Ursus americanus luteolus) was removed from the list of threatened and endangered species under the U.S. Endangered Species Act in 2016 and our objectives were to determine the most appropriate parameters and thresholds for monitoring and management action. Capture mark recapture (CMR) data from 2006 to 2012 were used to estimate population parameters and variances. We used stochastic population simulations and conditional classification trees to identify demographic rates for monitoring that would be most indicative of heighted extinction risk. We then identified thresholds that would be reliable predictors of population viability. Conditional classification trees indicated that annual apparent survival rates for adult females averaged over 5 years (Monitoring vulnerable species is critical for their conservation. Thresholds or tipping points are commonly used to indicate when populations become vulnerable to extinction and to trigger changes in conservation actions. However, quantitative methods to determine such thresholds have not been well explored. The Louisiana black bear ( Ecological monitoring is an important component of natural resource management and conservation of biodiversity . In receMonitoring threatened or endangered species during the recovery phase often focuses on factors such as population response to change in habitat quantity and quality, abatement of risks to the target species, or overall assessment of population status relative to recovery criteria. Because recovery criteria should be based on clearly justified demographic targets meant to alleviate threats to long-term persistence , recoverUrsus americanus luteolus) threatened status under the U.S. Endangered Species Act (ESA), listing loss and fragmentation of habitat as the primary threats [U. a. luteolus from the list of threatened species in March 2016 [In 1992, the U.S. Fish and Wildlife Service (USFWS) granted the Louisiana black bear ( threats . The 199 threats . Laufenb threats  performe threats  concluderch 2016 .A post-delisting monitoring plan was developed and implemented by the Louisiana Department of Wildlife and Fisheries (LDWF) and the USFWS . As is tOur general approach was comprised of 2 stages. First, we conducted a sensitivity analysis using stochastic population simulations combined with conditional classification trees to identify demographic rates most important to long-term extinction risk for bears of the TRB and UARB subpopulations. The second stage was to identify short-term demographic parameters that could be used as indicators of long-term population persistence  at the TRB and the UARB that could be estimated by modifying existing data collection protocols.To perform a sensitivity analysis, it was first necessary to develop a population projection model for calculating extinction rates. The CMR estimates used by Laufenberg et al.  were basN), annual apparent survival (φ), annual per-capita recruitment (f), annual realized population rate-of-change (λ), and weekly detection probabilities (p). To separate sampling variance from process variance for φ and f, annual values for each of those vital rates were treated as random variables coming from a common hyperdistribution using an appropriate link function. The hyperdistribution for φ was logit (φ) = μφ + εt where εt ~ Normal  and μφ was the overall mean annual apparent survival on the logit scale, εt was the annual deviation from the mean in year t, and σφ2 was the temporal process variance. Temporal process variation of f (σf2) was similarly modeled. To model density dependence in per-capita recruitment [f and N was defined as log (f) = β0f+ β1fNt + εt, where εt ~ Normal  and β0f and β1f were the intercept and slope parameters, respectively. Thus, demographic rates used in the projection model included μφ, σφ (standard deviation of σφ2), β0f, β1f, and σf (standard deviation of σf2). Apparent survival (φ) included mortalities and emigrants. Estimates of f included births and immigrants and, when summed with φ, produced the finite population growth rate (λ), which represented the annual realized rate of change in abundance as a function of births, deaths, immigration, and emigration.Laufenberg et al.  used a hruitment , we defiWe adopted a Bayesian approach to population viability analysis based on stochastic population projection methods to assess probability of persistence. A population projection simply is 1 of many possible population trajectories, some of which are more likely to occur than others based on a stochastic model with a number of simplifying assumptions that govern population dynamics. Probability of persistence can be inferred from those trajectory outcomes most likely to occur  while accounting for uncertainty caused by stochastic population processes. Our goal was to develop a set of models based on a range of biologically reasonable model parameters and assumptions to project subpopulation trajectories and characterize persistence probabilities.φ and f) than did Laufenberg et al. [We used Monte Carlo methods to include 2 types of random variation into each model: temporal  stochasticity in annual vital rates and demographic stochasticity in individual life-history events. One goal of the sensitivity analysis was to simulate population trajectories across a much wider range of demographic conditions  and annual values of λ, φ, f, and N.For each projection model, we simulated trajectories over a 100-year period. We generated 2,000 random combinations of projection model parameters from the above uniform distributions for the TRB and 2,000 for the UARB. We then used each of those combinations to assess stochasticity by simulating 500 population trajectories for 100 years resulting in 1,000,000 trajectories for each subpopulation. For each trajectory, we recorded the model parameter values used in the projection (N100) ≥ 1 and assigned a value of 0 otherwise. We then randomly selected 500,000 of the 1,000,000 simulated trajectories as a training data set for which we constructed random forests of conditional classification trees for the TRB and the UARB using the cforest function in the R package party [μφ, σφ, β0, β1, and σf). We restricted the size of individual trees by limiting tree depth to 4 levels to ensure interpretability of individual trees and to reduce suboptimal performance associated with overfitting when sample size is large and the number of explanatory variables is small [varimp function in the party package. We assessed model fit and predictive performance of the random forests by calculating the overall classification error rate and the error rate for incorrectly classifying a trajectory that went extinct as extant . We performed those calculations for a holdout sample independent of the training data set that was comprised of 10,000 trajectories randomly selected from those trajectories not selected for the training data set. We considered Type II error  to be the most appropriate for endangered species decision making.To identify the most important parameter values for predicting extinction risk, we chose recursive partitioning, a widely used method for nonparametric regression and classification. Recursive partitioning is a multivariable analytical method that creates a decision tree that attempts to classify members of the population by splitting them into subpopulations based on dichotomous independent variables. A type of recursive partitioning is known as random forests, which has been successfully applied in many scientific fields . Random ge party –30. Explis small . We set N, φ, and λ extracted from the training data set. We calculated the mean value for each variable observed during the first 5 . To evaluate which demographic parameters estimated over a 5-year time span best predicted extinction risk, we derived new variables based on values for Traditional statistical analysis methods  can be inadequate for revealing complex nonlinear relationships in high-dimensional ecological data, whereas machine-learning methods such as classification trees are well suited to such situations ,33. We uN and λ could range from 0 to ∞ and φ could range from 0 to 1.We were primarily interested in identifying demographic rate thresholds that would be reliable indicators of long-term population persistence  and could be measured over relatively short monitoring durations . Therefore, we constructed conditional classification trees for the TRB and UARB populations based on demographic rates consistent with our sensitivity analysis over a 5-year monitoring duration. The classification tree was focused on duration-specific average values derived from annual demographic rates generated by our population simulations. Those averages were defined based on rate-specific numeric scales where ctree function in the R package party [N100 <1 and EXTANT otherwise, to accommodate the data structure required by the ctree function. Again, we restricted the size of the trees to 4 levels, set the number of explanatory variables selected for determining splits to 3, and set the number of trees in the forest to 100; all other function arguments were left at default values. We assessed model fit and predictive performance based on a hold-out sample of 10,000 trajectories independent of the training data set as we did for the random forests analysis. We defined reliable demographic rate thresholds as scenarios  from our classification trees that resulted in persistence probabilities ≥95% for 100 years.We used the same training data sets and explanatory variables as our variable importance analysis and the ge party  to grow φ on the logit scale (μφ) was identified as the long-term rate with the greatest relative importance for predicting extinction of the TRB subpopulation (φ (σφ) was also a strong predictor for the TRB. Conversely, σφ had the greatest relative importance to extinction risk and μφ was only moderately important in the UARB  were classified as extant . Scenari00 years . At UARB00 years . Of the φ. This scenario represents a significant decrease in monitoring intensity at UARB compared with what was prescribed in the monitoring plan (116 traps sampled for 3 weeks/year)[Our simulations suggest that a threshold of R models  or CormaR models , both ofR models . Post-hoeks/year).φ, whereas the TRB subpopulation was most affected by the long-term mean of annual female φ. Greater importance of temporal variation to persistence of the UARB is reasonable given its relatively small size which makes it more susceptible to environmental stochasticity, even when long-term average female λ is positive [φ also was evident in our short-term demographic rate classification trees as it was included in every scenario that resulted in population persistence (≥95%). Error rates of short-term demographic rates were smaller than long-term rates for the TRB, possibly related to lower importance values of the explanatory variables in the former data set relative to the latter. Also, UARB had lower error rates than TRB, likely because the smaller initial abundance used in simulations for UARB caused its likelihood of extinction to be more sensitive to small perturbations in important demographic rates which translated into greater predictive power for those rates.A better understanding of the demographic rates that drive population dynamics is vital to long-term planning, especially for monitoring persistence, and our sensitivity analysis helped to provide that insight. For example, viability of the UARB subpopulation was most sensitive to variation in annual female positive ,38,39. Tφ identified by the classification trees has been reported to be a primary driver of black bear population dynamics based on parametric statistical analyses [The conditional classification tree methods that we employed were designed to focus on detecting and describing patterns in the data rather than allowing for insights into ecological process , which ianalyses ,43,44.We identified design improvements that would result in monitoring efforts that better track population persistence, were cost-effective, and maintained data collection protocols consistent with recovery monitoring. Discovering such improvements is the primary goal for Phase 4 of the “road map” for designing and implementing a monitoring program developed by Reynolds et al. . The traLindenmayer et al.  describe


This vaccine is based on pestivirus chimera “CP7_E2alf” that carries the main immunogen of CSF virus “Alfort/187”, glycoprotein E2, in a bovine viral diarrhea virus type 1 backbone (“CP7”). This review summarizes the available data on design, safety, efficacy, marker diagnostics, and its possible integration into control strategies.Due to its impact on animal health and pig industry, classical swine fever (CSF) is still one of the most important viral diseases of pigs. To control the disease, safe and highly efficacious live attenuated vaccines exist for decades. However, until recently, the available live vaccines did not allow a serological marker concept that is essentially important to circumvent long-term trade restrictions. In 2014, a new live attenuated marker vaccine, Suvaxyn The presented review gives an overview on published or otherwise available data  remains one of the most important viral diseases that affect sustainable pig production world-wide . To contountries . These vountries , 23, 32.ountries , 50 thatountries ; modifieountries  for markountries . It becaountries , 12 and The pestivirus chimera “CP7_E2alf” was constructed based on the infectious cDNA clone of the cytopathogenic bovine viral diarrhea virus (BVDV) strain “CP7”. In this backbone, the E2 coding region was replaced with that of CSFV strain “Alfort/187” . The parThe subsequent steps for the generation of the chimera are detailed by Reimann et al. . In briehttp://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Product_Information/veterinary/002757/WC500185867.pdf (visited August 7th 2016).The manufactured vaccine is now grown in porcine kidney cells (SK cells) in either roller bottles or bioreactors. The final bulk antigen is stabilized with a commercial stabilizer and formulated in a lyophilized way. The summary of product characteristics can be found at the EMAs webpage: In brief: a chimeric pestivirus was constructed based on the infectious cDNA clone of BVDV “CP7″. In this backbone, the E2 was exchanged with that of CSFV “Alfort/187”. The resulting virus “CP7_E2alf” replicates mainly in porcine cells.While safety and efficacy are crucial parameters for an emergency vaccine, it is also essential to demonstrate that a genetically engineered RNA virus is genetically stable, i.e. does at least not show higher mutational rates as its parental viruses.st and 11th passage of “CP7_E2alf”, PCR products spanning different fragments of the genome region encoding the structural proteins were cloned into plasmids . A minimum of eight clones per fragment taken from individual PCR runs were sequenced along with direct sequencing of the respective RT-PCR products [rns at positions H450R, and G479R, E1 at position L686H, E2 at positions M783T, S 973P, R1023H, and V1035E. Of these variations, the G479R exchange is of special interest, as it plays a major role for the interaction with membrane-associated heparan sulfate and thus cell culture adaptation [th passage of “CP7_E2alf” and the first GLP (good laboratory practice) produced batch [136 N), Erns (L478F), E2 (S981P), p7 (Y1071C), NS2 (V1561L) and NS4B (AM2457V), and two substitutions were found in the NS5B  encoding region. It is obvious that only one mutation concerned the immunogen E2, and it does however not affect the known B- or T-cell epitopes. The other changes in the backbone cannot influence the induction of neutralizing antibodies. Judging on the genetic stability, it should be kept in mind that the variability of the vaccine virus was narrowed down upon establishment of the master seed virus through end-point dilution of the pre-master seed virus.To generate reliable sequence data for construct characterization and comparison of the 1products . Under taptation . Only thed batch . In generns protein gene. Ten cell culture passages did not lead to additional changes in the consensus full-length sequence. On quasispecies level (high frequency variants), no substitutions were observed in the first five passages. In passage 10, five high frequency variants were observed, all in the backbone sequence outside the structural protein genes. Moreover, no recombination events could be demonstrated. The comparison of the vaccine virus and the virus in a tonsil from an animal trial revealed one substitution in the NS5B region of “CP7_E2alf”. Due to the low viral load, the latter comparison was only possible based on overlapping PCR products [Stability of the final vaccine formulation was addressed in in vitro and in vivo studies reported by Goller et al. . In brieproducts .In brief: “CP7_E2alf” proved to be highly stable both in vitro and in vivo. No indications exist for recombination events or an increase in mutational events compared to its parental viruses.Following the design and initial testing of “CP7_E2alf”, innocuousness for target and non-target species was investigated. For the assessment of the latter, high titres of “CP7_E2alf” were applied orally to calves, goat kids, sheep lambs, and young rabbits. Additional animals were left as sentinels for transmission. None of the animals seroconverted and vaccine virus was not found in any of the samples taken from both vaccinees and sentinels . In the With regard to virus distribution, König et al.  studied Supplementary studies were done to assess the presence of the vaccine chimera in faeces, urine, and organs of the male reproductive tract . To thisIn brief: “CP7_E2alf” proved to be innocuous and safe in target and non-target species. Limited replication occurs primarily in the tonsils and is detectable there over several weeks mainly through the use of real-time RT-PCR. The vaccine virus is not transmitted or shed through urine, faeces or semen.7 tissue culture infectious doses 50% (TCID50) of “CP7_E2alf” conferred full protection against challenge infection 28 dpv with the highly virulent CSFV strain “Eystrup”.In the framework of the initial studies reported by Reimann et al. , intramuEfficacy for oral immunization of wild boar was first addressed in the study reported by König et al. . It was 4.25 TCID50), and challenge after 28 days [Later on, it was demonstrated that full clinical protection against highly virulent challenge with CSFV “Koslov” was conferred 1 week after intramuscular and 2 weeks after oral vaccination . MoreoveSupplementary studies with very early challenge showed partial protection as early as 2 dpv . In thisLately, an additional pre-registration efficacy study with MDA negative piglets was published by Lévai et al. . This stAs part of the licensing procedure, duration of immunity had to be investigated. As mentioned in the OIE Manual and the Phr. Eur., duration of immunity after vaccination against CSF shall not be less than 6 months. This has to be demonstrated in at least 10 vaccinated pigs. A corresponding study  was undeTo address the request for an efficacy and cross-protection against all strains and types of field viruses, an efficacy trial with intramuscular and oral vaccination was carried out using challenge strains of more recent genotypes, namely 2.1  and 2.3  along with CSFV “Koslov” of genotype 1. The study demonstrated that solid protection was achieved against all employed genotypes and thus, broad applicability under relevant field conditions can be assumed .For the risk assessment towards (emergency) vaccination strategies that include breeding animals, the impact of maternally derived antibodies (MDA) is a crucial parameter. For “CP7_E2alf”, one study was so far published that addresses “CP7_E2alf” derived MDA . Two othIn the study reported by Rangelova et al. , pregnanVery recently, efficacy of intramuscular vaccination was also confirmed in animals with pre-existing BVDV-1 antibodies . The stuIn brief: “CP7_E2alf” showed repeatedly that protection is conferred within 1 week post intramuscular vaccination or even less, and the immunity lasts for at least 6 months. Oral vaccination and vaccination of MDA positive or very young piglets can slightly reduce the efficacy.Live attenuated vaccine strains such as the C-strain or “CP7_E2alf” show a very limited replication even in the target host  and thusrns antibodies. Animals vaccinated with “CP7_E2alf” will carry CSFV E2 but not CSFV Erns antibodies while field virus infected animals will also show CSFV Erns responses. At present, two CSFV Erns ELISAs are commercially available. One is the PrioCHECK CSFV Erns , the other the pigtype CSF Marker (Qiagen).The serological marker system of “CP7_E2alf” is based on the detection of CSFV-specific Erns response. In this case, a negative result is not caused by a lack of sensitivity but represents the true status of the sample [rns antibody responses under field conditions. Taken together, there is still room for improvement, and the test systems should target populations/herds rather than individual animals.In general, there are some biological issues that influence the performance of the discriminatory assays and limit their optimization. If for example solid and almost sterile immunity/protection is achieved, the lack of challenge virus replication might also lead to a missing Ee sample . Anotherrns antibody ELISA showed problems with sensitivity and cross-reactions with other pestiviruses [In a first inter-laboratory assessment of CSFV antibody detection assays that could be used for the differentiation of infected from vaccinated animals, the E2 antibody ELISAs showed highest sensitivity and specificity. Under these ringtrial conditions, the PrioCHECK CSFV Eiviruses . It has iviruses , 17, 48.iviruses .rns ELISA and would therefore score false positive. Based on the same assumptions, false positives in a negative (unvaccinated) population would be very unlikely (< 0.001% of samples tested in a comparable population). Eble et al. observed also weaknesses with regard to cross-reactivities with BVDV-2 and (to a lesser extent) Border disease virus (BDV) antibodies.In the comparative study published by Eble et al. , all “CPrns [Recently, another ringtrial was conducted among five international partners to review the current version of the PrioCHECK CSFV Erns . Based orns Ab, Qiagen) were published [rns -specific antibodies against CSFV isolates of different genotypes was 93.5% combined with a specificity of 99.7%. In combination with “CP7_E2alf”, the novel ELISA showed a sensitivity of 90.2% and a specificity of 93.8%. Cross-reactivity with antibodies against ruminant pestiviruses was observed. In the framework of the evaluation, it was confirmed that false positive reactions are rarely found in both available Erns antibody ELISA systems.Recently, data on the evaluation of the Erns—specific double-antigen ELISA  and specificity (97%). At least under the reported conditions, this indirect ELISA was superior to the commercial PrioCHECK CSFV Erns ELISA [Recently, yet another CSFV Ens ELISA .In brief: suitable tools are available for both genetic and serological DIVA. The latter has still some room for improvements and has to be embedded in a concerted control strategy.The onset of detectable E2 specific antibodies was reported between 11 and 15 days after oral immunization  and in t50 at sampling 1 week after birth. These estimates are in line with C-strain data, where the half-life was reported to be approximately 12 days [50. Recently, MDA kinetics were also investigated after single vaccination of pregnant sows 21 days before farrowing [In terms of MDA kinetics, a separate study group was monitored for the decay of MDA in the study published by Eble et al. . Based o 12 days . The corarrowing . The E2 Renson et al.  showed tIn brief: usually, “CP7_E2alf” induces antibodies within the first 2 weeks post vaccination and the antibodies persist for at least 6 months.rns antibody ELISA  was observed with a total of 85% of animals scoring correctly negative.Due to legal restrictions (prophylactic vaccination is prohibited within the EU), field studies were difficult to implement. However, it was possible to carry out a small study on oral immunization in two so-called “faunistic hunting farms” in Umbria, Italy . For thiIn brief: a first field trial gave promising results using the bait formulation routinely used for C-strain vaccination.With the licensing of “CP7_E2alf”, another tool of CSF control has become available and warrants revisiting the option of emergency vaccination of both domestic pigs and wild boar. Marker vaccines can only be part of a concerted control action and are no substitute for sanitary measures . Such meIn the recent past, it was demonstrated that oral vaccination of CSF affected wild boar populations can help to control the disease and prevent introduction into the domestic pig population , 52. So In brief: “CP7_E2alf” is a new instrument in the tool-box of CSF control. It can be used to revisit emergency vaccination scenarios.


Treatment of clavicular non- and malunion is still challenging. Current surgical procedures often result in frustrating functional outcome along with high-grade subjective impairment and increased rates of revision surgery. However, the combination of biological augmentation with vital bone graft and a biomechanically sufficient fixation system seems to be a promising concept of treatment.n = 11) and/or malunion (n = 3) of the clavicle were enrolled. All patients were surgically treated using an anatomical precontoured locking compression plate (LCP) and autologous iliac crest bone graft. Functional outcome was assessed using the age- and sex-specific relative Constant Score.In this retrospective study, 14 patients with a mean age of 44 years (26–67 years) suffering from non-union (p < 0.05). All patients showed bony union radiographically. One patient presented with a re-fracture of the clavicle nearly 3 years after revision surgery and 5 weeks after implant removal. Secondary fractures at the donor site of the anterior superior iliac spine were recorded in two patients.Mean follow-up was 27 months (range 12–44 months). The relative Constant Score significantly improved from preoperative 61 ± 8 (43–72) to 82 ± 10 (65–100) points at the final follow-up examination (Iliac crest bone graft and anatomic locking plate fixation allow for a safe and adequate stabilization and radiographical bony union in non- and malunions of the clavicle with a high degree of patient satisfaction. However, secondary fractures of the anterior superior iliac spine constitute relevant complications and the time of hardware removal should be considered carefully. Mal- and/or non-union of the clavicle result in several disabling problems for both patient and treating surgeon. Potential symptoms include pain, loss of full range of motion, cosmetic deformity, poor quality of sleep and loss of power. The clavicle is predisposed to non-union due to its subcutaneous position and the influence of fracture associated soft-tissue damage . IncidenAll consecutive patients who were treated for clavicular non- or malunion with a locking compression plate and an iliac crest bone graft between January 2010 and December 2014 were included in this retrospective study. The study protocol was approved by the local ethics committee .The preoperative symptoms included pain, limited range of motion, pain when sleeping on the affected side, skin irritation caused by dislocated screws, feeling of weakness of the arm and crepitation.Standard radiographs of the affected clavicle  were performed at the time of the initial presentation of the patient as well as during the routine follow-up examinations in our outpatient clinic. Preoperative planning included the performance of a magnetic resonance imaging (MRI) scan to estimate the non-vital parts of bone to be resected as well as the performance of a computed tomography (CT) scan of both clavicles to measure the original clavicle length to be able to adequately decide if a bone graft would be required. A tricortical iliac crest bone graft was used in all cases with bony defects larger than 15 mm due to the risk of abnormal shoulder biomechanics following clavicular shortening possibly resulting in pain, shoulder motion impairment and loss of strength .The Constant Score was used to assess the shoulder function and activity preoperatively as well as during the routine follow-up examinations in our outpatient clinic. Subsequently the original Constant Score values were converted according to Gerber et al.  to receiThe LCP (locking compression plate) superior anterior clavicle plate with the possibility of lateral extension  is an anatomically precontoured fixation system with three to eight medial shaft holes for 3.5 mm locking or 3.5 mm cortex screws and in case of lateral extension six lateral 2.7 mm divergent locking or 2.4 mm cortex screws. This implant was used for treatment of all enrolled patients.All patients underwent surgery placed in a beachchair position with the affected arm in a mobile position. A longitudinal skin incision was set below the clavicle with subsequent incision through the clavipectoral fascia also in longitudinal direction to allow for a later closure to ensure sufficient soft tissue coverage. After exposure of the non- or malunion a complex multidimensional osteotomy of the clavicle with medial and lateral axial correction up to vital bone was performed . Vital bRegarding postoperative rehabilitation primarily the affected arm was immobilized in a sling for 6 weeks. Patients started physiotherapy on the first postoperative day following a standard rehabilitation protocol: abduction and flexion were restricted to 30° for the first two weeks, to 60° for the week three and four postoperative and to 90° for week five and six postoperative. Full weight bearing was not allowed before week 12 postoperative. Contact sports were not allowed before 6 months postoperative. 3, 6, 12 and 24 weeks postoperatively radiographs were performed to evaluate bone healing.p-value <0.05 determined significance. Statistics were calculated using commercially available programs .Data is given in terms of the arithmetic mean ± standard deviation and the range in brackets. Preoperative and final relative Constant Scores were compared by calculating the Wilcoxon rank-sum test. A n = 12), limited range of motion (n = 2), pain when sleeping on the affected side (n = 2), skin irritation caused by dislocated screws (n = 1), feeling of weakness of the arm (n = 1) and crepitation (n = 1). In 13 patients the non- or malunion occurred in the midshaft of the clavicle whereas in only one patient the non-union was allocated at the lateral third of the clavicle. In this context Fig. Between January 2010 and July 2014, 14 consecutive patients  with a mean age of 44 years (26–67 years) suffering from 11 clavicular non-unions and 3 malunions respectively were enrolled in the study and treated using an iliac crest bone graft and the Synthes® LCP superior anterior clavicle plate .All three surgeons  who performed the surgical procedure are experienced upper extremity surgeons. CK performed surgery in 8, PB in 4 and SHW in 2 cases. Due to a defect size larger than 1.5 cm an iliac crest bone graft was used in all patients. Average duration of surgery accounted for 146 min (97–201 min) and the mean dose area product was 46 cGycmth (patient No. VIII) and 15th (patient No. IV) postoperative day respectively. In 5 of 14 patients the implant was removed on average 28 months (range 23–35 months) after surgery. Main reasons for hardware removal were the patients’ explicit request due to implant-associated skin irritation while carrying a heavy backpack or sensitivity to weather changes. Patient No. IX presented five weeks after implant removal as well as 33 months postoperatively, with a sudden onset of pain in her left shoulder during swimming. Radiographic control showed a re-fracture of the clavicle  (Table The mean age- and sex specific Constant Score significantly improved from preoperative 61 ± 8 points 43–72) to 82 ± 10 points (65–100) after a mean follow-up of 27 months (12–44 months) (–72 to 82p < 0.05). However two secondary fractures of the anterior superior iliac spine and one re-fracture after implant removal occurred in the presented patient collective.In the present study we report the clinical and radiological mid-term outcome following treatment of clavicular non- and malunions using iliac crest bone grafting and locking plate fixation. The relative Constant Score significantly improved from preoperative 61 ± 8 to 82 ± 10 points at a mean follow-up of 27 months (The presented study collective consists of 14 consecutive patients with a mean age of 41 years and a male–female ratio of 4:3 comparable to other outcome studies concerning age and gender , 15, 16.Major complications in the treatment of clavicular non- and malunion as performed in the presented study contain loss of plate fixation , persistIn 5 of 14 enrolled patients elective implant removal was performed by the patients’ explicit request because of complaints such as pain if carrying a heavy backpack or sensitivity to weather changes. In the current literature, routine removal of metal implants remains a controversial issue with a lack of evident guidelines in the surgical treatment of acute clavicle fractures as well as of clavicle non- or malunions. Due to the typical surgical risks and complications, implant removal should only be performed in symptomatic patients after a detailed discussion of potentially available treatment options. Compared to other bony sites of the upper limb, removal of metal implants is most commonly performed in the clavicle , most lip < 0.05; see Table Follow-up evaluation was performed during the routine follow-up examinations at the outpatient clinic by assessing the Constant Score. The original Constant Score values were converted according to Gerber et al.  to receiThe major drawbacks of the presented study are its retrospective nature without the assessment of a control group and the low number of included patients. However the most studies focusing on the outcome of clavicular non- or malunions contain a comparable number of cases due to the general low incidence of non-unions of the clavicle. The low incidence is also the reason that we found no comparative study published in the literature. Another drawback presents the postoperative rehabilitation that was performed on an outpatient basis. Thus it was not performed in a standardized way and although physiotherapy should be done according to a standard protocol we cannot guarantee the patient’s compliance.Iliac crest bone graft along with anatomic locking plate fixation allow for a sufficient biomechanical stabilization and lead to radiographical bony union in non- and malunions of the clavicle with a high degree of patient satisfaction. However, complications at the donor site such as secondary fractures of the anterior superior iliac spine constitute relevant complications. Hardware removal should be individually discussed and the time of removal should be considered carefully.


Staphylococcus aureus and Streptococcus pneumoniae, some hosts may carry the pathogen and transmit it to others, yet display no symptoms themselves. These asymptomatic carriers contribute to the spread of disease but go largely undetected and can therefore undermine efforts to control transmission. Understanding the natural history of carriage and its relationship to disease is important for the design of effective interventions to control transmission. Mathematical models of infectious diseases are frequently used to inform decisions about control and should therefore accurately capture the role played by asymptomatic carriers. In practice, incorporating asymptomatic carriers into models is challenging due to the sparsity of direct evidence. This absence of data leads to uncertainty in estimates of model parameters and, more fundamentally, in the selection of an appropriate model structure. To assess the implications of this uncertainty, we systematically reviewed published models of carriage and propose a new model of disease transmission with asymptomatic carriage. Analysis of our model shows how different assumptions about the role of asymptomatic carriers can lead to different conclusions about the transmission and control of disease. Critically, selecting an inappropriate model structure, even when parameters are correctly estimated, may lead to over- or under-estimates of intervention effectiveness. Our results provide a more complete understanding of the role of asymptomatic carriers in transmission and highlight the importance of accurately incorporating carriers into models used to make decisions about disease control.For infectious pathogens such as  We designate these asymptomatic hosts, ‘asymptomatic carriers’ or ‘carriers’ and reserve the term ‘symptomatic infectious’ for those with observable clinical manifestation, who are more readily identified and targeted by disease control efforts.The elusive nature of asymptomatic carriers has three important implications for understanding and controlling infectious diseases with carriers. First, incidence data typically only reflect symptomatic cases of infection, making the true extent of asymptomatic carriage for particular diseases difficult to assess. For example, estimates of the population-level prevalence of influenza carriage range from 5 to 35% . For EboSecond, the sparsity of direct evidence of asymptomatic carriers makes interpretation of the epidemiological record difficult, creating uncertainty when defining details of the carrier state and a pathogen’s more complex natural history of disease ,6. For eThird, the presence of asymptomatic carriers undermines control interventions that rely on identifying infectious cases, such as border monitoring or isolating and/or treating infectious cases . AsymptoStaphylococcus aureus (MRSA) [Mathematical modelling has helped to unravel some of the complexities of asymptomatic carriage and its implications for control ,12–14. Fs (MRSA) . Accordis (MRSA) . This unThere is a need to understand how the structure of a mathematical model, specifically, how a model depicts asymptomatic carriage, affects its behaviour. To address this need, here we review existing mathematical models of infectious disease transmission for the purpose of identifying the types of model structures that are used to describe transmission with asymptomatic carriers. Based on this review, we define a new model of pathogen transmission that enables us to systematically compare the behaviour of alternative model structures and how they influence the estimated impact of control interventions. First, we analyse this model to establish the contribution of asymptomatic carriers to measures of pathogen transmission, the prevalence of symptomatic infections and the total infection (both symptomatic and asymptomatic) prevalence. We then consider how excluding asymptomatic carriers from this model could lead to the misinterpretation of data and the conditions under which this obscures the most important drivers of pathogen transmission. Finally, we evaluate the impact of a range of interventions on transmission and the prevalence of disease, and discuss how the choice of model structure might affect the assessment of these interventions.Streptococcus pneumoniae vaccines. These vaccines only protect against about 10% of this pathogen’s 90 identified serotypes. Those included in the vaccine are selected for their predominance as causes of invasive pneumococcal disease, allowing less virulent (carriage) strains to persist [Our analysis reveals that interventions that alter the relative incidence of symptomatic infections compared to asymptomatic carriers are particularly vulnerable to being incorrectly assessed by models with inappropriate structure. Examples of this type of intervention are the multivalent  persist , essenti2.2.1.S. aureus (nine studies), hepatitis B virus (4), Neisseria gonorrhoeae (3), Chlamydia trachomatis (2), Neisseria meningitidis (2), hand-foot-mouth disease enterovirus (2), Mycoplasma mycoides subsp. mycoides small colony (2), S. pneumoniae (1), norovirus (1), Trichomonas vaginalis (1), rotavirus (1), Salmonella enterica serotype Typhi (1), Bordetella pertussis (1), Acinetobacter baumannii (1), Middle East respiratory syndrome coronavirus (1), A/H1N1 pandemic influenza virus (1) and HIV (1), while eight studies considered unidentified abstract pathogens.The literature review identified 132 articles presenting models of pathogen transmission accounting for asymptomatic carriers, of which 42 articles  met our inclusion criteria . Pathogens modelled in these studies include S); asymptomatic carriers (C); symptomatically infectious (I); and immune (R). Models with an exposed state (E) were collapsed in a way such that any exposed and adjacent infectious states (either C or I) were combined into single infectious states. For example, a pathway R, from symptomatic infection: R). These generic models were sorted according to whether or not hosts can experience infection more than once , and then whether disease progression  no transitions exist between C and I; (2)–(3) transitions can occur in one direction only; and (4) transitions can occur in both directions, as shown in We categorized the remaining 10 generic models according to the presence or absence of flows between the asymptomatic carriage state 2.2.S=N−C−I =β(ηC+I)/N, where β>0 is the pathogen’s transmission rate during symptomatic infection, η>0 is the relative transmission rate of asymptomatic carriers compared to symptomatically infected hosts, and N>0 is the total host population size (which is assumed to be constant).We consider the following model of transmission of a non-immunizing infectious disease with asymptomatic carriage:The model represents the dynamics of a pathogen where the time scale of disease is typically much faster than the time scale of host births and deaths, and where infection typically does not kill hosts, such as norovirus, an influenza virus or hand-foot-mouth disease. Hence, the demographic effects of the population are ignored in the model and there is no input or output from the host population. However, in electronic supplementary material, appendix C, we provide details of an equivalent model accounting for host demography that would better describe pathogens with longer time scales of disease.α and 1−α. Hosts may also regress to asymptomatic carriage from symptomatic infection or progress to symptomatic infection from asymptomatic carriage at respective per capita rates ω≥0 and τ≥0. The model allows natural recovery to occur from both symptomatic and asymptomatic infection, resulting in hosts returning to the susceptible class at respective per capita rates γ≥0 and ξγ≥0, where ξ≥0 is the relative recovery rate of asymptomatic carriers compared to symptomatic infectives. Hence, neither recovery from infection nor recovery from carriage confers protective immunity. A summary of these parameters and variables is provided in New cases of infection are either symptomatic or asymptomatic with complementary proportions τ and ω are non-zero. Specifically, models in category 1 are characterized by τ=ω=0, category 2 by τ>0 and ω=0, category 3 by τ=0 and ω>0, and category 4 by τ>0 and ω>0.In this framework, the four model categories identified in the literature review  are defi3.3.1.3.1.1.h1=τ/(τ+ξγ) of asymptomatic carriers progress to symptomatic infection, and a fraction h2=ω/(ω+γ) of symptomatic infections regress to asymptomatic carriage. Hence, a fraction h1 of newly infected asymptomatic carriers progress to symptomatic infection at least once, a fraction k times, spending an average of g1=1/(ω+γ) time units with symptoms per instance of symptomatic infection. Similarly, all newly infected asymptomatic carriers experience asymptomatic infection at least once, a fraction h1h2 experience asymptomatic infection at least twice and a fraction k times, spending an average of g2=1/(τ+ξγ) time units without symptoms during each instance of asymptomatic carriage. Thus a newly infected asymptomatic carrier spends on average β (from symptomatic infections) and ηβ (from asymptomatic carriers), and summing gives The basic reproduction number quations ,3.1R0=, which is stable when endemic equilibrium . If recovery is only possible from symptomatic infection (so that ξ=0), then γ=0 and γξ→a constant), then The relative reproduction potential of asymptomatic carriers 3.1.2.P(t) is the proportion of cases in a host population at a given time t accounting for both asymptomatic carriers and symptomatically infected cases. In our model P(t) is given byPC(t) is the prevalence of asymptomatic carriers and PI(t) is the prevalence of symptomatic infection at time t such thatI→C (both directly and indirectly via S), and from C→I (both directly and indirectly via S). In electronic supplementary material, appendix B, we show that The total prevalence of infection quations  to zero 3.2.When confronted with a sparsity of direct evidence of asymptomatic carriage, it may be tempting to assume asymptomatic carriers play a minimal role in pathogen transmission and to disregard them in transmission models. Here, we consider the consequences of such an assumption for estimating transmission from symptomatic infections and of a disease’s basic reproduction number and prevalence.βSIS and γSIS. Similar to our model, in this SIS model the pathogen will become extinct if Excluding asymptomatic carriers from our model transforms it into a standard susceptible-infected-susceptible (SIS) model. In the SIS model, the basic reproduction number ll known  and are γSIS and γ of recovery from symptomatic infection in both models should also be equivalent so thatβSIS and β of transmission from symptomatically infected hosts. Hence, these rates may vary between the two models. By combining equations , then the basic reproduction number will be equivalent in both models so thatquations  and , and larger if this is not the case . Even when less prevalent, asymptomatic carriers can be responsible for the majority of transmissions when c. A necessary but not sufficient condition for this to occur is that asymptomatic carriers are more infectious than symptomatic hosts so that η>1 .We illustrate these results in when A<1 a, and lathe case b,c. Even3.3.β. We also consider interventions that change η (e.g. by quarantining symptomatic infectives), γ (e.g. through a mass drug administration), ξ (e.g. by treating only symptomatic cases), ω , τ (e.g. by improving sanitation) and α .Now we assess how control interventions that target different aspects of the disease process may affect the basic reproduction number First, we determine whether the slope of x∈{η,β}, and never holds true for the parameters x∈{γ,ξ}, which makes intuitive sense because increasing the infectivity of either infection type (through increasing β or η) or prolonging their periods of infectiousness (through decreasing γ or ξ) will always result in more secondary infections on average per infectious case. For parameters controlling the relative frequency of appearance of asymptomatic carriers , the reproduction potential of carriers x∈{ω}, condition , the reproduction potential again plays a key role in determining whether τ or α, or increasing ω) will always result in a lower endemic prevalence of disease We also considered the effects of the same set of interventions on the endemic prevalence of disease τ, ω and α).Overall, our analyses highlight how the relative reproduction potential of carriers ω (a), the progression rate τ (b) or the symptomatic proportion α (c). The corresponding endemic prevalences of asymptomatic carriers, symptomatic infections and total prevalence before and after these interventions are shown in electronic supplementary material, figure S3. Accordingly, if estimates of In n rate ω a, the prn rate τ b or the ortion α c. The co4.The presence of asymptomatic carriers can hamper control efforts and make it difficult to uncover the true details of the natural history of an infectious disease and to estimate the total infection prevalence. Here, we have shown that if carriage is not correctly incorporated into mathematical models used to inform control decisions, there is a risk that these models may produce substantially misleading predictions.4.1.c, this intervention may actually increase transmission and disease prevalence.Our analysis revealed the possibility that certain interventions assessed as capable of interrupting transmission may actually lead to the opposite effect, or alternatively the effectiveness of an intervention may be underestimated, making it appear unviable in some circumstances. Specifically, poor estimates of a quantity which we interpret as the relative reproduction potential of carriers The same quantity also influences whether excluding asymptomatic carriers from our model either overestimates or underestimates the contribution of symptomatic infections to pathogen transmission. If 4.2.From our analysis it is clear that the threshold behaviour determined by the relative reproduction potential of carriers  of MRSA ,17,20–22athogens ,29, thenb).Therefore, the threshold behaviour due to Our results highlight the importance of accurately characterizing the relationship between asymptomatic carriers and symptomatic infections through a purposive process incorporating the clarification of the dominant modes of pathogen transmission, the conduct of carriage studies, seroepidemiology and active contact tracing to inform the correct attribution of infectious states.4.3.The threshold behaviour that characterizes our model has been identified in other models of pathogen transmission incorporating asymptomatic carriage ,30. In tsubcritical persistence, which can allow an infectious disease to persist in a host population post intervention under certain conditions where, otherwise, it would have disappeared. In the models described in [Another threshold behaviour identified in models with asymptomatic carriers and with implications for control is a phenomenon known as ribed in ,31, subcribed in .4.4.A key result of our study is that interventions that alter the frequency of symptomatically infected hosts can lead to unintuitive outcomes in the presence of asymptomatic carriers. Vaccines targeted against a multi-strain pathogen almost invariably target only a proportion of strains. This partial coverage has the potential to change the balance of asymptomatic to symptomatic infection episodes, with implications for population-level vaccine impact.Streptococcus pneumoniae vaccine, for example, targeted the seven most-prevalent strains associated with invasive disease when it was first added to routine childhood vaccination schedules in various populations. Numerous studies documented the subsequent declining rates of asymptomatic carriage of PCV7 serotypes and of overall invasive pneumococcal disease (IPD) among young children [S. pneumoniae vaccine [The PCV7 children ,33, althchildren . Overallchildren ,33, althchildren . The mecchildren ,36. Simi vaccine . Serotyp vaccine .Streptococcus pyogenes, for example, have mostly commensal interactions with humans and occasionally cause symptomatic skin infections. When such pathogens are transmitted in environments with high rates of host injury , it is  endemic . Our anaFor example, asymptomatic carriers are thought to be more important for the transmission of healthcare-associated MRSA (HA-MRSA) than symptomatically infected hosts, reflected in the effectiveness of patient decolonization and improved hand hygiene compliance of healthcare workers for control . OutsideStreptococcus pyogenes is characterized by a large difference in the prevalence of infections in high-income settings versus settings of poverty. In fact, children in remote Indigenous communities in northern Australia, New Zealand and the Pacific Islands have the highest prevalence of skin sores caused by S. pyogenes in the world [S. pyogenes has a much lower prevalence. From our analytical results, we can deduce that asymptomatic carriers of S. pyogenes have a lower reproduction potential than symptomatically infected hosts, because increasing the proportion of symptomatic infections across host settings corresponds to an increase in prevalence.he world . These c4.5.Mycobacterium tuberculosis and HIV [S. pneumoniae and S. pyogenes age-assortative mixing and strain diversity are likely to be key drivers of observed transmission dynamics [Our proposed model of infectious disease transmission with asymptomatic carriers has a number of limitations. First, it applies to only those pathogens for which asymptomatic carriers are infectious, clearance of infection is possible and does not confer lifelong immunity, and infection does not alter the death rate of hosts. These restrictions mean that our results are not relevant to two of humanity’s leading causes of death attributable to a single infectious agent –  and HIV . A naturdynamics ,42. Ther


Alligator sinensis) is an endangered freshwater crocodilian endemic to China, which experienced a severe bottleneck about 30 years ago. In this study, we developed locus-specific primers to investigate the polymorphism of 3 major histocompatibility complex (MHC) loci in 3 Chinese alligator populations, in combination with 6 neutral microsatellite markers as a contrast. We found the genetic trace for the bottleneck effect on the endangered Chinese alligator: the low allelic diversity , the low nucleotide substitution rate (no more than 0.009) at all sites, the deviation from Hardy-Weinberg Equilibrium/heterozygote deficiency, and the significant Tajima’s D values, indicating the MHC class I and class II loci being at different stages of bottleneck. We also obtained 3 pieces of evidence for balancing selection on this severely bottlenecked reptile: an obvious excess of nonsynonymous substitutions over synonymous at the antigen-binding positions, the mean synonymous substitution rate of MHC exons significantly higher than mean nucleotide substitution rate of introns, and the differentiation coefficient FST of MHC loci significantly lower than that of microsatellite loci. Consequently, we emphasize that the Chinese alligator holds a pretty low adaptive ability and requires scientific conservation strategies to ensure the long-term population development.Chinese alligator ( Alligator sinensis) is a very ancient species that had once been pushed to the fringe of extinction by massive hunting, territory deprivation and environment deterioration1. In the 1960s, the wild Chinese alligators become very scarce, and the complicated environments of wetlands and swamps made them even harder to be spotted2. The population of Chinese alligator didn’t stop dropping until 1979 when Changxing Yinjiabian Chinese Alligator Nature Reserve (CYCANR) and Anhui Research Center for Chinese Alligator Reproduction (ARCCAR) were founded to conserve this species, starting with 7 and 212 founder alligators respectively. As of today, there are more than 8,000 captive Chinese alligators at both reserve sites, making the ARCCAR and CYCNAR populations not only the last but also the largest captive populations in China. On the other hand, the number of the Chinese alligators in the wild is dropping 20% every year and the current number is predicted to be lower than 1203.The Chinese alligator  over synonymous (dS) substitutions. Hence, the true level of genetic diversity at single MHC genes of the Crocodylia is still pending for exploration.Judging from the population size alone, the Chinese alligators seem to be recovering quite well from the bottleneck event 30 years ago, and previous studies on the major histocompatibility complex (MHC) genes didn’t raise alarms on any of the repercussions of the bottleneck effect. The MHC genes are not only the most functionally polymorphic genes in vertebrates, but also mediate immune responses, thus making them good adaptive markers to assess the evolutionary potential of fighting pathogenset al. constructed a bacterial artificial chromosome (BAC) library that contains 6.8-fold genome equivalents15. Ye et al. constructed several contigs containing MHC genes based on the BAC library16. Wan et al. published the Chinese alligator genome as 2.3 Gb in size, and annotated over 22,200 genes16. These works enable us to design locus-specific primers to characterize MHC genes, hoping to investigate whether bottleneck event continues to impact the Chinese alligator population at the single gene level even after an exponential population growth, to unveil the actual polymorphism of a single MHC gene, and to elaborate the evolutionary forces that influence MHC genes.In our previous studies on the Chinese alligator, He A. sinensis genome and predicting potential MHC genes, we found 3 MHC loci with intact coding sequences and gene structures. The 3 MHC loci can be pinpointed to different A. sinensis genome scaffolds: scaffold1303_1 (NCBI accession number: NW_005842753), scaffold364_1 (NCBI accession number: NW_005842546) and scaffold184_1 (NCBI accession number: NW_005842983). The 3 MHC loci were also verified to be from 3 BAC clones that had no overlap sequences, suggesting that they were independent loci. The BAC clones 1327C2 and 20A2 contained 2 MHC I loci and the BAC clone 1085A9 contained 1 MHC II locus; these MHC loci were accordingly named I1327, I20 and Beta1085, respectively.By BLASTing the BAC-end and known MHC sequences to the The single strand conformation polymorphism (SSCP) results of these loci were all di-morphic: 2 alleles at each locus, which were confirmed by the sequencing results. Nonetheless, the gene I1321 is actually mono-morphic at the functional level due to the same amino acid sequence of the 2 alleles  in all the MHC loci (Table HWE) test results show a severe heterozygote deficiency at the Beta1085 locus in ZJ and AH populations. Differently, the Tajima’s D test results are strange  selection; and dN/dS = 1 indicates neutral (i.e. no) selection. As we can see in Table dN and dS values due to the low allelic and nucleotide diversity, and the Z-tests show no significant P value at any locus. The following trends, however, can be observed: at the MHC class I loci, the dN/dS ratios all exceed 1 at antigen binding sites (ABS), showing the sign of positive selection. Nonetheless, at the MHC class II locus, there is no nucleotide substitution at ABS positions  in introns of the MHC class I and II genes, and plotted them against the nucleotide position  with no differentiation at the MHC loci  in introns and the mean dS in exons are usually about equal – for their mutations are both selectively neutral to a gene – except when it comes to the MHC genes, the mean dS in exons is always much higher than the mean d in introns. They explained that exons in the MHC genes are very ancient because they have been maintained by balancing selection for a very long time, while the mutations in introns are controlled by recombination and genetic drift. When an ancient intron polymorphism is lost due to recombination or drift, it will be selectively neutral to the MHC gene. As time goes by, introns in the MHC genes will become evolutionarily younger on average than are the exons constantly maintained by balancing selection. The much older exons will consequently possess a much higher dS than that of the younger introns, hence mean dS > mean d in MHC genes22. Our findings in this study support Hughes and Yeager’s theory.Therefore, we need to compare the theoretically balancing-selection-influenced sites to those non-balancing-selection-influenced sites to reveal the presence of balancing selection. The comparison between exons and introns would be an effective tool. Hughes and Yeager25. Even if the Chinese alligator populations are sustainable at present, they would still be susceptible to new pathogens when reintroduced to the wild in the future. While the balancing selection is working on the MHC genes, proper conservation strategy should be devised to protect this endangered species.The environmental factors could be another cause of the similar MHC genes among populations. Both AH and ZJ populations are intensively managed as captive populations, and their habitats are modified to natural wetlands surrounded by farming countryside. The isolated habitats may provide an environment with low level exposure to pathogens, and cause the MHC genes lack motivations to change; similar situations happen to many other speciesAll experiments were carried out in accordance with the guidelines issued by the Ethical Committee of Laboratory Animal of Zhejiang University, and all experimental protocols were approved by the Ethical Committee of Laboratory Animal of Zhejiang University.A. sinensis samples  by BLASTing the end sequences of the target BACs and the known MHC sequence against the A. sinensis genome11 using default parameters of a local BLAST tool downloaded from NCBI (ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/). Then, we used Augustus (http://augustus.gobics.de/) to predict exon positions for each MHC sequence, and manually translated each exon to amino acid sequences in order to search for MHC loci with intact coding sequences and gene structures. All the MHC genes from the BAC and the genome were manually checked for gene structural integrity to rule out pseudogenes. The MHC genes with a complete gene structure were selected for subsequent studies. We gave prefix ‘Alsi’ to all the MHC genes.We screened the BAC clones using the universal MHC primers of Ye http://www.premierbiosoft.com/), and the primers’ binding positions are illustrated in Supplementary Fig. 17.We then designed locus-specific primers to amplify the antigen binding domains of the MHC loci . Intron 2 sequences were also amplified and sequenced to ensure the correctness of genotyping as well as to be a useful comparison material (see Supplementary Table The PCR amplifications were performed in a 10 μl reaction system containing 1 L of template DNA (30–50 ng/µL), 0.5 U rTaq DNA polymerase (TaKaRa), 1 µL of 10 × ExTaq buffer (TaKaRa), 1 µL of 2.5 mmol/L dNTPs, 0.2 µL of each primer (diluted to 10 mol/µL), and 6.5 µL of double-distilled water. The PCR was programmed as follows: initial denaturation at 95 °C for 5 min, followed by 35 cycles of 95 °C for 30 s, 63 °C for 30 s, and extension at 72 °C for 30 s, with a final extension of 72 °C for 5 min.27 to genotype each individual and screen 3 Chinese alligator populations in order to examine the level of adaptive genetic variation in this endangered reptile. In the SSCP-HD analysis, we added 5 μl of 2X loading buffer  for each 10 μl of PCR product. And the mixture was denatured at 95 °C for 5 min, and swiftly transferred onto ice for cooling down. Then, the PCR products were run in an acrylamide gel consisting of 12% 37.5:1 acrylamide/bisacrylamide with 2.5% crosslinking, and sequences were separated in 0.5 × TBE running buffer at 16 °C by 150 V for 6.5 h on the Decode System (Bio-Rad). Finally, the SSCP gel was fixed in 10% acetic acid for 30 min, washed with dH2O, and achieved silver staining pictures. We repeated the PCR-SSCP process for 3 times at each locus to make sure the banding pattern was stable and consistent, and obtained the allele sequences by sequencing the homozygous individuals with unique banding patterns in BGI, Shanghai. Microsatellite loci were amplified and genotyped as described by Ma17.We adopted Single-Strand Conformation Polymorphism - heteroduplex (SSCP-HD) techniques28 for sequence alignment. We used DnaSP29 to calculate the haplotype diversity (Hd), the nucleotide diversity (π), and Tajima’s D values. The non-synonymous (dN) and synonymous (dS) substitution rates of the MHC exons were calculated using Mega 5, and we used K-estimator30 with Kimura-2p method to compute nucleotide substitution rate of intron 2 (d) as well as the dS of exon 2 and exon 3, which were then plotted against nucleotide position using a sliding window size of 15 base pairs and steps of 3 base pairs. We used Kaufman’s study31 as a reference to annotate antigen-binding sites.We used Lasergene 7 (DNASTAR) for nucleotide sequence editing and Mega 5HO), expected heterozygosity (HE) and HWE, as well as pairwise FST among 3 populations were calculated in Arlequin 3.532.Allelic frequency, observed heterozygosity (The genomic sequences of MHC genes are collected from the scaffolds 1303_1 (GenBank accession number: NW_005842753), 364_1 (NW_005842546) and 184_1 (NW_005842983) of the Chinese alligator genome (GCA_000455745.1). The new nucleotide acid sequences obtained in this study are available in the supplementary Data Supplementary Information


Such atomically thin materials have great potential for use in electronics, and are thought to make possible the first real applications of spintronics. Here in, a facile and reproducible method of producing wafer scale atomically thin MoS2 layers has been developed using the incorporation of a chelating agent in a common organic solvent, dimethyl sulfoxide (DMSO). Previously, solution processing of a MoS2 precursor, ammonium tetrathiomolybdate ((NH4)2MoS4), and subsequent thermolysis was used to produce large area MoS2 layers. Our work here shows that the use of ethylenediaminetetraacetic acid (EDTA) in DMSO exerts superior control over wafer coverage and film thickness, and the results demonstrate that the chelating action and dispersing effect of EDTA is critical in growing uniform films. Raman spectroscopy, photoluminescence (PL), x-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM) and high-resolution scanning transmission electron microscopy (HR-STEM) indicate the formation of homogenous few layer MoS2 films at the wafer scale, resulting from the novel chelant-in-solution method.It is of paramount importance to improve the control over large area growth of high quality molybdenum disulfide (MoS Graphene has played a central role in 2D materials research due to its high carrier mobility but its lack of a band gap hinders its potential for applications in the IC industry12. Unlike graphene, MoS2 possesses a film thickness dependent bandgap in the optical range 14. The change in bandgap energy is accompanied by a transition from indirect in bulk to direct in single layer MoS2, which has been observed using photoluminescence (PL) spectroscopy15. These properties make thin-layer MoS2 films suitable for field effect devices and optical applications, which further accentuate the need to achieve thin layered films over large areas. Here, we report an advancement towards full wafer scale synthesis of MoS2 by using a facile solution based synthesis.2D materials have been intensively studied for the past few decades with ever-growing interest. Large area growth of 2D materials remains a challenge that must be overcome for commercial applicationset al. first reported such a process using ammonium thiomolybdate ((NH4)2MoS4), which could be utilized for thin film applications as a single precursor with no need for sulfur addition18. Liu et al. produced MoS2 films by dip-coating silicon dioxide (SiO2) substrates in a solution prepared by dissolving (NH4)2MoS4 in dimethylformamide (DMF)19. This method, however, is difficult to scale up due to the unpredictability of dip coating and precise control of layer thickness. An alternative method -spin coating- has been shown as a more efficient way to obtain controlled layers based on the solution concentration viscosity and spin coating parameters20. George et al. performed a spin coating process by dissolving (NH4)2MoS4 in n-methylpyrollidone (NMP) and obtained various thicknesses based on solution processing20, which provided a new approach to obtain wafer scale TMD layers. However, further refinement to reduce the density of surface defects, dewetted areas, and to improve large area uniformity was still needed20.Solution based methods have been emerging as novel techniques for obtaining wafer scale 2D thin films. Many have taken advantage of either dip coating or spin coating but so far uniformity across large areas have been difficult to achieve. Putz 3) and tungsten trioxide (WO3) have alternatively been utilized to obtain MoS2, WS2 and other TMD domains and layers23. In these techniques, mostly triangular domains associated with CVD processing have been observed but their dimensions were very much limited6. Similar research done towards large area growth or large grain growth using either oxide powder or hydrothermally synthesized oxides indicated slightly larger features, but not exceeding 200 μm across the domains with most reports averaging in the range 10–20 μm24. Alternatively, work done by Kang et al. has been promising for large area synthesis of 2D materials utilizing MOCVD processing which could be scaled up and implemented at an industrial scale, but yet to be realized for wafer scale electronics applications25. Research and development for growing 2D materials will still be ongoing in the coming years to develop further novel approaches.Oxide precursors such as molybdenum trioxide  in Dimethylsulfoxide (DMSO) was first prepared before dissolving the (NH4)2MoS4 precursor. To enhance the wettability of our substrates, an RCA clean was performed followed by an HF wash. Large area films were synthesized by tuning and optimizing the ratio of precursor concentrations and spin coating parameters. Homogeneity of as synthesized MoS2 films were characterized by Raman spectroscopy, Photoluminescence (PL), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR) atomic force microscopy (AFM) and high-resolution scanning transmission electron microscopy (HR-STEM).Here, we demonstrate a uniform non-sulfur assisted method of wafer-scale synthesis of MoS4)2MoS4 dissolved in DMSO; curves in green represent EDTA dissolved in DMSO; and curves in blue represent EDTA and (NH4)2MoS4 dissolved in DMSO. Figure SO spectral region for EDTA, molybdenum precursor salt and a combination of these species in DMSO solutions. Altogether, the data identifies the regions exhibiting significant observable changes. The measurements are primarily focused on the DMSO’s S=O stretching regions and their changes in the presence of EDTA salt and a combination of external intermolecular interaction stimuli. The primary focus was the S=O region spectra since C-H region were relatively stable in spectral shape and position for DMSO even when the molybdenum salt was introduced. Figure −1 and 1018 cm−1 in the presence of any other additives in DMSO. The intensity of the band at 1041 cm−1 reduces while the intensity of the band at 1018 cm−1 increases. The additives do not change the frequency for 1040 cm−1 and shows a slight red shift from 1018 cm−1 to 1016 cm−1. No noticeable intensity changes are observed for the CH3 peak at 950 cm−1 in the presence of EDTA or only molybdenum precursor salt addition. At 697 cm−1 and 667 cm−1, no noticeable changes were observed for both intensity and frequency of the vibrational bands.FTIR data from the prepared solutions were obtained to study potential molecular interactions. In Fig. −1 to 1018 cm−1 indicate interactions of additives with the primary solvent DMSO, including changes in hydrogen bonding interactions with the solvent26. The high absorbance in this range suggests that DMSO-DMSO interactions are the strongest interactions in this system26. A more subtle effect is observed due to EDTA in this mixture, which shows a stretch behavior characteristic of aliphatic amines and carboxylic acid groups interacting with dissolved metal cations in the mixture28.The IR data provided offers an explanation for the interactions taking place in the pre-spun mixtures. Intensity changes in the region 1041 cm−1 and 1300–1200 cm−1 representing the C=O and C–O bonds, respectively28. The carboxylate ions have antisymmetric modes in the range 1650–1510 cm−1 and relatively strong symmetric COO− stretching in the 1400–1280 cm−1 range. Stretching vibrations at 1661 cm−1 in the DMSO-EDTA solution are assigned to the EDTA`s asymmetric COO− stretching and the high intensity peak at 1400 cm−1 is attributed to the symmetrical COO− stretching. The peaks in the 1360–1100 cm−1 region are attributed to NH+ stretching. The addition of a molybdenum precursor salt shifts the asymmetric COO− stretch from 1661 cm−1 to 1634 cm−128. Coordination modes of these carboxylic acids are generally in three common groups and can be distinguished in the IR spectra by their separations between the carboxylate stretch bands (Δν). The band separations are in the range of 350–500 cm−1 for monodentate binding, 150–180 cm−1 for bridging, and 60–100 cm−1 for chelating28. A separation, Δν, was observed to be 228 cm−1 in this experimental setup.The IR spectra of ketones and carboxylic acids are generally characterized by absorptions in the ranges 1750–1660 cm− asymmetric stretching region. Overall, our observations suggest that EDTA and molybdenum salt solutions in DMSO form a network, which is primarily driven by hydrogen bonding in which EDTA forms a mixture of monodentate, and bridging interactions with the molybdenum salt.The result of ATR-FTIR studies overall indicate that the addition of a molybdenum salt or EDTA separately results in the breaking of S=O. Hydrogen bonding thus disturbs the dimer-like or larger polymer-like hydrogen bonding that drives the self-assembly. Addition of all these species at once showed similar a trend for the S=O stretching region, however, a combination of all indicated significant changes for the COO−1 to 1634 cm−128. The lowered frequency of the upfield C=O stretch can be explained by a σ-donation of the acid group of EDTA to the cationic Mo(VI) metal center28. The IR spectrum of MoS42− in DMSO with EDTA is also indicative for the presence of uncoordinated carboxylic acid groups as a consistent peak at 1724 cm−1  peak to the triply degenerate silicon mode at 520 cm−1. These images confirm the presence of MoS2 over large areas while the small variation of intensity ratios demonstrates the relative uniformity of the film. While monolayer regions were observed intermittently primarily at the center of the wafer, the Raman images show that thickness varies across the wafer due to the nature of the spin coating process. To further probe the uniformity of the MoS2 films, the difference between the A1g and the E12g peaks were mapped out for the same two regions and are displayed in Fig. −1, which corresponds to the spectral resolution of the measurement system, indicating a relatively uniform film.The uniformity and consistency of the sample was tested using Raman mapping over a 25 × 25 micron area with a spectrum acquired every 333 nm. Shown in Fig. 2 region of the film. Figure 2 as Raman imaging shows small dots of high MoS2 intensity  across a 1.5 μmSee Fig. . The ove2 films. Figure 5/2 at 229.3 eV; at 232.4 eV corresponding to the Mo 3d3/2; and at 226.6 eV corresponding to the sulfur peak (S 2S) binding energy30. These characteristics are a match to the Mo4+ state, noted in previous investigations30. Binding energy for the spin orbit couple S 2p3/2 and S2p1/2 are 162.2 eV and 163.4 eV, respectively, as shown in Figure 30. The uniformity and intensity of the peaks validate the quality of the material and are consistent with the reported values for MoS2 crystal structure.XPS was used to determine the chemical composition of the as synthesized MoS2 can be observed along the [001] zone axis31. The lattice parameter of MoS2 measured in the image is 3.2 A, which is in agreement with previous findings32.To characterize the atomic structure of the solution grown film, a representative sample was transferred onto a quantifoil TEM grid  in preparation for STEM imaging. Figure 2 field effect transistors (FET) were fabricated on a highly doped Si (100) wafer with 285 nm thick SiO2 dielectric layer using 20 nm and 200 nm Ti and Au contacts. The MoS2 film was passivated with a 50 nm thick HfO2 layer in order to keep the MoS2 film from deteriorating (oxidation) over time from exposure to atmospheric conditions. Measurements were conducted at room temperature in a dark box setup with the bottom gate grounded. Figure d versus Vd curves (Id-Vd) for a top gated MoS2 FET with the top gate voltage (Vtg) ranging from −10V to 5V. Figure 2 FET, where three of the plots are the Id-Vtg curve, the applied Is-Vtg current profile and the leakage current from the top gate, Ig-Vtg. Although there was some leakage current through the top gate, it is apparent that the majority of the current is from the contribution of Id-Vtg. The current through the bottom gate (Ibg) is also shown to demonstrate that there was no leakage through the bottom gate. The total measured current for both Id-Vd and Id-Vtg are on the order of 1 pA, indicative of a resistive MoS2 film. The overall resistive behavior could be due to the presence of small domains in the MoS2 film, and/or high resistance (non-Ohmic) of the Ti/Au contacts.Double gated MoSEnhanced wetting capability of DMSO-EDTA resulted in a uniform spin-coated layer of the mixture solution on the wafer surface. To assess the effect of protonated carboxylic acid groups on wettability, a solution of tetra-methylated EDTA and ammonium tetrathiomolybdate in DMSO was prepared and spin coated according to the techniques described in the methods section. The resulting sample was found to be too uneven in quality to anneal and barely retained any of the DMSO solution on the wafer surface. Our observations suggest that the protonated carboxylic acid groups play a crucial role in retaining a uniform precursor solution on the silanol functionalized surface.42− anions when dissolved in DMSO. Primarily the EDTA/MoS4 solution is spin coated onto SiO2/Si wafers, spreading the coordinated metal ions evenly over the surface27. Heat from the first step of the annealing sequence evaporates DMSO and evolves H2S and NH3 gases leaving an equivalent of MoS3 coordinated by one equivalent of EDTA33, which then evaporates at elevated temperatures, leaving MoS3 evenly distributed on the silanol functionalized surface. Mo(VI) trisulfide is then reduced to the crystalline Mo(IV) disulfide product and generates S0 as a byproduct. The films formed using EDTA solutions were optically uniform, with thicknesses ranging from single-layer to a few-layers. As shown in Fig. 42− ions; the adsorption of EDTA to the hydroxyl-terminated SiO2 substrate; and, coordination with MoS3 prior to conversion to MoS2. In the as-prepared solution, EDTA forms multiple hydrogen bonds (H-bonds) with both the DMSO solvent molecules and the MoS42− ions. Simultaneously, the surface of the substrate, composed of hydroxyl-terminated SiO2, interacts with EDTA via H-bonds. Thus, a layer of EDTA forms on the SiO2 surface, with at least one carboxyl group from EDTA interacting with a hydroxyl group from SiO2. This can be described as anchoring the precursors to the substrate. Ryczkowski et al. identified the interactions between EDTA and γ-Al2O3 supports via FTIR/PAS measurements34. Their studies concluded that there was a high dispersion of Ni metal on the surface of the alumina supports due to interactions of the chelate with the surface hydroxyl groups34. The interactions between EDTA and the hydroxyl groups support the idea that EDTA both chelates MoS42− ions and distributes them on the hydroxyl-terminated SiO2 substrate surface via an anchoring action. We propose that, with the H-bond network established, ramping to a temperature of 100 C causes DMSO to evaporate, thereby decreasing the concentration of DMSO and increasing the concentration of MoS42−, which continues to interact with EDTA. After a film of EDTA-anchored MoS42− ions forms on the substrate, the temperature is then ramped up to 450 C. In this step, the MoS42− converts to MoS3 in the temperature range 120–260 C, according to reports by Brito et al.35. During the annealing step up to ~260 C, EDTA continues to coordinate with MoS3 until the EDTA decomposes. The EDTA-MoS3 complexation was demonstrated by Badoga et al., leading to dispersed MoS3 crystallites27. Here we demonstrate a similar process with MoS3 in the temperature range of 120–260 C, which generated a film of highly dispersed EDTA-anchored MoS3 by action of chelation27. As the temperature is ramped from 450 C to 800 C, the well-dispersed MoS3 converts to MoS2. This reaction was previously confirmed by Furimsky and Amberg, who reported the highest degree of crystallinity at 800 C, showing strong x-ray signals for hexagonal MoS236. We drew from the aforementioned strategies and chelation-synthesis mechanism to help explain the functionality of EDTA in effectively dispersing the MoS2 precursor crystallites (MoS3), as well as the reactions that take place at various temperature ranges, which ultimately convert MoS42− to MoS2. Thereby, exploitation of the related chelating properties of EDTA has led to a novel contribution to the field of TMD thin film synthesis.Further studies on molecular interactions are presented in Fig. 2 growth sites by incorporating a common chelating agent, EDTA, with ammonium tetrathiomolybdate in DMSO solvent as a spin-coating solution for MoS2 thin film growth. The thickness of the MoS2 layers can be tuned depending on the EDTA and thiomolybdate concentrations, and large-area coverage of MoS2 is made possible via the chelating action of EDTA with the thiomolybdate ions and subsequent intermediates to MoS2. Raman, XPS, and FTIR analysis confirmed the effect of using a chelating agent on MoS2 film quality and substrate coverage. Our methods could pave the way towards solution processing of a variety of TMD thin films and their heterostructures at the wafer scale for applications in electronics, optoelectronics and spintronics.In conclusion, we have demonstrated a novel approach for improving the dispersion of MoS2 thin films are as follows: First, a 4-inch diameter silicon substrate with a 300 nm thick SiO2 film (Si/SiO2 substrate) was washed with acetone and IPA for several times, followed by an cleaning in an RCA-1 solution (5:1:1 v/v H2O:NH4OH:H2O2) at 100 C for 20 minutes. After a follow up wash in DI H2O, the substrate surface was then partially etched using a dilute HF solution (1:5 v/v HF:H2O) for 20 seconds. After that, the wafer was cleaned for several times in DI H2O, then rinsed in IPA and statically dried. A 5 mL solution of 0.1 M ethylenediaminetetraacetic acid (EDTA) was prepared in Dimethylsulfoxide (DMSO), to which 60 mg of ammonium thiomolybdate ((NH4)2MoS4) (Sigma Aldrich high purity 99.99%) was added and dissolved. The solution was stirred for 2 hours, then filtered with a 0.2 μm polycarbonate filter. Several drops of this solution were dropped onto a cleaned Si/SiO2 substrate, followed by spin coating for 1 minute at 3000 rpm to establish a thin and homogenous coating on the substrate. Subsequently, the substrate was placed in a quartz tube furnace for thermal processing, which was purged for multiple times with Argon under 500 Torr vacuum. Then the temperature was ramped from room temperature to 100 C over 30 min and held for 30 min, followed by ramping to 450 C over 30 minutes and held for 60 minutes, and finally ramped to 800 C over 30 minutes and held for 10 minutes. The system was then slowly cooled, and the sample was removed for characterization.Procedures for growing single layer and few-layer MoS2 layer to release the grown MoS2 films. Samples were then sectioned and fished onto a TEM grid followed by an acetone and chloroform wash, then kept in acetone for at least 30 minutes before drying.For TEM imaging, samples cleaved from a wafer were first coated with PMMA using a spin coater at 3000 rpm and baked for 5–10 min on hotplate at 100 C. The samples were then placed into a 5% HF solution in order to etch and undercut the SiO−1. X-ray photoelectron spectroscopy (XPS) characterization was conducted using a Kratos AXIS ULTRADLD XPS system equipped with an A1 Kα monochromatic X-ray source and a 165 mm mean radius electron energy hemispherical analyzer along with a vacuum pressure of 3 × 10−9 Torr. FTIR measurements were taken using a Nicolet 6700 FTIR system having ATR accessory with a resolution of 0.500 cm−1. Scanning transmission electron microscopy (STEM) imaging of the MoS2 films were conducted using a FEI Titan G2 60–300 X-FEG aberration-corrected and STEM equipped system with a CEOS DCOR probe corrector. ADF-STEM images (2048 × 2048 pixel2) were acquired on the STEM operating at 200 keV using a dwell time of 3–6 µs per image pixel at a camera length of 130 mm. The beam convergence angle αobj was measured to be 23 mrad. The ADF detector inner and outer angles of collection were measured to be 54 mrad and 317 mrad, respectively. Under these conditions, the measured probe size was ~0.8 Å.Raman and PL spectroscopy were conducted using a Horiba LabRAM HR instrument with a laser wavelength of 532nm. Raman imaging was performed using a WiTec alpha300R system with a 532 nm light source (333 nm spot size) and a spectral resolution of +/−1 cm


The prevalence of dog erythrocyte antigen (DEA 1) in canine population is approximately 40–60%. Often data are limited to a small number of breeds and/or dogs. The aims of this study were to evaluate frequency of DEA 1 in a large population of purebred and mongrel dogs including Italian native breeds and to recognize a possible association between DEA 1 and breed, sex, and genetic and phenotypical/functional classifications of breeds. Frequencies of DEA 1 blood group collected from screened/enrolled blood donors and from healthy and sick dogs were retrospectively evaluated. The breed and the sex were recorded when available. DEA 1 blood typing was assessed by immunocromatographic test on K3EDTA blood samples. The prevalence of DEA 1 antigen was statistically related to breed, gender, Fédération Cynologique Internationale (FCI) and genotypic grouping.Sixty-two per cent dogs resulted DEA 1+ and 38% DEA 1-. DEA 1- was statistically associated with Dogo Argentino, Dobermann, German Shepherd, Boxer, Corso dogs, the molossian dogs, the FCI group 1, 2 and 3 and the genetic groups “working dogs” and “mastiff”. DEA 1+ was statistically associated with Rottweiler, Briquet Griffon Vendéen, Bernese mountain dog, Golden Retriever, the hunting breeds, the FCI group 4, 6, 7 and 8 and the genetic groups “scent hounds” and “retrievers”. No gender association was observed.Data obtained by this work may be clinically useful to drive blood donor enrollment and selection among different breeds. Blood groups are defined according to antigenic and species-specific genetic markers localized on the surface of erythrocyte cell membranes , 2. CaniDal [Antigens other than DEA were discovered in dogs, i.e. Dal –12, Kai Dal .The canine blood typing for the red blood cell antigens, other than DEA 1, can be done only by a laboratory. In clinical practice, for in-house canine blood group DEA 1 determination, commercial point-of-care typing tests i.e. typing cards and immunochromatographic strips, using murine monoclonal anti-DEA 1 antibodies, are available , 13–15.The general prevalence of DEA 1 in canine population is approximately 40–60% , 16–18. The aims of this study were (1) to evaluate frequency of DEA 1 in a large population of purebred and mongrel dogs including Italian native breeds and (2) to recognize a possible association between DEA 1+ and DEA 1- blood groups and breed, sex and genetic and phenotypical/functional breed grouping.n = 354), the Department of Veterinary Medicine, Unit of Transfusion Medicine at the University of Perugia  (n = 313), the Veterinary Laboratory “San Marco”  (n = 242) and the Department of Veterinary Medicine at the University of Milan  (n = 128).This is a cross-sectional study without an “a priori” definition of the sample. It includes data collected from January 2013 to December 2015 and obtained from dogs screened and/or enrolled as blood donors and from healthy or sick dogs evaluated during clinical activity. DEA 1 blood typing and breed were available in all the dogs, and sex in the majority of the cases. Data were obtained from the Canine Blood Bank at the Istituto Zooprofilattico Sperimentale delle Venezie   according to the manufacturer’s instructions. Based on the test results, the dogs were classified as DEA 1+ and DEA 1–. For DEA 1+ dogs the band strength was not assessed.http://www.fci.be/en/) breed classification (10 groups); ii) based on genetic grouping (10 groups) as reported by vonHoldt et al. (2010); iii) breeds with hunting function included in FCI group 6 (Scent hounds and related breeds), 7 (Pointing dog) and 8 (Retrievers-Flushing dogs-Water dogs) compared to non-hunting breeds; iv) molossian dogs belonging to both FCI group 2 (Pincher and Schnauzer-Molossoid breeds-Swiss Mountain and Cattle dogs) and group 9 (Companion and Toy dogs) compared to non-molossian dogs.The differences of DEA 1 prevalence in relation to sex and breed were tested by chi-square test or Fisher’s exact test when appropriate. Data of the breeds represented by at least 15 dogs were used for breed statistical analysis referred to frequencies. Moreover for breed comparison, dogs were grouped following different criteria: i) based on Fédération Cynologique Internationale  and their 95% confidence interval (95% CI) were calculated for each comparison made. In case of zero value, a unity was added to each number in the calculation of the ORs. The mongrels group was chosen as the reference group. For data concerning FCI and genetic group classification, every group was compared with the others and not with the mixed breed dogs.Chi-square value, http://www.winepi.net/uk/index.htm) [p < 0.01 were considered significant.Statistical analyses were performed using the freely available online software WinEpi 2.0 (dex.htm) . Values n = 205) and purebred (n = 832), male (n = 498) and female (n = 523). The sex was unknown for 16 dogs. Purebred dogs belonged to 88 breeds. Thirty-eight percent (396/1037) of all the dogs tested DEA 1- and 62% (641/1037) DEA 1+. The percentages remained the same considering the mongrel and purebred subgroups. Dogs belonging to 14 breeds were statistically analyzed. In Tables In the present study, 1037 dogs were evaluated. They were mongrels n = 05 and puNo difference in the prevalence of DEA 1 antigen was observed in relation to gender .n = 79) belonging to an Italian breeds are reported in Tables Data about DEA 1 frequency of dogs  resulted statistically associated with DEA 1+ . The molossian dogs  resulted statistically associated with DEA 1- .The hunting dogs (n = 468) were classified according to the genetic groups proposed by vonHoldt et al. (2010). Based on FCI classification dogs belonging to groups 1 “Sheepdogs and cattledogs, except swiss cattledogs” , 2 “Pinscher and Schnauzer-Molossoid and swiss mountain and cattledogs”  and 3 “Terriers”  were more likely to be DEA 1- (p < 0.01). Dogs belonging to groups 4 “Dachshunds” , 6 “Scent hounds and related breeds” , 7 “Pointing dogs” , and 8 “Retrievers – Flushing dogs – Water dogs”  were more likely DEA 1+ (p < 0.01). Dogs belonging to genetic groups “working dogs”  and “mastiff-like”  were significantly more likely to be DEA 1- (p < 0.01) while dogs belonging to genetic groups “scent hounds”  and “retrievers”  were significantly more likely to be DEA 1+ (p < 0.01).All purebred dogs were classified based on FCI groups and dogs belonging to 42 breeds (The present study reports data about DEA 1 frequency obtained from a large canine population including purebred dogs belonging to 88 breeds and tested with a commercial immunochromatographic kit. Few reports are available on breed-specific DEA 1 frequencies, often involving a limited number of dogs and breeds. They were reported in Japan , Brazil The DEA 1 antigen was present in the 62% of the dogs included in this study without difference between mongrel and purebred subgroups. In agreement with our results, canine population frequencies range between 53% in Switzerland  and 61% Considering breed-specific frequency results, DEA 1- group was statistically associated with Dogo Argentino, Dobermann, German shepherd, Boxer and Corso dogs. Frequencies obtained agree with those previously reported (Table DEA 1+ blood group resulted statistically associated with Rottweiler, Briquet Griffon Vendéen, Bernese mountain dog and Golden Retriever breeds. Moreover dogs belonging to English setter, Labrador Retriever, and Ariégeois resulted prevalently DEA 1+ with frequencies ranging from 73 to 94%. Our results were in agreement with data reported elsewhere for Golden retriever  and BernThe discrepancies observed in our frequencies referred to mongrel and some purebred dogs compared to other studies could be related to the geographical different expression of the antigen as previously hypothesized , 33. TheThe FCI nomenclature splits purebred dog into ten groups based on morphology, current use and historical criteria. Recently, genetic diversity of dog breeds was evaluated and surprisingly a correspondence between genetic and phenotypical/functional breed grouping was observed with some exception . Dogs weFurthermore, molossoid dogs resulted statistically associated to DEA 1- blood group while dogs belonging to hunting breeds were significantly associated with DEA 1+ blood group. More data have to be collected to confirm this observation.Frequency of only DEA 1 antigen was studied in the present work. Extended canine blood groups assessment including DEA other than 1 should be performed on candidate blood donor but it was not easily available for clinicians. In the clinical practice only DEA 1 kits are commercially available and could be used before the transfusion to test donor and recipient. Consequently the resulted reported in the present large canine population study could be exploited by clinicians to choose the most suitable dog to test in order to find the right candidate blood donor quickly.The present study provides data on the frequency of DEA 1 antigen in a large number of dogs and confirms a broad variability among breeds. In fact, some breeds show high frequency of DEA 1- subjects resulting more susceptible to immunization when they receive blood from an incompatible donor.In the routine activity of clinicians and of canine blood bank, our results may be valuable to drive blood donor enrollment and selection among different breeds.