remporA gene (NEIS1364) was absent in 10 genomes (1.3%), while four genomes had a deleted PorA VR1 loop, two genomes had a deleted PorA VR2 loop, and one genome had an incompletely assembled NEIS1364 gene and its PorA VR1 loop could not be assigned. Additionally, six genomes (0.8%) were lacking FetA VR assignment: five were missing the NEIS1963 (fetA) gene and one had a non-coding protein sequence. The most prevalent antigen fine-types in the dataset (n ≥ 10) included: 7–2:4:F1–5 (8.0%); 7:16:F3–3 (5.2%); 5:2:F3–3 (4.7%); 22:14:F5–5 (3.7%); 18–1:3:F3–9 (3.1%); 5–1:10–8:F3–6 (3.0%); and 5–2:10–1:F4–1 (3.0%).The antigenic fine-type data (PorA VR1:PorA VR2:FetA VR) were assessed for each of the isolates’ genome assemblies. The finetype antigen profile was complete in 778 of the isolates (97.4%). The nhba) or the NEIS1969 (nadA) gene. The most common 12 BASTs were present at a frequency of at least 1.0% (n = 8) and an additional 32 BASTs were represented by at least three to seven genomes each  vaccine contains four different peptide components and is represented by the peptide profile BAST-1  . There wmes each .nadA was absent in 64.2% (n = 513) of 799 genomes and 229 of the BASTs. The BAST of each genome was assessed for direct matches to the BAST-1 components for prediction of potential vaccine coverage. Predicted coverage, based on sequence identity, was most frequently associated with the PorA VR1 7–2  and fHbp 1  vaccine components  with two representative genomes each and 238 profiles (71.7%) that occurred once. Overall the two most common profiles were BAST-223  and BAST-4 . The gene encoding mponents .The genomes were also assessed for exact peptide matches for the bivalent rLP2086 (Trumemba) vaccine composed of fHbp variants 45 and 55. The fHbp variant 55 was not observed, while variant 45 was present in 27 (3.4%) genomes from eight countries: the Netherlands (n = 8), Poland (n = 7), France (n = 4), Germany (n = 3), Slovakia (n = 2), and one each in Austria, Denmark, and Finland. There was no overlap of coverage between the vaccines, 52.4% (n = 419) of the genomes had exact matches to BAST component and an additional 3.4% (n = 27) of the genomes contained the fHbp variant 45.gyrA gene), penicillin , and rifampicin  [The pubMLST.org/neisseria database contained some phenotypic antimicrobial data in the form of minimum inhibitory concentration (MIC) ranges. These data were limited to a subset of isolates used for targeted gene sequencing to define susceptibility for ciprofloxacin  ,16. AllerpoB alleles were present in the dataset, 23 of which were associated with a susceptible MIC (≤ 1µg/mL). Most genomes  contained one of four rpoB alleles: 18, 2, 4, or 34, and an additional eight alleles were present in at least 1% of the genomes . The next most prevalent, allele 184 , had no MIC associated testing data and was found in Germany (n = 3) and Poland (n = 2). There were 18 additional alleles, each present in the dataset once, with no MIC associated testing data.Forty-one penA gene and no allele assignment and 44 genomes (5.5%) did not have the penA gene. There were 67 penA alleles in the dataset, 13 of which had MIC values that were generally sensitive, ≤ 0.06µg/mL . Thirty-eight penA alleles were associated with MIC values of intermediate sensitivity (> 0.06 to 1µg/mL). There were 16 additional alleles with no MIC associated testing data, 15 of which were found once in the dataset and one allele was found eight times, from Germany (n = 4), Poland (n = 3) and Hungary (n = 1).There were 141 genomes (17.7%) with an incompletely assembled gyrA alleles and 642 genomes (80.4%) contained allele 4, 2, or 3. Eight genomes (1.0%) had an incompletely assembled gyrA gene. Five additional alleles were found in at least 1% of the isolate genomes and represented an additional 16.5% of the genomes (n = 132). All eight alleles had an associated susceptible MIC test result (≤ 0.03µg/mL) and half of the alleles had at least one isolate record where the MIC result was intermediate (> 0.03 but < 0.12µg/mL). Only three of the eleven remaining alleles had MIC associated data and sequence comparison of the eight alleles with known MIC data was not discriminatory.There were a total of 19 Meningococcal lineage nomenclature was used as the fundamental unit of analysis for the genetic relationships at the genome level . This isAdditional groups were evident, including a cluster of genomes from Poland (n = 7), Germany (n = 7), and Hungary (n = 2), containing eight STs not associated with a cc. Members of this new lineage, while sharing up to five of seven MLST alleles with members of cc174 (lineage 14), were distinct using cgMLST (data not shown). A number of lineages formed larger groups or clades including: lineages 11 and 8; and lineages 2 and 35 with lineage 5. Lineage 11 and lineage 23 contained distinct sub-lineages,  as previously described . The vacNeisseria genomes contain repeat regions, homopolymeric tracts, and paralogous loci [ous loci ,23; all ous loci ,13, and The sporadic nature of IMD, combined with the unpredictable occurrence of epidemics and the diversity of meningococci, and especially of meningococcal antigens, makes accurate surveillance an essential element of effective public health measures . The welNeisseria loci, including 38 sequence-based typing loci such as antigen genes, seven locus MLST, and cgMLST loci. This permits the rapid genetic characterisation and comparison of > 93% of the content of future meningococci genomes added to the genome library with minimal additional curation. Within pubMLST.org/neisseria these loci are grouped into different schemes: currently 40 such curated schemes are publically available, including those for metabolic pathways, plasmid and phage components, typing profiles, and genomic regions [de novo WGS assembly, cgMLST analysis ameliorates these problems as, in a high-quality draft genome, such problems affect a minority of loci which are unlikely to materially alter an analysis [To maximise international public health impact, the EMSC-GL is hosted within the pubMLST.org/neisseria web-accessible database, which also hosts the MRF-MGL, and allows members to submit genome data and the isolates associated provenance information. This provides publicly-accessible sequence data, described with a uniform nomenclature and linked to relevant provenance and phenotype information. As of early 2017, the continually-expanding pubMLST.org/neisseria database comprised over 2,400  regions . The WGS regions ,29. It i regions . For exa regions . While eanalysis .csw) and NEIS2163 (csy) genes and, while there is no evidence of multiple capsule expression, horizontal genetic transfer of region A capsule genes has been described for other isolates [Given its importance in invasion and as a vaccine antigen the meningococcal capsule, which defines the serogroup, is one of the most important isolate characteristics required for disease surveillance. The capsule encoding genome regions are analysable by WGS, resolving anomalies that can arise from phenotypic data and enabling the characterisation of meningococci that do not express capsule. The EMSC-GL contained a small number of isolates with indeterminate W/Y expression and isolates containing genes of more than one capsule type . The difisolates ,32. The isolates . Finallyisolates ,34.Analysis of cgMLST data defined 13 lineages, had good ccs concordance as defined by conventional MLST, and identified a new lineage. Vaccine antigens were also rapidly extracted, including those used to assign BAST types, demonstrating a strong association between vaccine antigen variants and cc among the EMSC-GL. This association was consistent with previous studies in other countries such as Ireland, the Netherlands and the UK, and is important when considering the likely impact of protein-based vaccines because prevalent lineages change over time likely causing changes in disease incidence ,19,35.The deposition of future meningococcal genome data in the EMSC-GL will permit the resolution of the epidemiology of IMD at the whole genome across the entire continent, and as long as the European Union (EU) maintains funding the infrastructure costs are minimal. The inclusion of additional isolates in close to real time will enable the rapid identification of outbreaks. The past 40 years have seen the spread of successive hyperinvasive meningococci throughout Europe and, while disease levels remain low in most European countries most of the time, epidemics, hyperendemics, and localised disease outbreaks occur regularly across the continent, including transnational outbreaks ,26,36-38https://pubmlst.org/databases/).In conclusion, the detailed meningococcal isolate characterisation available thought the EMSC-GL will be essential for European IMD surveillance, leading to the improvement of control strategies and disease prevention across the continent. This model is generalisable to other bacterial pathogens, many examples can be found on the pubMLST.org website (