We assessed the three-dimensional (3D) pattern of the physiologic drift of the remaining adjacent teeth after premolar extraction due to orthodontic reasons and the associated factors. Data were collected from 45 patients aged 17.04 ± 5.14 years who were scheduled to receive a fixed appliance after maxillary premolar extraction. Seventy-five drift models were obtained and digitalized via 3D scanning. The average physiologic drift duration was 81.66 ± 70.03 days. Angular and linear changes in the first molars, second premolars, and canines were measured using the 3D method. All the examined teeth had tipped and moved towards the extraction space, leading to space decreases. Posterior teeth primarily exhibited significant mesial tipping and displacement, without rotation or vertical changes. All canine variables changed, including distal inward rotation and extrusion. The physiologic drift tended to slow over time. Age had a limited negative effect on the mesial drift of posterior teeth, whereas crowding had a limited positive effect on canine drift. Thus, the mesial drift of molars after premolar extraction may lead to molar anchorage loss, particularly among younger patients. The pattern of the physiologic drift of maxillary canines can help relieve crowding and facilitate labially ectopic canine alignment, whereas canine drift is accelerated by more severe crowding. When teeth are missing due to caries, periodontal disease, trauma, or extraction, the equilibrium will be broken, and the remaining dentition will spontaneously move to establish a new equilibrium.Based on the theories of Weinstein3 and Begg4 reported it over 60 years ago. The most commonly extracted teeth for orthodontic purposes are the first premolars5. Although controversial7, extraction is commonly used to correct the tooth discrepancy8, affect the vertical dimension9, retract incisors10 and relieve crowding11. After extraction, the missing premolars can induce a spontaneous adjustment of dentition.Extraction has been a very common strategy in orthodontic treatment since Tweed12 termed this adjustment “physiologic drift”. Because this is a common phenomenon in cases with missing teeth, several animal experiments have been performed since the 1960s to confirm the drift25, establish an experimental animal model23, and explore the mechanism25.Bourdet26 and PASS27. However, the benefit of long-term physiologic drift—including the spontaneous and uncontrolled movement of dentition—with current treatments need to be evaluated.Physiologic drift is important in orthodontic treatment. Some orthodontic techniques use the drift to simplify the treatment, including the methods of Alexander33. After the extraction of lower premolars, the researchers observed mesial movement of the first molar and the distal movement of the canines, including tipping and translation. Extraction sites also tend to close, and the incisors spontaneously migrate to reduce crowding. Hence, studies have proposed a delay in the bonding of the appliances for the lower arch to take advantage of the physiologic drift. The first molar is usually the most important anchorage tooth during orthodontic treatment34, and its mesial movement could contribute to a potential risk of losing anchorage. However, studies36 have found that the movements of these lower first molars are minimal and only account for a small proportion of closure of the premolar extraction space during the drift.Previous clinical research has primarily focused on mandibular dentition42, but only a few studies42 have assessed the physiologic drift of maxillary dentition, and even fewer have addressed this issue in orthodontics. The maxillary arch usually requires stronger anchorage34 for orthodontic treatment. Thus, identification of the characteristics of physiologic drift of the upper arch following tooth extraction may help to control upper anchorage in the duration between extraction and bonding application, or even during the early stage of treatment using very soft wires. Hence, it is vital to design an appropriate research study to assess the problem.Physiologic drift does not only occur in the lower arch37 have used manual measurement with lateral cephalometric radiographs and dental cast models to evaluate dentition drift after premolar extraction. However, accurate relative three-dimensional (3D) tooth displacement during physiologic drift has not been evaluated. In the present study, we aimed to quantify the physiologic drift of untreated adjacent teeth following first premolar extraction during the permanent dentition stage using 3D digital technology. To our knowledge, this is the first study to assess the 3D pattern of the physiologic drift of the maxillary remaining adjacent teeth after first premolar extraction for orthodontic reasons and the associated factors.Most previous studiesSex: 10 male and 35 female;Age: 17.04 ± 5.14 years, from 12 to 29 years, and the 95% confidence interval was 15.97 to 18.12 years;43: Angle I, 16; Angle II, 13; Angle III, 6;Angle ClassificationCrowding prior to extraction: 3.69 ± 2.41 mm, from 0 to 12 mm, and the 95% confidence interval was 2.96 to 4.41 mm.The descriptive data on the characteristics of the 45 participants were as follows:X were as follows:Sex: 30 male and 120 female;Age: 15.79 ± 4.49 years, from 12 to 29 years, and the 95% confidence interval was 15.06 to 16.51 years;43: Angle I, 62; Angle II, 50; Angle III, 38;Angle ClassificationCrowding prior to extraction: 1.79 ± 1.64 mm, from 0 to 6 mm, and the 95% confidence interval was 1.52 to 2.05 mm.The average drift duration was 81.66 ± 70.03 days.Distribution after grouping based on the months of drift at Tx: 1 month, 50; 2 months, 23; 3 months, 24; 4 months, 11; 5 months, 10; 6 months, 11; more than 6 months, 19.The descriptive data on the characteristics of the 150 extraction sides at TTable The abovementioned markedly changed variables were selected, and the average monthly change was estimated and depicted using line charts Fig. . Only a U3 markedly changed in all directions. U3 inclined lingually and distally, and showed distal inward rotation, extrusion, and distal movement. U6 and U5 had tipped and displaced mesially, and U6 also showed changes in lingual inclination. The adjacent teeth near the extraction site tipped into the space; hence, the extraction space decreased over time.The monthly average velocities of the marked changes in variables were calculated Table  and usedU6i and VU3r. Age and crowding may have a moderate effect on certain velocities. VU6a, VU6m, VU5a, VU5m, and VSC may decrease with age. More severe crowding prior to extraction may lead to an increase in VU3a, VU3i, VU3m, VU3v, and VSC. However, these factors may have a limited influence on the velocity of physiologic drift because the R² of all the linear regression models only ranged from 0.136 to 0.225.Multiple linear regression models Table  showed t37 of dentition drift to assess tooth movement. However, only certain aspects of tooth movement were analyzed. Tipping and displacement of the teeth in the mesial-distal direction were often considered37. Closure of the extraction space was also discussed by some researchers33. Only a few studies focused on the change in other directions such as transversal angulations32 and rotation37. A reason for this may be because cephalometric measurement may only reflect the changes in the sagittal and vertical directions, and the manual measurement of pre- and post-extraction cast models could not assess the relative 3D changes accurately using 2 independent models. However, a rare 3D study of the physiologic drift40 of maxillary dentition also did not measure rotation and vertical changes. Most of the physiologic drift occurred in the first 6–12 months37, but the observation intervals were usually longer than this quick period of drift37, making it difficult to evaluate the continuous and specific characteristics of physiologic drift during this period. The use of a 3D laser scanner to digitalize the cast provides a new and convenient option to measure tooth displacement that is considered accurate and reliable46. Using 3D methods48 in this study, we conducted comprehensive and accurate measurements of 3D changes in the teeth after maxillary first premolar extractions at about 1-month interval.Manual measurement, via lateral cephalometric radiographs and dental cast models, was used in most previous studies37. Additionally, the present study measured the changes in labiolingual inclination and rotation for each tooth using 3D methods. U6 and U3 showed lingual inclination, whereas U5 showed no significant inclination . The difference in the drift pattern between the posterior teeth and canines may result from the lack of occlusal interference with the canines. U3 showed significant extrusion and rotation, although U6 and U5 did not, possibly because these teeth had more occlusal contact16 and were multirooted.Table 49 and Murphy13 found that transseptal fibers can self-repair and reconnect adjacent teeth after destruction of the periodontal ligament or extraction. Many previous studies50 also proposed that the reconstruction of transseptal fibers is the main cause of physiologic drift. There are some other potential causes for movement of the teeth into the extraction space. The position of the teeth may be influenced by the soft tissues surrounding them, and they remain stable in the equilibrium state of forces of the stomatognathic system52. Ogushi52 found that lip and cheek muscle pressure was highest at the canine. Hence, it is possible that, after extraction, the arch continuity may have been broken and the posterior support of canines was missed, leading the canines to move distally under the effect of the lip muscle. Moreover, Southard et al.54 measured an anterior component of occlusal force and reported its influence on dental alignment, which could also be a reason for the mesial drift of the upper molars after first premolar extraction.The findings from the present study and previous research have shown that tipping is the main style of movement during physiologic drift after extraction, and this phenomenon could partially prove the hypothesis that transseptal fibers, which are embedded in the cementum between adjacent teeth and extend interproximally over the alveolar bone crest, play an important role in physiologic drift. Kusters31 observed that mandibular dentition drifted approximately 10 months after lower first premolar extraction and found that the lower first molars had completely moved mesially by approximately 1.19 mm and had tipped forward by approximately 1.90°. Weber’s research35 showed that the lower first molars moved mesially by approximately 1.6 and 2.6 mm after the first and second premolar extraction, and the canines moved distally by approximately 4.4 mm over the 2.5 years. Additionally, Mamopoulou37 found that the lower first molar had moved forward by 2.5 mm at 1 year after second premolar extraction. Stephens29 observed that the extraction space decreased by approximately 1.6 mm in women and 3.23 mm in men during the first 6 months after the first premolar extraction. Swessi32 reported that the lower first molar did not markedly move during the long-term observation after the first premolar extraction. The present study found that the maxillary extraction space decreased by an average of 0.792 mm/month after extraction, leading to an approximately 4-mm decrease in the extraction space over 6 months. U6 also showed faster tipping (1.127°/month) and movement (0.386 mm/month) over the 6-month period  could have a higher risk of physiologic anchorage loss, relative to the lower ones after premolar extraction, particularly among younger orthodontic patients during the period of growth and development.Alexanderiod Fig. , Table 2iod Fig. . This reMolars can drift for at least 6 months after extraction Fig. , indicat27 considers both aspects. The XBT buccal tube could prevent the continuous mesial tipping movement of the upper first molar initially, and the low friction design at the canine brackets can minimize the prevention of the distal drift, thus achieving both objectives with one option.For canines, distal tipping, distal movement, and extrusion are beneficial for alignment, particularly among cases with labially ectopic canines or those with anterior teeth crowding. Therefore, the physiologic drift of the canines can be used during treatment. An appliance technology can better offer the potential to control the mesial drift of the upper first molar and could help exploit the distal drift of the canines simultaneously. PASS technology8 or crowding11. Additionally, extraction is a commonly used strategy to affect the soft tissue profile10 and skeletal vertical dimension9. However, according to some recent studies69, the effects on the skeletal and soft tissue of extraction orthodontic treatment could not be confirmed and might be overestimated. Kirschneck’s retrospective cohort study67 reported that premolar extraction in juvenile borderline patients did not lead to a significant reduction in the skeletal and vertical dimensions compared with non-extraction treatment in the short-term. Kouvelis’ systematic review68 also concluded that the extraction treatment protocol aiming to reduce or control the vertical dimension did not seem to be an evidence-based clinical approach. Both Kirschneck’s67 and Iared’s69 studies showed that differences between the changes in the patients’ facial profiles resulting from orthodontic treatment with and without extraction were clinically irrelevant or not significant. The duration of orthodontic treatment was significantly longer in patients who underwent extraction, and Iared69 suggested the cephalometric parameter, Li-E, could help decision making in borderline cases. Combined with the results in the present study that maxillary premolar extraction could lead to molar anchorage loss to some extent by inducing the mesial drift of the first molars, the decision on extraction for orthodontic reasons should be made more carefully. More individual factors and evidence-based implications should be considered when making an integrated treatment plan.Orthodontists usually decide to extract healthy premolars for patients with severe tooth discrepancy48 at each interval because the 3D dental scanner  can register numerous coordinates in three dimensions with ±20 μm precision of the scan. The generalizability of these results might be limited by the characteristics of the study sample because this single-center study included mostly teenage and young adult patients seeking orthodontic treatment. There are some bias factors in the characteristics of the participants because most of the patients with long-term observation required mild or medium anchorage. Some of the observed results from this study showed high individual variation, consistent with some other research37, indicating that, although the general trends of the physiologic drift could be summarized, some individual differences exist in the drift of patients with different patterns of malocclusion. Some factors significantly influencing the physiologic drift were filtered by multiple linear regression procedures, while the R² of all the linear regression models only ranged from 0.136 to 0.225, indicating that these factors might only have limited contribution to the physiologic changes in these samples. Thus, the average velocities of these changes generally remained stable for at least 6 months after extraction in this study. However, the prolonged observation time would impede the therapy because the uncontrolled tooth movement during physiologic drift could lead to maxillary molar anchorage loss and affect the treatment outcomes. Although some possible biasing influences on the rate of tooth movement, such as prescription medication, smoking and alcohol abuse, were eliminated in the patient cohort, it was uncertain whether the participants started the administration of drugs, nicotine or alcohol during the drift. Additional factors70 may also influence physiologic drift, in addition to those considered in this study, such as occlusal influences, muscle pressure, drift of the lower dentition, and mandible growth. For patients with different patterns of malocclusion, it is difficult to control these factors and explore their influence on physiologic drift with this sample size. Hence, further research with a larger sample size and more comprehensive analysis is needed.The general trends of the physiologic drift of the remaining adjacent teeth after first premolar extraction could be described in this cross-sectional study, but this study had some limitations. Errors may be caused due to cast preparation, cast digitalization and measurementDespite these limitations, this study explored the continuous and specific three-dimensional characteristics of physiologic drift of the upper arch following tooth extraction and assessed some factors influencing the drift. We believed that it had certain clinical value in orthodontics and could fill the gaps of previous studies to some degree.All the measured teeth showed a similar tipping movement toward the extraction spaces, leading to a decrease of the extraction spaces. The maxillary first molars and canines tended to move into the spaces at a similar speed, indicating that the mesial drift of molars after premolar extraction may cause molar anchorage loss to some degree and should be prevented as soon as possible after premolar extraction in cases in which maximum control of molar anchorage is needed. Moreover, the pattern of physiologic drift of maxillary canines after premolar extraction helps relieve crowding and labially ectopic canine alignment. Additionally, drift time, age and crowding can influence physiologic drift. Physiologic drift tended to slow over time. In younger patients, posterior teeth tended to drift mesially at a faster rate and the extraction spaces tended to close faster. Crowding may have a positive effect on the drift of canines and space closure, but sex and the angle classification did not significantly affect drift.This study was designed to explore the 3D characteristics of physiologic drift of the upper arch following maxillary first premolar extraction and associated factors. To gain as many generalizable results as possible, we planned to evaluate teenage and young adult (Under 30) patients, who made up the largest group of patients seeking orthodontic treatment in our department. For these patients, the decisions on bilateral maxillary first premolar extraction were made by senior orthodontists. To reduce the possible bias, the patients were enrolled based on the following inclusion criteria: (1) permanent dentition, (2) no molar crossbite or buccal crossbite, (3) no orthodontic history, (4) no congenital missing teeth, and (5) a complete crown. The exclusion criteria were as follows: (1) systemic disease, including chronic or metabolic diseases requiring the long-term administration of prescription medication, (2) congenital craniofacial deformity and syndromes, including cleft lip and palate, (3) maxillary surgical history or trauma history, (4) impacted teeth, (5) moderate to severe gingivitis or periodontal disease, (6) periapical periodontitis of the posterior teeth, and (7) smoking or alcohol abuse.40, while no researcher explained how the study size was determined. To obtain as much data as possible, we decided to enroll all the available patients. This study was approved by the institutional review board of Peking University School of Stomatology (PKUSSIRB-201310072). Written informed consent was obtained from all the subjects before the study. The methods were carried out in accordance with the relevant guidelines and regulations.There were 1,527 patients in total who started treatment in our department between October 2015 and September 2018. Six hundred fourteen patients underwent bilateral maxillary first premolar extraction, and 116 of them met the inclusion criteria. The observation time was from the extraction sites generated to the day for fixed appliance bonding. Different drift durations were determined for each patient, ensuring that their treatment was not affected. Because the observation could probably extend the period of treatment, finally 45 patients  agreed to be the subjects  was obtained prior to extraction and the inter-observation casts (Tx) were obtained after extraction and before fixed appliance bonding. The dental and periodontal status of each patient was monitored by dentists during the observation. Next, 120 dental casts (45 casts at T0 and 75 casts at Tx) were obtained and scanned with a 3D dental scanner  and were virtually constructed into digital casts using the Rapidform 2006 program . Crowding prior to extraction in each patient was measured using Proffit’s method47   Fig. . The 3D 72, the first measurement was used.Intraclass correlation coefficients (ICC) for the measurements of the angular variable, linear variable, and space closure were computed to assess intra-examiner reliability (repeatability). The results were 0.94, 0.91, and 0.95 for measurements of the angles, the linear distances, and extraction space closure, respectively. Because the assessment showed an excellent ICC valueX were summarized based on descriptive statistics, and the data were used for further analyses.The characteristics of the 150 extraction sites at TFirst, the Pearson’s correlation coefficients between all the changes in the variables and durations of drift (days) were calculated to test the significance of the change over time. Next, the markedly changed variables were selected for further analyses.Changes in the selected variables between Tx and T0 were grouped based on the months  of drift at Tx. Thus, they were divided into 7 groups, including 1-month to 6-month groups, and the over 6-month group. The average significant changes in each month was calculated to create a line chart to directly observe the amount and tendency of drift.X = SX*30/T; V: average velocity; S: change in the angular or linear variable; T: days between extraction and observation, 30 days as a month; X: variable of tooth movement) was calculated. Two-tailed single-sample t-test was used to test the significance of the velocity , and the 95% confidence interval was calculated.The average velocity of movement over 1 month  was set as a dummy variable and was entered first. A forward stepwise linear regression procedure was then performed for the other factors.The results of the Kolmogorov–Smirnov test showed that all the data were approximately normally distributed. Statistical significance was determined at the 1% and 5% levels of confidence. We used IBM SPSS Statistics 22  for statistical analyses.


In this work, we fabricated three carbon nanoplume structured samples under different temperatures using a simple hot filament physical vapor deposition (HFPVD) process, and investigated the role of surface morphology, defects, and graphitic content on relative humidity (RH) sensing performances. The Van der Drift growth model and oblique angle deposition (OAD) technique of growing a large area of uniformly aligned and inclined oblique arrays of carbon nanoplumes (CNPs) on a catalyst-free silicon substrate was demonstrated. The optimal growing temperature of 800 °C was suitable for the formation of nanoplumes with larger surface area, more defect sites, and less graphitic content, compared to the other samples that were prepared. As expected, a low detection limit, high response, capability of reversible behavior, and rapid response/recovery speed with respect to RH variation, was achieved without additional surface modification or chemical functionalization. The holes’ depletion has been described as a RH sensing mechanism that leads to the increase of the conduction of the CNPs with increasing RH levels. The detection and control of humidity are important considerations for different applications including food quality monitoring, meteorology and medical equipment functioning. Recently, carbon nanomaterials have been investigated as a promising candidate for sensing applications due to their comparatively high conductivity, high mechanical stiffness, large sensing surface area, and high chemical inertness ,2,3. SpeThe morphological features and structure include: surface area, graphitic contents , number Usually carbon adsorbs numerous oxygen derivations on the surface, which makes the carbon surface hydrophilic, and enhances water vapor adsorption . Upon adThe originality of this work involves providing a comparison of the sensor response of carbon nanostructures synthesized at different temperatures with a simple OAD technique by hot filament physical vapor deposition (HFPVD). Dinh et al.  recently−3 mTorr, and then Ar gas was fed into the chamber until the ambient pressure was attained, followed by pressurizing down to 10−3 mTorr again. This process was repeated three times in order to keep air out and obtain as good a vacuum as possible. A direct current (DC) power supply was used to heat the filament to promote the carbon gas phase activation. For all the experiments, the incident angle of the plasma deposition was controlled at approximately 45° and the deposition duration was kept at 20 min. The sputtered Cr/Au double-layer electrodes were grown on both sides of the carbon films by using shadow masks, after which the carbon samples were transferred to a quartz tube furnace to anneal at 1000 °C for one hour in a nitrogen atmosphere and then slowly cooled down to 25 °C at a rate of 1.7 °C/min in order to reduce the stress and strain in the films as much as possible. The three CNPs samples prepared by this process at 600, 700 and 800 °C will henceforth be referred to as CNPs-600, CNPs-700, and CNPs-800, respectively.Carbon nanostructures were synthesized on 1 cm × 1 cm Si substrates in a HFPVD chamber. A similar fabrication process has been described in our previous publication . In orde2) cooled charge-coupled device was used to collect and process the scattered data .The morphologic surface and the cross section of the samples obtained were investigated by SEM and TED. Room temperature (RT) Raman spectroscopies were measured by the Jobin-Yvon T64000 Triple-mate system with the coherent argon ion laser emitting radiation of 514.5 nm. A liquid nitrogen  of the sensor to RH is defined as:The sensitivity (The comparison of sensing performance, including sensitivity and response/recovery time, between our work and previous reports about carbon nanomaterial based humidity sensors ,29,30,31The carbon nanostructures are normally hydrophilic due to the numerous adsorbed oxygen derivatives . ConsequIn conclusion, three CNPs samples have been obliquely deposited using the HFPVD techniqueunder different temperatures. The morphologies, defect sites and graphitic content, develop with temperature, which has been exhibited by SEM and TED images, and Raman spectra. The Van der Drift model and OAD technique has been attributed to the growth mechanism of the CNPs. The optimum temperature of growing nanoplumes with large surface area, high defect and low graphitic content was 800 °C, which induces best sensing performances by comparing the sensing characteristics among three CNPs samples.The detection limit of the CNPs-800 based sensor was investigated with a RH as low as 5%, and the response of the CNPs-800 was calculated as 703% at 60% RH. The transient response behavior indicates high response, good reproducibility, rapid response and recovery speed of the sensor to RH. Furthermore, for the small hysteresis characteristic, a maximum value of 7.40% was obtained. The holes’ depletion was caused by the electron donation released by the adsorbed water molecules and consequently increased the impedance of the CNPs-800 samples. The comparison of the sensing performance between the CNPs-800 based sensor and other carbon nanomaterial-based humidity sensors indicates that the CNPs samples synthesized by the HFPVD process are a promising candidate for RH sensing applications.


Strong positive genetic correlations were found between anhedonia and major depressive disorder, schizophrenia and bipolar disorder; but not with obsessive compulsive disorder or Parkinson’s Disease. Polygenic risk for anhedonia was associated with poorer brain white matter integrity, smaller total grey matter volume, and smaller volumes of brain regions linked to reward and pleasure processing, including orbito-frontal cortex. In summary, the identification of novel anhedonia-associated loci substantially expands our current understanding of the biological basis of state anhedonia and genetic correlations with several psychiatric disorders confirm the utility of this phenotype as a transdiagnostic marker of vulnerability to mental illness. We also provide the first evidence that genetic risk for state anhedonia influences brain structure, including in regions associated with reward and pleasure processing.Anhedonia is a core symptom of several psychiatric disorders but its biological underpinnings are poorly understood. We performed a genome-wide association study of state anhedonia in 375,275 UK Biobank participants and assessed for genetic correlation between anhedonia and neuropsychiatric conditions . We then used a polygenic risk score approach to test for association between genetic loading for anhedonia and both brain structure and brain function. This included: magnetic resonance imaging (MRI) assessments of total grey matter volume, white matter volume, cerebrospinal fluid volume, and 15 cortical/subcortical regions of interest; diffusion tensor imaging (DTI) measures of white matter tract integrity; and functional MRI activity during an emotion processing task. We identified 11 novel loci associated at genome-wide significance with anhedonia, with a SNP heritability estimate (h It constitutes a core symptom of several neuropsychiatric disorders, including major depressive disorder (MDD), schizophrenia, bipolar disorder and obsessive compulsive disorder (OCD), as well as Parkinson’s Disease (PD)6. Along with a direct negative association with quality of life and subjective wellbeing, anhedonia is associated with multiple negative health-related behaviours, such as smoking, illicit drug use, and low physical activity, even in the absence of psychiatric disorder8. In line with a Research Domain Criteria (RDoC) approach9, state anhedonia can be measured and studied as a dimensional psychopathological trait.Anhedonia refers to reduced capacity to experience pleasure in situations that individuals would normally enjoy, and has been a focus of psychiatry research for decades11. Measures of anhedonia have been associated with altered functional activity during reward-based tasks within frontal cortical regions  and subcortical striatal regions (caudate and putamen)12, and with reduced volumes in a similar set of frontal and striatal regions14. Anhedonia is also associated with reduced white matter integrity16.Anhedonia has been closely linked to the function and structure of reward circuitry in the brain . These neurobiological associations are consistent with the view that anhedonia reflects dysfunction in reward processing18. However, to date only small underpowered GWAS studies of anhedonia have been published. A study of 759 patients with MDD identified 18 SNPs associated with an ‘interest-activity’ measure of anhedonia19. The largest study to date is a mega-analysis of three studies of young people from the UK and Sweden, with a total sample size of 6579; a single locus was associated with anhedonia in the discovery sample, but not in the replication sample20. A Finnish study examined genetic associations with physical and social anhedonia, as assessed with the Chapman scales21, in 3820 individuals but no genome-wide significant loci were identified22. Genetic loci associated with anhedonia have therefore not yet been reliably identified in large clinical or general population samples23, and association tests of genetic risk for anhedonia with brain structure and function have not yet been performed.The genetic underpinnings of anhedonia are largely unknown. Several GWAS of disorders where anhedonia is a feature have been reported, such as MDD and schizophreniaHere we report a large GWAS of state anhedonia within the UK Biobank cohort. We also use a polygenic risk score (PRS) approach to assess whether genetic loading for anhedonia is associated with brain structure and brain function.24. The cohort was designed to assess how genetic, environmental and lifestyle factors influence a range of morbidities in middle and older age. Baseline assessments occurred over a 4-year recruitment period (from 2006 to 2010) across 22 UK centres. These assessments covered a wide range of social, cognitive, lifestyle and physical health measures. Informed consent was obtained from all participants, and this study was conducted under generic approval from the NHS National Research Ethics Service  and under UK Biobank approvals for application #6553 ‘Genome-wide association studies of mental health’ (PI Smith).UK Biobank is a large cohort of over half a million UK residents, aged between 39 and 73 years at baseline assessment22. Pre-imputation quality control, imputation (both 1000Genomes and HRC Reference Panels) and post-imputation cleaning were conducted centrally by UK Biobank .In March 2018 UK Biobank released genetic data for 487,409 individuals, genotyped using the Affymetrix UK BiLEVE Axiom or the Affymetrix UK Biobank Axiom arrays , which have over 95% of content in common“Over the past two weeks, how often have you had little interest or pleasure in doing things?” (Data field 2060). Respondents could choose from the following answers: “not at all”; “several days”; “more than half the days”; and “nearly every day”. These responses were coded as 0, 1, 2 and 3 respectively. This question on anhedonia is derived from the Patient Health Questionnaire-9 (PHQ-9), a well-validated screening instrument for MDD25. To maximise numbers available for downstream magnetic resonance imaging (MRI) analyses, we excluded from the primary GWAS those participants with any available MRI data . Additional exclusion criteria for the GWAS included individuals in whom: over 10% of genetic data were missing; self-reported sex did not match genetic sex; sex chromosome aneuploidy was reported; where heterozygosity value was a clear outlier; and participants not of European ancestry .As part of the comprehensive baseline assessment participants were asked: 27, which accounts for population structure and sample relatedness by including a genetic relatedness matrix within the models. Models were further adjusted for age, sex, and genotyping array. SNPs included in the analysis were filtered by MAF > 0.01, Hardy–Weinberg Equilibrium p > 1 × 10–6, and imputation score > 0.3. BOLT-LMM was also used to provide a SNP-heritability estimate and an estimate of λGC.Genetic association with the measure of anhedonia was performed using BOLT-LMM28 was carried out using LDSC on GWAS summary statistics from several published studies, to obtain genetic correlations with psychiatric disorders where anhedonia is known to be a feature , as well as Parkinson’s Disease32. We left the intercept unconstrained to allow for sample overlap between the sample used here and the outputs of the other GWAS.Linkage Disequilibrium Score Regression (LDSR)33. LDpred differs from the more common pruning and threshold (P&T) PRS because it generates a single risk score for the trait of interest derived from as many loci as possible. A training set was created to obtain the LD structure by using 1000 unrelated Biobank participants who had passed the same genetic QC as those used in the GWAS (but who were excluded from the GWAS because they did not respond to the anhedonia question and had not provided brain imaging data). These training data were then used for the construction of anhedonia PRS in those participants for who brain imaging data were available.Polygenic risk scores (PRS) were created using LDpred34. These measures are: total volume (mm3) of brain grey matter, white matter and cerebrospinal fluid (CSF), each normalised for head size; volumes  of 15 cortical and subcortical regions of interest (ROIs); diffusion tensor imaging (DTI) measures of white matter integrity  and mean diffusivity (MD)); and functional MRI activity during an emotion processing task (the Hariri face shape task)35 in an amygdala mask and group-defined mask consisting of occipito-temporal and amygdala regions. For a more detailed description of these variables, and for details of MRI acquisition and pre-processing and IDP selection, please see A number of structural and functional brain MRI measures have been made available by UK Biobank as Imaging Derived Phenotypes (IDPs)n = 2479) or who self-reported a developmental or neurological disorder at either the baseline assessment or the imaging visit (n = 575)  were normalised for head size prior to adjustment for the above covariates. ROI analyses were additionally adjusted for total brain volume  volume); and fMRI analyses were also adjusted for head motion during the emotion processing task. False Discovery Rate (FDR) correction was applied37.Models were adjusted for age at MRI visit, (age at MRI)“not at all”; 60,212 (16.0%) reported “several days”; 9405 (2.5%) reported “more than half the days” and 6876 (1.8%) reported “nearly every day”.The GWAS was performed on 375,724 UK Biobank participants, of whom 203,322 (54.1%) were female. The age of the sample ranged from 39 to 73 and the mean age was 57 years (S.D. = 8.01). In response to the anhedonia question, 299,232 (79.64%) answered “not at all”, 2469 (14.7%) reported feelings of anhedonia for “several days”, 297 (1.8%) for “more than half the days”, and 207 (1.2%) “nearly every day”.MRI analyses were conducted in 17,120 participants, 52.4% (8978) of whom were female. The mean age (at the time of MRI) of these participants was 62.7 years . Of the 16,783 participants with available MRI data who answered the anhedonia question, 13,810 (82.3%) responded p < 5 × 10–8), and, of these, represented 11 independent loci on 9 different chromosomes , however considering the sample size this is expected to have had a negligible impact on findings. There was evidence for a polygenic component  and no evidence for undue inflation of the test statistics due to unaccounted population stratification.Some inflation of the GWAS results was observed (λp = 0.015). This would not survive correction for multiple testing. Of the 11 top hit SNPs, 8 of 11 coefficients were in the same direction as in the discovery set .Additionally, we determined if the PRS of anhedonia in those excluded from the GWAS was associated with their state anhedonia scores. The model was adjusted for the first 4 genetic principle components, genotyping chip, age, sex and Townsend score. We detected a small but highly significant association between the anhedonia PRS and the anhedonia phenotype scores , schizophrenia  and bipolar disorder  but not with OCD or Parkinson’s disease (q = 0.6 and q = 0.97 respectively)  or fractional anisotropy (FA) Table . PRS assThese analyses represent the largest genetic association study of state anhedonia performed to date. We identified eleven genetic loci associated with anhedonia in the UK general population. Within each associated region there were a number of genes that could have a functional impact on anhedonia and the pleasure cycle (considered in detail below). Consistent with an RDoC approach focusing on transdiagnostic symptoms and traits, we found strong genetic correlations between anhedonia and MDD, schizophrenia and bipolar disorder, but not between anhedonia and OCD or Parkinson’s Disease. Despite the lack of correlation with Parkinson’s Disease, several of the loci identified in our GWAS include genes with known association with Parkinson’s Disease (see below). We also report the first investigation of associations between genetic loading for anhedonia and both brain structure and brain function.RGS genes, most notably RGS1 and RGS2, encoding regulators of G-protein signalling that show prominent expression in the brain38. RGS2 has previously been identified as a modulator of LRRK239 expression, a gene known to be a genetic cause of Parkinson’s Disease. RGS2 has also been associated with symptom severity in schizophrenia40 and lower expression of RGS2 may be related to depression-like behaviours in animal models41.There are multiple genes in each QTL identified in the GWAS. Here we discuss potentially relevant genes based on their known function in the scientific literature. Within the chromosome 1 locus there are multiple EPHB1 on chromosome 3 encodes an ephrin receptor tyrosine kinase identified in a GWAS of antidepressant response42, and is associated with symptoms of schizophrenia in Chinese Han populations43, and with susceptibility to Parkinson’s Disease44.GRM5 encodes a metabotropic glutamate receptor that has been extensively studied in relation to MDD46 and schizophrenia48. Assessments in mice have also found that agonists of GRM5 attenuate Parkinsonian motor deficits via striatal dopamine depletion49. The Genotype Tissue Expression (GTEx) database38 shows prominent expression of GRM5 in the brain, especially within the nucleus accumbens, a region with a major role in the prediction of reward51.At the more centromeric locus on chromosome 11, DISC1FP1. DISC1 (Disrupted in Schizophrenia 1), a gene located on chromosome 1, is part of a chromosome 1:11 translocation that increases risk of schizophrenia, schizoaffective disorder and bipolar disorder52. DISC1FP1 is disrupted by this translocation, impacting on both intracellular NADH oxidoreductase activities and protein translation53.Another likely candidate gene at this chromosome 11 locus is NCAM1  has been implicated as a potential link between depressive symptoms and brain structure54, specifically decreased FA. NCAM1 has been reported to be present at increased levels in the amygdala38, another brain region associated with the pleasure cycle, in depressed subjects55. However, we did not find any association between PRS for anhedonia and either amygdala volume or functional activity in the amygdala during an emotion processing task. It is possible that NCAM1 may exert its effects via expression in other brain regions.At the more telomeric locus on chromosome 11, DRD2, encoding the dopamine D2 receptor. There is an extensive literature on the importance of DRD2 for the psychopharmacology of both schizophrenia and MDD57.Another gene of interest at this second chromosome 11 locus is LOC and LINC genes. These encode different classes of functional RNA but little is known about their function beyond possible post-transcriptional regulation of other gene products.The chromosome 12 locus contains only PRKD1, which encodes a serine/threonine-protein kinase identified in a GWAS of schizophrenia18. The more telomeric locus on chromosome 14 contains the gene SLC8A3, a gene involved in maintaining Ca2+ homoeostasis within a variety of tissues, including neurons. SLC8A3 may also play a role in Parkinson’s Disease58.The more centromeric locus on chromosome 14 contains ISLR2 on chromosome 15 is involved in neurodevelopment59 but its role in psychiatric and neurological traits is not well characterised. Another candidate gene within this locus, NRG4, encodes a neuregulin protein that activates type-1 growth factor receptors. Recent work has shown that NRG4 acts a regulator of the growth and elaboration of pyramidal neuron dendrites in the developing neocortex60. Pyramidal neurons have been directly implicated in the pathophysiology of schizophrenia61.DCC (the most significant hit) encodes a netrin 1 receptor with a role in axon guidance, and has been previously reported to be associated with anhedonic phenotypes in mice and humans62 and potentially with schizophrenia pathogenesis63. We have previously identified DCC in GWAS of mood instability64, suicidality65 and multisite chronic pain66.Finally, on chromosome 18, 71. The regions most consistently implicated include nucleus accumbens, caudate, putamen, medial frontal cortex and orbitofrontal cortex.Our findings on the relationship between genetic loading for anhedonia and brain structure and function are of considerable interest. Greater levels of anhedonia, in healthy and clinical populations, have been linked to altered functional activity (and less consistently to reduced volume) in frontal/striatal regions involved in reward or pleasure processing72. We also noted uncorrected associations of PRS for anhedonia and smaller volumes of nucleus accumbens  and medial frontal cortex (linked to pleasure processing69), but these associations did not survive correction for multiple comparisons. These cortical/subcortical volume findings are therefore consistent with an association between genetic risk for anhedonia and reward/pleasure processing73.We found that increased genetic risk for anhedonia was associated with smaller volumes of the orbitofrontal cortex the Fig.  a region74) and fusiform cortex. Most ROIs in our analysis were selected on the basis that they were associated with smaller volumes in MDD vs. healthy controls in the most recent MDD brain structure meta-analysis75. Our findings are consistent with two scenarios: either, genes for anhedonia partly mediate the association between the ROI volumes and MDD, or these genes exhibit horizontal pleiotropy and affect both phenotypes via separate mechanisms. Consistent with our finding of widespread associations of genetic risk for anhedonia with cortical/subcortical volumes, we also found an association with reduced total grey matter volume, adjusted for head size.Increased genetic risk for anhedonia was also associated with smaller volumes of insular cortex , in most individual tracts, but not in a general factor of MD. Several tracts also showed reduced FA.Evidence of reduced white matter integrity (FA) in several tracts in individuals scoring higher on measures of anhedonia has been reportedWe did not, however, find any association between PRS for state anhedonia and functional brain activity. UK Biobank ROIs were selected based on average responses during an emotion processing task; it is therefore plausible that effects would emerge with the use of a reward processing task, or by applying whole-brain voxel-wise analyses, to include further reward/pleasure processing regions.77. This idea is reinforced by the significant genetic correlations with other psychiatric disorders. Additionally, we used an ordered ordinal phenotype, resulting in more power to detect associations than with the more common dichotomised (‘non-anhedonic’ vs. ‘anhedonic’) analyses78. Identification of loci associated with population-level anhedonia may be important from a personalised medicine perspective, for example, in terms of developing stratified medicine approaches to identify individuals at high-risk of developing psychiatric disorders.This study is the largest GWAS of state anhedonia to date, and substantially contributes new knowledge on the biology of this important transdiagnostic symptom. We conducted analyses within a large population-based cohort. Our primary analysis included individuals with mental health histories, but these represent only a small proportion of the total sample, which is an order of magnitude larger than any previous study of this kind. Subclinical anhedonia is common, and associated with increased risk of later mental illness79, so reported levels of anhedonia in this sample may be lower than the population rate. Therefore, it is plausible that the strength of associations we identified may be an underestimate of the true population value; there may also be an inflated type 2 error rate.Notably, however, the UK Biobank cohort has a degree of selection bias. In general, volunteers are typically healthier and of higher socioeconomic status and higher education level than the general population81. However, we have assumed here that despite transient environmental and physical factors, individuals who are prone to trait anhedonia will be more likely to report higher frequency of anhedonia at any given time point, and it seems likely that a recent anhedonia phenotype will be enriched for individuals with stronger genetic predisposition towards anhedonia. In line with this assumption, the anhedonia item of the PHQ-9 at a single time point has demonstrated utility in predicting longitudinal brain structural change82.The measure of anhedonia employed here was a single question from a depression screening instrument, the PHQ-9, assessing frequency of symptoms anhedonia within the preceding two weeks. This item therefore measures state anhedonia at a single time point, and clearly could be influenced by environmental factors, such as season or current health statusAdditionally, our attempts at replication of the GWAS results met with limited success, with no real replication of the top hits. However, the PRS analysis was able to detect a significant correlation with state anhedonia in the replication sample. We attribute the lack of replication success in the GWAS to lack of power  and small effect sizes for individual SNPs.The GWAS treated the ordered ordinal responses to the frequency of anhedonia question as linear. This approach is likely to have had a minimal impact on the BOLT-LMM GWAS results. Although the distance between points on the anhedonia scale is not evenly spaced, each point can reasonably be considered to be at least several days of anhedonia in the preceding two weeks than the point before it.21, or into anticipatory vs. consummatory components of pleasure83. Future studies using more detailed anhedonia scales may be of use in examining the extent of genetic overlap between anhedonia subtypes.The anhedonia measure did not enable determination of specific anhedonia subtypes. Existing validated instruments typically divide anhedonia into physical and social subscalesWe report the largest GWAS to date of state anhedonia, a common symptom associated with several psychiatric disorders. We identified 11 novel genetic loci and our findings indicate substantial genetic overlap between anhedonia and several psychiatric disorders including MDD, schizophrenia and bipolar disorder. PRS analyses revealed association between genetic loading for anhedonia and smaller volumes of several brain regions, and poorer white matter integrity. Taken together, these findings provide important insights into the neurobiology of an important but under-studied psychiatric symptom and strongly support the proposition that genetic predisposition to anhedonia influences brain structure and function.https://data.bris.ac.uk/datasets/3074krb6t2frj29yh2b03x3wxj/UK%20Biobank%20Genetic%20Data_MRC%20IEU%20Quality%20Control%20version%201.pdf. Genotyping and quality control information – http://www.ukbiobank.ac.uk/wp-content/uploads/2014/04/UKBiobank_genotyping_QC_documentation-web.pdf.UK Biobank genetic data release information - supplementary legendssupplementary methodsTable S1Table S2Figure S1Figure S2Figure S3Figure S4Figure S5Figure S6Figure S7Figure S8Figure S9Figure S10Figure S11Table S3Table S4Table S5Figure S12Table S6Table S7Table S8Table S9


Aedes aegypti (Linnaeus) mosquitoes presents unique challenges. For example, during transport from the rearing facility to the release site and during the actual release in the field, damage to male mosquitoes should be minimized to preserve their reproductive competitiveness. The short flight range of male Ae. aegypti requires elaborate release strategies such as release via Unmanned Aircraft Systems, more commonly referred to as drones. Two key parameters during transport and release are storage temperature and compaction rate. We performed a set of laboratory experiments to identify the optimal temperatures and compaction rates for storage and transport of male Ae. aegypti. We then conducted shipping experiments to test our laboratory-derived results in a ‘real-life’ setting. The laboratory results indicate that male Ae. aegypti can survive at a broad range of storage temperatures ranging from 7 to 28°C, but storage time should not exceed 24 h. Male survival was high at all compaction rates we tested with a low at 40 males/cm3. Interestingly, results from our ‘real-life’ shipping experiment showed that high compaction rates were beneficial to survival. This study advances key understudied aspects of the practicalities of moving lab-reared insects into the field and lies the foundation for further studies on the effect of transport conditions on male reproductive fitness.Sterile insect technique (SIT) is a promising, environmentally friendly alternative to the use of pesticides for insect pest control. However, implementing SIT with  Aedes aegypti (Linnaeus), is a highly anthropocentric disease vector species with worldwide distribution  is a targeted, environmentally friendly tactic for insect control where large quantities of sterile males are released into targeted areas, where they compete with the wild male population for mating opportunities with females . MatingsCochliomyia hominivorax Coquerel [Diptera: Calliphoridae]), which was eradicated from southern United States, Mexico, and all of Central America (Ceratitis capitata Wiedemann Wiedemann [Diptera: Tephritidae]), eradicated from Central America and Mexico (Glossina austeni Newstead [Diptera: Glossinidae]), which was eradicated from the island of Zanzibar in Tanzania  the d Mexico ; and 3) Tanzania .Several critical components are required for a SIT program to be successful. The rearing program must be able to raise large numbers of healthy insects that exhibit normal behaviors. Sterilization, or inherited sterility, must be highly efficient with little or no negative impact on the fitness of the males so they can compete successfully for wild females . InsectsAe. aegypti in particular, poses several unique challenges. A key challenge with Ae. aegypti is their short flight range. The average flight range of Ae. aegypti, regardless of sex, is reported to be only ca. 25–200 m (Ae. aegypti requires us to consider different release strategies to effectively cover a given area when administering a mosquito SIT program with this species. Mosquito releases using Unmanned Aircraft Systems (UAS), also known as drones, carrying a mosquito storage and release system can provide a solution to this problem. By utilizing UAS, the distribution area can be increased compared with ground release strategies . Water with 20% sucrose was offered in 25-ml Eppendorf flasks with a cotton wick, and the males were left to recover for no longer than 2 h. Mosquitoes that were unable to fly were counted as dead.Males were anesthetized by placing the aspirator tubes on ice for approximately 10 min and transferred to the surface of a petri dish that was placed on ice. Males were separated from females using pointed feathers and tweezers, counted, and placed into 50-ml Falcon tubes . Holes to allow for airflow were drilled into the tube caps and in the bottom of the tubes using 16-gauge injection needles . Tubes were placed in an Incufridge incubator  set to 80% humidity and at 7, 14, 21, or 28°C. For these experiments, a replicate consisted of a tube containing 20 male mosquitoes with 3 were constructed from 50-ml Falcon tubes by cutting them with a hand saw at the 40-ml line. The cut end of the Falcon tubes was sealed off using a fine mesh attached with a hot glue gun . Holes were drilled into the lids using 16-gauge needles to allow for air flow. Males were anesthetized by chilling on ice (as described earlier), counted, and placed into the compaction chambers. The compaction chambers were then placed in an incubator set to 80% humidity and at 7, 14, 21, and 28°C, and held for 24 h. At each temperature, four levels of compaction were created by adding 100, 200, 400 and 800 males per compaction chamber. After 24 h, the mosquitoes were transferred to BugDorm cages  provisioned with sugar water as described earlier and allowed to recover for 2 h. Mosquitoes unable to fly after 2 h were counted as dead. For each temperature, four replicates were performed.Compaction chambers with a volume of 10 cm3) at numbers of 10, 20, 30, 40, 50, 60, 70, 80, 160, and 240 male mosquitoes. A second compaction study was performed using 5-ml syringes. Male mosquitoes were compacted at the same volume  were compacted in the 10-ml syringes to a volume of 1 cmmark see . The syrKaplan–Meier survival analysis tests were conducted using XLSTAT  to determine the statistical significance in differences in survival of male mosquitoes under different temperatures and compaction rates. A Mann–Whitney analysis was used to determine the statistical significance for temperature and cooling and compaction assays .3 containers in groups of 20 males. We started this study by determining suitable storage temperatures for male mosquitoes that were stored in 10 cm3); however, there was a statistically significant decrease in survival at 800 mosquitoes/10 cm3  and different compaction numbers  in the above-described setup. When observing mosquito survival at 7°C, there was no significant difference between the different compaction rates  stayed relatively consistent (over 70%) regardless of the number of males placed in the volume (P < 0.05).To confirm the results shown earlier, we conducted syringe assays at 14°C. Survival of males in a 10-ml syringe compacted down to 1 ml , whereas none of the survivors at the lowest compaction rate were missing scales  ; Cydia pomonella Linnaeus [Lepidoptera: Tortricidae]) demonstrate the efficacy of a single UAS deployed to release sterile insects. In larger-scale pest programs deploying SIT as a component of AW-IPM, the use of swarm technology is currently under development  . The facprograms . We propelopment . During In most realistic scenarios, the rearing/sterilization sites and release sites of SIT insects are located at considerable distances. Previously, packing for transportation of the screwworm fly has included low-temperature immobilization ; carbon Ae. aegypti males can tolerate a wide range of storage temperatures for the first 24 h. Mortality at the 48-h time point and all later time points was significantly increased at all temperatures we tested. We therefore recommend to avoid transport and storage times longer than 24 h for male mosquitoes.The results of our incubator studies  suggest 3 chambers suggested that a compaction rate of 40 mosquitoes/cm3 results in better survival than the higher compaction rate of 80 mosquitoes/cm3  resulted in the highest survival rates (Based on our findings, we decided to perform a real-life shipping assay using syringes as transport chambers and determine survival as well as damage to the mosquito males that were shipped at different compaction rates. The results of the shipping assay clearly show that the highest compaction rate we tested (240 males/cmal rates . AlthougIn summary, our results show that lower compaction rates produce less individual damage but significantly higher mortality rates in our real-life overnight shipping assay. Mosquito survival at the highest compaction rate we tested was 85%. This is good news because many professional courier delivery services offer ‘overnight shipping’ within the United States, thereby providing a suitable transport chain for sterile male mosquitoes from rearing facilities to their release sites.Supplemental FileClick here for additional data file.


Leishmania infantum–to reduce infantile clinical visceral leishmaniasis (VL).To assess the effectiveness of community-wide deployment of insecticide–impregnated collars for dogs- the reservoir of A pair matched–cluster randomised controlled trial involving 40 collared and 40 uncollared control villages , was designed to detect a 55% reduction in 48 month confirmed VL case incidence. The intervention study was designed by the authors, but implemented by the Leishmaniasis Control Program in NW Iran, from 2002 to 2006.The collars provided 50% (95% C.I. 17·8%–70·0%) protection against infantile VL incidence (0·95/1000/yr compared to 1·75/1000/yr). Reductions in incidence were observed across 76% (22/29) of collared villages compared to pair–matched control villages, with 31 fewer cases by the end of the trial period. In 11 paired villages, no further cases were recorded post–intervention, whereas in 7 collared villages there were 9 new clinical cases relative to controls. Over the trial period, 6,835 collars were fitted at the beginning of the 4 month sand fly season, of which 6.9%  were lost but rapidly replaced. Collar coverage (percent dogs collared) per village varied between 66% and 100%, with a mean annual coverage of 87% . The variation in post-intervention clinical VL incidence was not associated with collar coverage, dog population size, implementation logistics, dog owner compliance, or other demographic variables tested. Larger reductions and greater persistence in incident case numbers (indicative of transmission) were observed in villages with higher pre-existing VL case incidence.L. infantum.Community–wide deployment of collars can provide a significant level of protection against infantile clinical VL, achieved in this study by the local VL Control Program, demonstrating attributes desirable of a sustainable public health program. The effectiveness is not dissimilar to the community-level protection provided against human and canine infection with  Leishmania infantum. Dogs are the proven reservoir. The disease is of global health significance, and usually fatal unless treated. There are limited options to reduce transmission. Insecticide-treated dog collars have been shown to protect dogs against infectious bites from sand fly vectors, resulting in reductions of new infections in both dogs and humans. However, there have been no studies to demonstrate the public health benefits of this approach i.e. the impact on clinical VL incidence. This study assessed the effectiveness of community-wide deployment of insecticide–impregnated collars on dogs to reduce the incidence of clinical visceral leishmaniasis in children, the high risk age-group. Collars were fitted to dogs in 40 endemic villages over 4 consecutive years by the regional public health authorities in NW Iran. The case incidence of infantile visceral leishmaniasis in these villages was compared to that in 40 untreated villages at the end of the intervention period. The community-wide deployment of collars proved to provide a 50% reduction in the development of the disease in children. This effect was achieved under the operational conditions of the regional routine health authorities. We conclude that the implementation of insecticide-impregnated collars should be considered in strategic scale-up operations against zoonotic visceral leishmaniasis.Zoonotic visceral leishmaniasis is a sand fly-borne disease of humans and dogs caused by the intracellular parasite  Leishmania donovani or L. infantum, characterized by prolonged fever, wasting, splenomegaly, and hepatomegaly, and >95% case–fatality in the absence of treatment[Leishmania are transmitted by female phlebotomine sand flies. Leishmaniasis is a Neglected Tropical Disease (NTD) strongly associated with poverty and malnutrition[Visceral leishmaniasis (VL) is a protozoan vector–borne parasitic disease of humans following infection with treatment. Leishmanutrition, resultinutrition. Difficunutrition–5.L. donovani is anthroponotically transmitted, whereas VL due to L. infantum is a zoonosis involving infectious domestic dog reservoirs[VL due to eservoirs, and unceservoirs, in maineservoirs. Otherwieservoirs–14, altheservoirs, 16.Regional VL control programs focus on human case detection and treatment, adult sand fly vector control, and for dogs, optional strategies including canine vaccination, topical insecticide protection, chemotherapeutic treatment, or euthanasia–19. AnthVector and animal reservoir control are necessary to reduce zoonotic VL incidence as treatment of human clinical cases, though necessary, is unlikely to impact on the transmission cycle. Insecticides topically applied to dogs as slow release insecticide–impregnated collars have been extensively tested in different regions, showing that they are effective against sand fly vectors–28, and In collaboration with the regional VL control program authorities in NW Iran, and with access to VL case records provided by the Ministry of Health (MoH), we had the opportunity to evaluate the impact of community–wide distribution of collars against clinical VL incidence, and as conducted under operational conditions. The principal aims of the study were (i) to measure the efficacy of the collar intervention against VL case incidence; (ii) to evaluate the operational logistics of collar implementation; and (iii) to assess likely causes for the variation in intervention effectiveness.The study was conducted in the rural communities of the Kalaybar and Ahar administrative districts of East Azerbaijan province, NW Iran  from 2002 to 2006. Villages were located at altitudes of 369-1305m . The maiL. infantum infection incidence measured by Direct Agglutination Test (DAT) seroconversion was 2·4% compared to 1.9% by Leishmanin skin test (LST) reaction [Phlebotomus sand fly vectors and infected dogs[Leishmania seropositivity is associated with dog ownership, village dog density , and the dog to human ratio[Phlebotomus (Larroussius) perfiliewi transcaucasicus and Ph. (L.) kandelakii, are likely vectors in this region, being seasonally active for 4 months (late June to October)[In the region, 100–150 new VL cases were reported annually between 1998 and 2005, representing 45% of the total VL cases in Iran, the vast majority being children <10yrs of age, 38. At reaction . Canine reaction , with rereaction , 38, 39.reaction  comparedcted dogs. The ris October), 44.khaneh behdasht) where resident Health Promoters (behvarz) are responsible for disease surveillance, control implementation, and facilitate suspect VL cases attendance at the RHCs. The RHCs provide free VL diagnostic testing and treatment services, and refer those needing more specialist hospitalization to the DHCs or district hospital. Clinical VL is a notifiable disease.The VL control program hinges on the District Ministry of Public Health (DTARH) which is responsible for the District Health Centers (DHCs) in Kalaybar and Ahar. These centers coordinate activities of 12 provincial Rural Health Centers (RHCs), which are run by trained medical staff and health officers. Each RHC supervises about ten village health posts  or control (no collars) group by tossing a coin in the presence of observers. All subsequent pairs were similarly assigned alternately to either the treatment or control group, resulting in 40 control and 40 pair-matched villages.Leishmania control program, during the trial period neither insecticide spraying, canine test–and–slaughter, nor VL health education programs were conducted in the enrolled communities. However, 27 stray dogs were removed by the authorities .The pre–intervention DTARH house–to–house survey indicated that the collar and control villages (clusters) were demographically well balanced . AlthougDog collars impregnated with 40mg/g deltamethrin , were fitted to household dogs in the 40 treatment villages in late June/early July in each of four consecutive years (2002–2005). The Scalibor collar label claimed 5–6 months insecticidal activity, 26 whicThe intervention was implemented by DTARH operational DHCs in Kalaybar and Ahar. The collars were donated by Intervet, and given to the DHCs who distributed them to the village Health Promoters, ensuring that sufficient collars and replacements were supplied each year. Health Promoters conduct routine house–to–house visitation forming part of the DTARH public health advisory and monitoring system. On these visits, they fitted the collars to dogs, promoted the correct use of the collars, recorded the daily numbers of collars fitted, lost, replaced, or refusals to fit collars (defined here as non-compliance), and identified any adverse clinical effects reported by dog owners. The authors helped train the Health Promoters to systematically record these events.Leishmania in clinical aspirates. All confirmed cases received treatment following WHO guidelines[Leishmania tropica is also transmitted in the region. However, this parasite typically causes cutaneous infections, does not normally induce DAT seroconversion, and the distinctive clinical signs are known by the DHCs.Confirmed clinical VL case numbers were extracted from secondary records provided by DTARH. At the time of the study, the DTARH protocol stated that suspect VL cases were considered confirmed if they presented clinical signs including prolonged fever, lymphadenopathy, and hepatosplenomegaly, associated with a positive DAT (threshold ≥1/3200 titer), and/or microscopic detection of uidelines. Based ouidelines, LeishmaDTARH collated details of confirmed VL incident cases which were provided to the authors anonymised accompanied by records of the patient’s age, sex, village of residence, and date when they first presented at the medical center.Lacking placebo collars, the village Health Promotors were not blinded to the intervention, but the clinicians and DTARH data managers who collated the case data were.year × and end of Juneyear ×+1 were assigned to transmission seasonyear ×. This interval allowed for a ≥9 month incubation period which exceeds the usual 2–6 months incubation period reported in Iran and elsewhere[The primary outcome for analysis was the cumulative number of incident VL cases per village by the end of the 48 months follow–up period. The transmission season of infection was attributed based on the date of the patient’s initial presentation: dates falling between Julyelsewhere.The study was statistically powered to detect a 55% reduction in the 48 month clinical VL incidence in collar clusters compared to control clusters, from baseline 0.004 (~3.978 cases per 1000 children ≤10yrs old/yr) calculated for the 4·5 yr pre–intervention period in the 80 trial clusters, and a surveyed harmonic mean of 106 children per cluster. Data from the previous collar trial in the region, gave an10 transformed childhood baseline village incidence, and the pair–matching structure (as a cluster term). The number of children ≤10yrs per village, being the well documented high risk group, was set as the model offset parameter. The variance in cluster ratios of children to total population size were similar between treatment arms (The intervention effect was computed using random–effects Poisson regression to test differences in the cumulative numbers of incident VL cases per cluster (= village), expressed as an incidence risk ratio (IRR). The model included variables describing the trial design: the attributed year of infection, the logent arms . Additiot-test where the results of each cluster are given equal weighting[A secondary analysis following tested tweighting. DiffereThe relationship between post-intervention VL incidence differences between pair-matched villages, and pre-intervention incidence, was examined by linear regression .The trial protocol was approved by the Regional Committee of Medical Ethics, Tabriz University of Medical Sciences, Iran. The University of Warwick’s Biomedical and Scientific Research Ethics Committee (BSREC) confirmed that the trial raised no significant ethical concerns due to its use of secondary anonymised human case data. There was no local animal ethical committee at the time of the study. Informed written consent was obtained from village leaders, and from dog owners to fit collars, and dogs were monitored on a regular basis by village Health Promotors for any adverse reactions to the collars.In the 4·5 years prior to the intervention (January 1998– June 2002), 113 confirmed clinical VL cases were reported in a population of 6,562 children in the 40 control villages, with an average annual case incidence of 3·83 per 1000. In the 40 collar villages during the same period, 118 cases were recorded amongst 6,341 children, an average incidence of 4·14 per 1000. These rates were not significantly different .2(1) = 7·83, p = 0·0051). The trial design–adjusted incidence risk ratio (IRR) was 0·50   = 25·7, p<0·0001), equivalent to 50%  protection against clinical VL.During the 48 month intervention period, 24 compared to 46 clinical VL cases were recorded in the collar and control arms, respectively. The corresponding incidences were 0·95/1000/yr and 1·75/1000/yr. Accounting for a general decline in VL case numbers over the years of observation in both trial arms , there wvs control villages  (Supplementary S1). The non–parametric sign rank test of these residuals was also significant (p = 0·008).The intervention effect was further confirmed by showing a significantly lower than expected number of cases in collared 2(1) <1·92, p>0·187).Additional demographic variables , with M:F sex ratios of 25:21 and 12:12 in control and collar arms, respectively. There were no significant differences in case age distributions between trial arms , nor between intervention years .The annual point estimates of the intervention effect suggested that the protective effect steadily increased with time under intervention, though the case numbers were low and the errors broad .2(1) = 7·27, p = 0·007).By the end of the intervention, 12 (30%) of the 40 collar villages, compared to 24 (60%) of the 40 control villages, presented ≥1 clinical VL case, indicating a reduced risk also at the village level  collared villages showed reductions in incidence compared to the pair-matched control villages , with 11The difference in post-intervention VL incidence between pair-matched villages attributed to the intervention was negatively associated with their pre-intervention baseline incidence  . This suIn the control arm, there was a positive association between village pre–and post–intervention incidence  . This re2(3) = 0·260, p = 0·967), although it varied significantly between villages (range: 65·7%–100%)  = 0·122, p = 0·0001) . Of these, 6.9%  were lost per year, respectively 7·3% , 6.4% (110/1722), 6.9% (117/1689) and 7.0% (122/1742). The mean annual coverage  was 87%  being consistent across years  . Coverag2 = 0.008); similar coverage was observed in villages with no incident VL cases, as those with >0 incident VL cases  of supply to the first village, all collars were fitted in the 40 villages within one month.Based on the most complete daily records (2002 data), collars were distributed by the DHCs to all village Health Promoters within 16 days at the start of the study. Aligned from the date  from collar provision, with >70% in place within eight days. The number of dogs per village did not influence either the total fitting time  or daily fitting rates . Fitting rates were similar for guard and shepherd dogs .Collars were lost in 35 of the 40 collar villages, 2–147 days post-fitting. A median 3  collars were lost per village. However, these were rapidly replaced: 59% within 4 days, and 91% within 8 days.The effectiveness of insecticide–impregnated collars against human clinical VL had not been trialled as a potential public health control option. Community–wide collar coverage over 4 transmission seasons reduced the relative risk of infantile VL by 50% , and reduced the total VL case numbers by 48% during the intervention period.L. donovani infection[L. infantum VL case age distributions are appearing[The protective effect was clear despite a 70% general decline in reported case numbers over the study period, the decline being consistent with national trends from the late 1990s to 2014 in Iran, with spinfection, and in infection. Traditiappearing.L. infantum[cf. clinical disease) incidence of 43%  in ≤10yr old children[Phlebotomous perniciosus sand fly bites for up to 12 months[Leishmania antibody titers, observed in seronegative collared dogs[All eight published studies designed to evaluate the efficacy of Scalibor collars, demonstrated reduced canine seroinfection incidence by 46%–86% within 1–2 years of use, 37, 48. children. What wa children, 26. Mor12 months. These e12 months, 51; colared dogs, supportThe annual effect estimates in this study suggest that one to two years of continued intervention achieved a similar outcome to the 4-year cumulative effect. However, the point estimate errors were broad as the study was not statistically powered to detect significant changes in any single transmission season. Interpretation of the effect estimates over time was further hindered by the very low VL case numbers in both arms and inevitable stochastic events in the latter years of the study. A different cyclical pattern of VL between control and intervention villages is unlikely to have occurred due to the randomization of trial clusters.Clinical VL cases continued to be notified in villages during the years of collar use, indicating that collars were not wholly effective in interrupting transmission. Villages with higher pre–intervention incidence were more likely to experience continued transmission, despite achieving greater relative reductions in disease incidence compared to villages of low pre–intervention incidence. This raises the possibility that the collar coverage was insufficient in some villages.Collar coverage showed significant variation between villages with some being consistently low. Changes in village VL incidence was not associated with village coverage, nor indeed with any demographic variable. In three of the seven collared villages where the intervention failed to prevent a higher number of incident cases compared to controls, annual collar coverage was >84% (average c.90%); and in all except one of the 7 villages, the minimum annual coverage was >77%. Therefore, we do not attribute these failures to lower collar coverage levels. Nor do we attribute it to collar losses or to non–compliance (owner refusing to have collars fitted to dogs). Short–term allergic dermal reactions to deltamethrin, reported in 2%–16% of collared dogs in two Brazilian studies, 37, usuDogs ≤3m old were not collared (following label instructions) but represented <3% of the canine population. The period of latency to infectious onset far exceeds the 4 month transmission season, thus ruA significant finding of this study was that the intervention effect was achieved under the operational conditions as implemented by the DTARH VL control program. After ensuring that the design and village randomisation processes met CRT analytical requirements, the authors had minimal contact with authorities. The authors supplied known numbers of collars to the DHCs who distributed them to the villages, and collated details of the collar implementation. This provided the opportunity to measure the logistics and likely sustainability of programmatic scale–up.2, and total collars being fitted within one month from collar provision to the first village.Collars were fitted within only 15 days of village supply, and collar losses were rapidly replaced, suggesting coverage levels were maintained during the transmission seasons. Neither coverage level nor time to complete collar fitting were related to the total number of dogs eligible for a collar, concluding that lower village coverage levels were not due to time constraints. Feasibility of a regional–level scale–up was indicated by DTARH taking only 16 days to deliver collars to all 40 villages spread over an area of 15,000kmCollars were socially accepted, visibly distinguished treated dogs, and should not require replacement every 3–4 weeks, unlike alternative topical formulations. Coupled with the short transmission season, effective collar duration, and rapid replacement rate, the feasibility of effective sustainable scale–up is indicated. Collar losses in this region of between 1% per month and 1.7%Sustainability will be governed also by costs and cost–effectiveness. Considering the cost of collars alone, in this study, collars were donated by the manufacturer. However, based on €14 per collar, the annual material cost would have equated to approximately €24,000, or €96,000 over 4 years. That is €4,364 per VL case averted compared to case numbers in the control arm. This value is for a period with 70% relative decline in reported case numbers from the late 1990s to 2014[One key knowledge gap is how best to implement dog collar programs: currently unknown is the minimum community coverage level necessary to reduce transmission, equivalent to vaccination coverage thresholds calculated to maintain herd immunity. MathemaOf relevance to NTD elimination strategies, our data suggest that greater initial reductions in incidence may be achieved in communities with higher compared to lower preexisting transmission pressure. One prediction is that by reducing transmission, infection in the lower-prevalence settings becomes more clustered, thus more stable and more resilient to interventions. ApproacIn conclusion, the results of this study promote the community-wide application of insecticide–impregnated collars as a public health tool to reduce clinical VL burdens, not just human and canine infection. The observed level of impact was achieved working within the existing MoH infrastructure, demonstrating many desirable attributes of a potentially sustainable control program that could be scaled–up with minimal additional technical capacity training.S1 Table(PDF)Click here for additional data file.


Escherichia coli (EHEC) in raw milk and molecular characterization of isolates using multiplex polymerase chain reaction (PCR).The aim of the present investigation was to study the epidemiology of enterohemorrhagic A total of 125 raw milk samples were subjected to isolation, identification, and confirmation of virulence-associated genes by multiplex PCR (mPCR). The samples were collected from a milk cooperative society of Thrissur district, Kerala. For further epidemiological investigation, samples such as dung (126), hair coat of cow (60), udder swab (60), udder wash (60), milking utensil wash (36), Milker’s hand wash (36), water (36), soil (36), and feed (36) were collected from the households from which the raw milk tested positive for EHEC.stx 2 gene while 53.33, 36, and 36% of isolates were encoded by stx 1, eae A, and hly A genes, respectively. On epidemiological survey, the multiple risk factors accountable for occurrence of EHEC in raw milk were found to be the quality of water used, improper and inadequate udder preparation, unhygienic hands of Milker’s, use of insufficiently cleaned milking utensils, and using common utensil for washings of udder and milking purposes.The occurrence of EHEC in individual raw milk samples was found to be 8.8%. The major source of contamination to raw milk was found to be dung (19.84%) followed by udder swab (16.67%), hair coat of cow (15%), Milker’s hand and milking utensils and water (11.11% each), and udder wash and soil (8.33% each). For identification of virulence genes, all the isolates were subjected to mPCR, of 75 isolates 73.33% of isolates harbored The result of the present study signifies that raw milk was contaminated with EHEC and possesses a high public health threat. As dairy cattle and its environment serve as a potential niche for EHEC, hygienic milking practices should be adopted to curb the occurrence of EHEC in raw milk. Safe and nutritious food is the key to attain and promote good health. Besides this with a change in human food habits, the lifestyle foodborne diseases are showing an upward trend, resulting in high morbidity and mortality across the world. It has been reported that the diarrheal diseases contribute to more than half of global foodborne disease burden .Escherichia coli (EHEC) in particular EHEC O157:H7 is an important emerging pathogen responsible for gastrointestinal illnesses in children and elderly people. The very low infectious dose of EHEC ranging from 10 to 100 colony forming units makes them highly virulent and increases the risk of infection. In humans, EHEC cause infections ranging from mild diarrhea to life-threatening complications, namely hemorrhagic colitis and hemolytic uremic syndrome [Among various foodborne pathogens, enterohemorrhagic syndrome ,3. Milk syndrome ,5. HenceEscherichia coli can be detected as well as differentiated into separate pathotype and serotypes using mPCR. Hence, application of mPCR is considered to be a rapid approach for early and easy detection of EHEC [Various conventional culture techniques described for its isolation are time-consuming and are mainly based on the phenotypic characteristics. Polymerase chain reaction (PCR) based on genotypic characteristics are considered to be a rapid and promising technique for molecular confirmation of pathogens. Hence, multiplex PCR (mPCR) can be applied to food matrices as it aids in the rapid detection of multiple pathogens or multiple virulence factors of a single pathogen.  of EHEC ,7. In EH of EHEC . PreventThe objective of the present research work was to study the epidemiology of EHEC in raw milk and retrospective trace backing of the source of contamination and route of entry into raw milk.The present study did not involve any invasive procedure and hence no ethical approval is necessary. The samples were collected as per the standard collection procedure. However, animal owners who do not wish to participate were respected and not considered for the study. Confidentiality of the collected data was maintained. Verbal informed consent was obtained from each animal owner.The EHEC reference culture procured from the repository of Indian Veterinary Research Institute, Izzatnagar was used as a standard culture in the study.The entire study was carried out in a milk cooperative society belonging to Thrissur district of Kerala. To select one milk cooperative society, pooled raw milk samples obtained from randomly selected milk cooperative societies belonging to Thrissur districts were screened for EHEC. From the selected milk cooperative society, positive milk samples were subjected to epidemiological study.In the selected cooperative society of total 230 cattle, 125 cattle were in lactation, and hence 125 raw milk samples were collected from the society and were screened for the presence of EHEC. To determine the source of contamination and route of entry of EHEC into raw milk an epidemiological study was performed in each of the EHEC positive households. The following samples were collected at the time of milking from the animal such as dung (126), hair coat of cow (60), and udder swab (60). The samples were also collected considering the milking practices and environmental parameters existing in and around the household premises which included udder wash (60), milking utensil wash (36), Milker’s hand wash (36), water (36), soil (36), and feed (36) samples. The above-mentioned samples were collected from all the positive houses for 6 times at an interval of 2 weeks and examined for the presence of EHEC.All the samples were collected during the study period from August 2016 to May 2017. The aseptically collected samples were brought to the laboratory under the refrigerated condition and processed for the analysis within 24 h.An epidemiological survey was conducted through a well-designed questionnaire to know the various risk factors involved in milking practices and their influence on occurrence of EHEC in raw milk samples. At the time of milk sample collection, a total of 92 farmers were interviewed, and the data on hygienic milking practices adopted by the farmers such as hand, udder, and milking utensil hygiene practices that had an indirect bearing on the quality of milk were collected.All the collected samples were processed in Quality Control Laboratory, Department of Veterinary Public Health, College of Veterinary and Animal Sciences, Thrissur. About 25 g of solid samples were transferred into 225 ml of trypticase soy broth (TSB) and homogenized for 180 s . The 25 ml liquid samples were pre-enriched in 225 ml of TSB while swab samples were pre-enriched in 45 ml of TSB.et al. [E. coli colonies with a metallic sheen on EMB agar were streaked onto Cefixime Tellurite-Sorbitol MacConkey agar (CT-SMAC) supplemented with novobiocin, cefixime, and potassium tellurite followed by incubation at 37°C for 18 h. To detect β-glucuronidase activity, the isolates were streaked onto 4-methylumbelliferyl β D-glucuronide (MUG EC) agar as described by Fujisawa et al. [Samples were subjected to isolation and identification of EHEC as per Meng et al.  with cera et al. . Non-fluE. coli O157:H7. Virulence gene profile of the isolated EHEC strains was analyzed using mPCR with primers specific against stx 1, stx 2, eae A, and hly A genes, producing amplicons of size 180, 255, 409, and 526 bp, respectively . The primers used and their corresponding product size are enlisted in A mPCR was standardized against virulence genes of EHEC using EDL 933 strain of ectively . The pri2 (25 mM), 2.5 µl dNTP mix (2 mM each), 0.75 µl taq DNA polymerase (5 units/µl), 1 µl each forward and reverse primers of stx 1, stx 2, and eae gene, and 0.5 µl primers of hly A gene. The final volume was adjusted to 30 µl with nuclease-free water. The mPCR cycle consisted of initial denaturation at 95°C for 5 min followed by 35 cycles of denaturation at 95°C for 35 s, annealing at 60°C for 45 s, and elongation at 72°C for 2 min. The final extension was carried out for 10 min at 72°C. The primers used and their expected product size are enlisted in The bacterial DNA was subjected to mPCR with a reaction volume of 30 µl consisting of 2.5 µl template, 3 µl 10× PCR buffer (200 mM), 2 µl MgClThe data obtained were statistically analyzed using the SPSS version 21.0. Since the sample size was more than 30, the occurrence of EHEC between various samples collected from dairy cattle rearing environment was compared using Z-test. The differences between frequencies or proportions in the occurrence of virulence genes in EHEC isolates were tested by Cochran’s Q-test. Fisher’s exact test and Z-test were used to study the influence of various risk factors involved in milking practices followed by farmers on occurrence of EHEC in raw milk.stx 1, stx 2, eae A, and hly A genes were detected in 27.27, 90.91, 18.18, and 27.27% of isolates. Statistical analysis using Cochran’s Q test revealed a significant difference (p<0.05) in occurrence of stx 2 gene from that of all the other virulence genes among isolates from raw milk  from that of udder wash samples. However, occurrence of EHEC in all the other samples examined from dairy cattle rearing environment did not differ significantly (p<0.05) .stx 1 and hly A gene in isolates from dung samples while the isolates from udder swab differed significantly with respect to occurrence of stx 2 and hly A gene  in the occurrence of y A gene .stx 2 gene was more predominant (73.33%) against stx 1 gene (53.33%) while hly A and eae A genes were detected in 36% of isolates. The distribution of virulence genes among the positive isolates from different sources is as shown in On mPCR all the 75 isolates obtained by conventional culture technique, coded either of virulence genes . Among tAssociation of hygienic milking practices followed by farmers with the occurrence of EHEC was studied using a well-designed questionnaire, and the details are given in It was observed that isolation rate of EHEC in raw milk was highest among the households using plain water for washing of hand, udder, and milking utensils against those households who used an antiseptic solution such as potassium permanganate solution. The raw milk from the houses wherein the same milking utensil was used for washing of udder and milking purposes were highly contaminated with EHEC. Hence, the major risk factors governing the occurrence of EHEC in raw milk were identified as the health status of animal, contaminated udder, unhygienic milking utensil, and Milkers’ hands.Milk is considered to be sterile when secreted from the udder of an apparently healthy animal. Thereafter, it is prone to contamination with pathogenic bacteria at various stages from production until consumption. Raw milk consumption is still practiced by some sector of population with an assumption that nutritional qualities and health benefits will be higher for raw milk than the boiled or processed milk which intensifies the risk of acquiring EHEC infection.et al. [et al. [et al. [et al. [From this study, it is evident that raw milk serves as a potential source of EHEC as it was isolated from 8.8% of samples. Similarly, in the previous studies Neher et al. , Garbaj  [et al. , and Set [et al.  had isol [et al.  and Ntul [et al.  who repoet al. [Among the different samples collected from dairy cattle rearing environment, the highest occurrence of EHEC was seen among the dung samples indicating that the dairy cattle can serve as reservoir and void EHEC inconsistently. Similar findings were reported by Cobbold et al.  wherein et al. . Hence, et al. [et al. [et al. [et al. [Other sources of contamination to raw milk were externally contaminated udder, hair coat of cow, milking utensil, Milker’s hand, and water and soil samples. Similarly, Abdissa et al.  and Anu et al.  had isol [et al.  and Msol [et al.  found th [et al.  reportedThe occurrence of EHEC in milking utensil rinses and Milker’s hand washes proved that insufficiently cleaned milking utensils and Milker’s hands contribute various microorganisms into raw milk. In 2016, Msolo  had repoet al. [et al. [et al. [et al. [et al. [Results of the present study also proved that the samples from dairy environment such as soil and water were contaminated with EHEC. While results were not in accordance with Wetzel and LeJeune  who recoet al.  who repo [et al.  had isol [et al.  who coul [et al. , while;  [et al.  had isolstx 2 was higher followed by stx 1, eae A, and hly A gene. Detection of virulence genes confirmed the sensitivity of conventional culture technique. In 2006, Fremaux et al. [stx genes in 80 fecal and 38 environmental isolates from French dairy farm. The author reported a similar trend in the distribution of virulence genes as observed in this study where stx 2 (84%) was frequently detected followed by stx 1 and eae A genes .Among the isolates recovered, the frequency of detection of x et al.  detectedet al. [et al. [The highest occurrence of EHEC among those cattle that were grazed within premises can be attributed to the contaminated pasture where the manure was used for agriculture purpose within the premises. Similarly, Hancock et al.  have fou [et al.  and by T [et al. , respectE. coli and EHEC in raw milk samples possess a great health risk to raw milk consuming population. Detection of both the primary virulence factors (Shiga toxin-encoding gene) and associated virulence factors (intimin and hemolysis) showed that dairy environment serves as a potential niche for EHEC and the dairy farmers were continuously exposed to this pathogen. Hence, this study demands a nationwide education of farmers regarding hygienic milking practices and its benefits in assuring the safety of raw milk to the public. Furthermore, awareness should be created among the dairy farmers on various zoonotic disease transmissions from dairy environment and the preventive measures.The results of the present epidemiological investigation on occurrence of EHEC in raw milk samples collected from cooperative society belonging to Thrissur district of Kerala indicated that raw milk was contaminated at primary production level itself. The major risk factors governing the occurrence of EHEC in raw milk were identified as the health status of animal, contaminated water, soil, udder, unhygienic milking utensil, and Milkers’ hands. Presence of VHD collected the samples, carried out the laboratory work. SC and LC supervised the research work, reviewed the manuscript, and guided research work. VHD, SC, and LC contributed to the molecular work of the study and revised the manuscript. All authors read and approved the findings of the manuscript.