SCN), with diminution of circadian peak dopaminergic activity at the SCN facilitating development of the seasonal obese insulin‐resistant condition. The present study investigated whether such an ancient circadian dopamine‐SCN activity system for expression of the seasonal obese, insulin‐resistant phenotype may be operative in animals made obese amd insulin resistant by high‐fat feeding and, if so, whether reinstatement of the circadian dopaminergic peak at the SCN would be sufficient to reverse the adverse metabolic impact of the high‐fat diet without any alteration of caloric intake. First, we identified the supramammillary nucleus as a novel site providing the majority of dopaminergic neuronal input to the SCN. We further identified dopamine D2 receptors within the peri‐SCN region as being functional in mediating SCN responsiveness to local dopamine. In lean, insulin‐sensitive rats, the peak in the circadian rhythm of dopamine release at the peri‐SCN coincided with the daily peak in SCN electrophysiological responsiveness to local dopamine administration. However, in rats made obese and insulin resistant by high‐fat diet (HFD) feeding, these coincident circadian peak activities were both markedly attenuated or abolished. Reinstatement of the circadian peak in dopamine level at the peri‐SCN by its appropriate circadian‐timed daily microinjection to this area  abrogated the obese, insulin‐resistant condition without altering the consumption of the HFD. These findings suggest that the circadian peak of dopaminergic activity at the peri‐SCN/SCN is a key modulator of metabolism and the responsiveness to adverse metabolic consequences of HFD consumption.Among vertebrate species of the major vertebrate classes in the wild, a seasonal rhythm of whole body fuel metabolism, oscillating from a lean to obese condition, is a common biological phenomenon. This annual cycle is driven in part by annual changes in the circadian dopaminergic signalling at the suprachiasmatic nuclei ( The seasonal obese condition is coupled with the hyperinsulinaemic, insulin‐resistant state similar to the human metabolic syndrome.We postulated that this ancient circadian control system for whole body regulation of fuel metabolism may modulate the sensitivity of the body to the obesity/insulin resistance‐inducing effects of a high‐fat diet (HFD) and, as such, play an important role in regulating the metabolic syndrome‐inducing effects of the westernised diet of modern man. This clock system for the regulation of metabolism is sensitive to seasonal changes in nutrient quality 22.1Laboratory rats  (10‐12 weeks of age) were housed individually and maintained in a temperature controlled room on 14 hour daily photoperiods (14 : 10 hour light/dark [LD] cycle) during all experimental study periods. Animals were allowed to feed either a HFD  or regular rodent chow (RC) diet  and to drink ad libitum throughout the study period. All animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Experimental Animals and also with the protocols approved by the Institutional Animal Care and Use Committee of VeroScience, LLC.2.2‐1 body weight, i.p.) and secured on a stereotaxic apparatus. Retrograde tracer fluorogold (FG)  was microinjected using a glass micropipette (inner tip diameter 20‐25 μm) driven by a Nanoject injector  into the SCN area . Ten days after FG microinjection, the rats were sacrificed under anesthesia by transcardiac perfusion with 4% paraformaldehyde. Brains were post‐fixed in 4% paraformaldehyde overnight and cryoprotected in 30% sucrose/phosphate‐buffered saline (pH 7.4). Free floating coronal brain sections (30 μm) were cut from frozen brains on a cryostat and sequentially collected in a freezing solution containing 30% ethylene glycol, 25% glycerol and 0.05 m phosphate buffer saline (pH 7.4) and kept at −20°C until use. For the dual‐labelling of fluorogold and TH, brain sections were first treated with 3% hydrogen peroxide for 10 minutes to quench the endogenous peroxidase activity followed by 1 hour of incubation in animal‐free blocker . Then, the sections were immunolabelled at 4°C overnight with a rabbit polyclonal antiserum for FG  followed by 1 hour of incubation at room temperature with a goat anti‐rabbit biotinylated secondary antiserum . The FG immunoreactivity was amplified by an avidin‐biotin complex (ABC) system and revealed as dark brown 3,3′‐diaminobenzidine (DAB) punctates. Following thorough washing, the sections were re‐blocked with animal‐free blocker and then sequentially incubated with the mouse monoclonal antiserum for TH  at 4°C overnight and a horse anti‐mouse biotinylated secondary antiserum  for 1 hour at room temperature. Following amplification with the ABC system, the TH immunoreactivities were revealed as diffuse blue‐grey staining in the cytoplasm . The dual‐labelled cells were examined under a light microscope . Images were captured by a digital camera and processed using photoshop .Female SD rats weighing 225‐275 g, maintained under LD 14 : 10 daily photoperiod and fed a RC diet were anaesthetised with ketamine/xylazine  and mounted on a stereotaxic apparatus  for implantation of microdialysis probes. In separate studies, a 30‐gauge stainless steel guide cannula  was permanently implanted aimed either at the top of the right SCN at coordinates 1.3 mm posterior to bregma, 0.25 right lateral to the midsagittal suture and 8.3 mm ventral to the dura, with the incisor bar set 3 mm below the interaural line or at the top of the ventromedial hypothalamus (VMH) at coordinates 2.6 mm posterior to bregma, 0.6 mm right lateral to the midsagittal suture and 9 mm ventral to the dura. The guide cannula was anchored firmly to the skull with three stainless‐steel screws and cemented in place with dental acrylic. Animals were allowed 1 week to recover prior to the initiation of microdialysis experimentation. During microdialysis, each animal was placed in an acrylic bowl with free access to food and water. A 32‐gauge dialysis probe with a 1‐mm long tip of semi‐permeable membrane (20 000 molecular weight cut‐off) was inserted into the guide cannula and the probe membrane protruded 1 mm outside the guide cannula into the SCN at 8.3‐9.3 mm. Using a microinjection pump , filtered Ringer's solution  was continuously perfused through the probe at a rate of 0.2 μL min‐1. The probe was connected to the microinjection pump by microbore Teflon tubing through a counterbalanced 2‐channel liquid swivel arm  attached to the rim of the bowl, thus permitting the animal to move freely without the tubing becoming tangled during the experimental period. Microdialysis samples (0.12 μL min‐1 flow rate) were collected into 300‐μL vials (containing 2 μL of 0.1 n perchloric acid solution) at hourly intervals through an automated refrigerated fraction collector  over a 24‐h period when animals were maintained on a 14 hour daily photoperiod and allowed free access to food and water.Animals (SD rats) utilised in microdialysis studies were anaesthetised with ketamine/xylazine  for peak integration, quantitation and analysis via external standard calibration. Standards of the neurochemicals were prepared in 0.1 n perchloric acid. The sensitivity of detection for each of the neurochemicals measured ranged from 500 fg to 1 pg. Microdialysis samples were analysed for extracellular dopamine metabolites  (measure of dopaminergic activity)The hypothalamic dialysis samples were analysed by HPLC with electrochemical detection . The Coulochem III was equipped with a guard cell  set to +350 mV and placed in‐line immediately preceding the analytical cell . The stationary phase was a commercially available 150 × 2 mm column packed with C‐18  and held at 35ºC, through which the mobile phase  was forced at a rate of 0.2 mL min2.5n perchloric acid solution) was injected into the system using a refrigerated autosampler (ESA 540). At the end of the experiment, animals were perfused under anesthesia with 4% paraformaldehyde and brains were collected at 4 and 16 hour after light onset (Zeitgeber time [ZT]4 or ZT16), respectively, to quantify c‐Fos and TH double‐immunopositive neurones in the SuMN.Female Sprague‐Dawley rats (14 weeks of age) were maintained on 14 hour daily photoperiods and allowed to feed and drink ad libitum. Rats were randomly divided into two groups (N = 10 per group) that were fed either a RC diet or HFD to induce weight gain (25% more than RC fed rats) for 6 weeks. A glucose tolerance test (GTT) was then performed on all rats and then, after a 3‐day rest, in vivo microdialysis was used to study daily extracellular profiles of dopamine metabolites in the SCN of rats. Microdialysis samples from the SCN of free‐living rats held under a 14 hour daily photoperiod and allowed to feed and drink ad libitum during the sampling were collected every 1 hour continuously over a 24‐hour period. Samples were assayed via HPLC for HVA and DOPAC. A sample of 5 μL from a total dialysate sample of 14 μL , respectively, to quantify c‐Fos and TH double‐immunopositive neurones in the SuMN. The brains were similarly processed as described above (in section 2.2) for fluorogold tracing and the 30‐μm coronal sections were stored at −20°C until dual immunohistochemistry of c‐Fos and TH was performed. Briefly, the brain sections containing the SuMN were first labelled with a rabbit polyclonal anti c‐Fos antibody  followed by a goat anti‐rabbit biotinylated secondary antiserum . c‐Fos immunoreactivity was revealed as black Ni‐DAB punctates in the nucleus. Subsequently, the brain sections were labelled with the mouse monoclonal antiserum for TH  followed by a horse anti‐mouse biotinylated secondary antiserum . TH immunoreactivity was revealed by DAB as brown staining in the cytoplasm. The c‐FOS/TH dual‐labelled cells were examined under a light microscope (Nikon). The images were captured with a digital camera and processed using photoshop without altering the ratios of signal from comparative sample regions. Dual immunopositive neurones for c‐Fos and TH at the SuMN and the adjacent posterior hypothalamus (PH) region were identified and counted manually on sequential sections across the whole SuMN in a double‐blind manner. The dual positive numbers obtained from sequential coronal sections of each brain in the same treatment group were used to generate a dual positive number per SuMN/PH test area. The between group difference in dual positive number per SuMN/PH area was then analysed by two‐way analysis of variance (anova). As a control experiment, a parallel set of coronal brain sections at the level of SuMN was processed for dopamine β‐hydroxylase (DBH) immunostaining and TH/DBH double‐immunofluorescence labelling. The coronal brain sections at the level of SuMN were labelled with rabbit polyclonal anti‐dopamine β‐hydroxylase antibody  followed by biotinylated goat anti‐rabbit IgG . The signals were amplified by the ABC complex and revealed by DAB chromogen. To further confirm that TH‐immunopositive neurones detected in the SuMN are dopaminergic but not noradrenergic neurones, double‐immunofluorescence labelling were sequentially performed on the same brain section using rabbit polyclonal anti‐dopamine β‐hydroxylase antibody /DyLight 488‐conjugated donkey anti‐rabbit IgG and mouse monoclonal anti‐TH antibody /RRX‐conjugated donkey anti‐mouse IgG. The TH‐immunoreactivity was detected by red RRX fluorescence and DBH‐immunoreactivity was detected by green DyLight448 fluorescence. Overlay red and green fluorescence signals showed no co‐localisation of DBH labelling with TH labelling.At the end of the above‐described microdialysis experiment, HFD and RC fed SD rats were sacrificed under anesthesia by perfusion with 4% paraformaldehyde at either 5 or 16 hours after light onset  and mounted in a stereotaxic apparatus. The core body temperature was maintained at 37°C with a heating pad. The skull was removed from the area overlying the right side SCN. A silver electrode was implanted at the coordinates: 1.3 mm posterior to bregma, 0.2 mm lateral and 9.2 mm ventral to the dura. The injection cannula was targeted to a region just exterior  to the SCN lateral edge (peri‐SCN), whereas the electrode was placed within the SCN itself. After basal neuronal activity had stabilised, test chemicals at various doses were infused for 1 minute, with rest periods of 25 minutes between each subsequent increased dosage. Electrical signals were passed through an amplifier and surveyed using a Bio Amp ML136 . Analyses of electrophysiological activities were conducted off line with the use of labchart, version 6 (ADInstruments) to isolate spike potentials from the background data. The dose‐response curves were analysed via the Hill equation. All data are expressed as the mean ± SEM. Statistical analysis was performed using Student's t test and anova to determine the treatment difference in dose‐response. A P < .05 value was considered statistically significant.Female SD rats were anaesthetised with thiobutabarbital (120 mg kg2.8m glutamate (loaded cannula concentration) was injected at the peri‐SCN at 14 nmol per 0.2 μL to evoke neuronal activity.m loaded cannula concentrations)m glutamate stimulation (14 nmol per 0.2 μL), and then dopamine (5 mm) infusion (1 nmol per 0.2 μL) with and without various doses of D1 specific receptor antagonist SCH‐23390  (0‐1.24 nmol per 0.2 μL range) or D2 specific receptor antagonist Eticlopride  (0‐1.06 nmol per 0.2 μL range) applied at the peri‐SCN in 1 minute. The SCN neuronal electrophysiological responsiveness to peri‐SCN applied dopamine (1 nmol per 0.2 μL in 1 minute) following similar glutamate stimulation was similarly studied in the presence of presynaptic dopamine D2 receptor antagonist AJ76  (1.86‐11.1 nmol per 0.2 μL range in 1 minute) both with or without dopamine (5 mm) applied.Female SD rats (12 weeks old) were maintained under 14 hour daily photoperiods and allowed to feed ad libitum for at least 1 week before the initiation of experimentation. To study time of day dependent differences in neuronal responses to dopamine at the SCN, electrophysiology recordings were conducted at 14 hours after light onset (ZT14)  or at ZT5 (sleep time of day). After basal neuronal activity had stabilised, 70 m2.9‐1 body weight i.p.) and placed in a stereotaxic frame (David Kopf Instruments). A 26‐gauge guide cannula  was permanently implanted aimed at the lateral edge of the SCN area at the following coordinates: 1.3 mm posterior to bregma, 0.4 mm right lateral to the midsagittal suture and 7.2 mm ventral to the dura. Cannula placement was confirmed by post mortem injection of 1 μL of black ink. The guide cannula was then filled with a stylet and anchored firmly to the skull with three stainless‐steel screws and cemented in place with dental acrylic. This guide cannula allows for the subsequent peri‐SCN/SCN drug administration in unanaesthetised and freely‐moving animals. Rats were allowed to recover for 1 week following the surgery. On each treatment day, the stylets were removed and a 28‐gauge injecting cannula connected to a Hamilton syringe mounted on an infusion pump was inserted into the guide cannula. The infusion cannula was protruded 2 mm from the guide cannula. Vehicle  or drug  was given in a total volume of 0.2 μL with a flow rate of 0.2 μL min‐1 for 1 minute. A further 60 s were allowed before removing the injection cannula. The dosing (2 nmol in 0.2 μL) was chosen based upon previously published data demonstrating strong physiological responses to paraventricular nuclei (PVN) targeted administration of such dopamine doseSurgery was performed on SHR rats at 16 weeks of age. Rats were anaesthetised with a mixture of ketamine/xylazine (80/12 mg kg2.10‐1 for 1 min once daily at 13 hours after light onset (ZT13) for 2 weeks. GTTs were performed after 2 weeks of treatment at 48 h prior to sacrifice for assessments of liver lipid, body fat store level, and plasma leptin and NE levels. In a subsequent study, we examined the effect of circadian timing of dopamine administration to the peri‐SCN area on glucose tolerance in SHR rats maintained on the HFD. Rats were divided into four groups and two groups of rats received dopamine (2 nmol in 0.2 μL) or vehicle at the onset of locomotor activity (ZT13), whereas two additional groups of rats received either the same dose of vehicle or dopamine infusion at the same peri‐SCN area but at ZT19 as described above. Again, GTTs were performed after 2 weeks of such treatment. At the end of the experiment, animals were killed and brains were collected and stored at −80°C for neurotransmitter analyses within the VMH and PVN.Twelve‐week‐old male SHR rats were maintained on a HFD  when held on a 14 hour daily photoperiod for 4 weeks until the SCN dopamine administration experiments began. Such rats at 16 weeks of age were continued on the HFD and a 14 hour daily photoperiod assigned to one of 2 treatment groups that were infused unilaterally at the peri‐SCN area with either vehicle  or dopamine solution (2 nmol) at a rate of 0.2 μL min2.11Frozen serial coronal brain sections were cut at a thickness of 300 μm on a cryostat maintained at −8°C. VMH and PVN tissues were punched out and placed in 40 μL of 2% trichloroacetic acid, sonicated and centrifuged. Supernatant was immediately analysed by HPLC with coulometric electrochemical detection (ESA) for NE and the NE metabolite MHPG content and quantified against a standard curve for each. The signal was analysed by EZChrom Elite data processing software (Agilent). Next, 10 μL of supernatant was injected into the system using a refrigerated autosampler (ESA 540). The results are expressed as pg per 10 μL of sample.2.12‐1 body weight) and blood samples were taken from the tail vein before glucose administration, 30, 60, 90 and 120 min after glucose injection for plasma glucose and insulin analyses.GTT was performed 6 h after light onset. A 50% glucose solution was administered i.p. . Plasma insulin and NE were assayed by an enzymeimmunoassay using commercially available assay kits utilising anti‐rat serum and rat insulin and NE as standards . Liver tissue was homogenised in 5% NP‐40, heated, centrifuged and supernatant assayed for triglyceride content using a Triglyceride Determination Kit .2.14m Tris‐HCl, 120 mm NaCl, 5 mm KCl, 1 mm CaCl2, 1 mm MgCl2 and 5.7 mm ascorbic acid (pH 7.4), and then incubated with dopamine D2‐receptor selective antagonist [I125]‐iodosulpride  prepared in Tris‐ions assay buffer for 30 min at room temperature. The slides were drained, washed twice in ice‐cold assay buffer (2 min each) and dipped in ice‐cold water and air‐dried immediately. Nonspecific binding was evaluated by treating a parallel set of slides with the same concentration of titrated [I125]‐iodosulpride plus an unlabelled competitor haloperidol (10 μm) to displace [I125]‐iodosulpride binding. Similar procedures were implemented for D1 dopamine receptor binding of prepared brain sections. The first step was equilibration of brain sections for 15 min at room temperature in Tris‐ions assay buffer excluding ascorbic acid. The next step was incubation with dopamine D1‐receptor selective antagonist [I125]‐SCH23982  prepared in Tris‐ions assay buffer in the presence of 2‐HT2A/2C antagonists (50 nm ketanserin and 100 nm mianserin) for 30 min at room temperature. Finally, washes (2 min × 2) were performed in ice‐cold assay buffer, followed by dipping in ice‐cold water and air‐drying. The nonspecific binding was determined by the replacement of [I125]‐SCH23982 binding with a saturation concentration of unlabelled dopamine D1 selective antagonist R‐(+)‐SCH23390 (10 μm). Finally, the dried slide‐mounted tissues were exposed to a film in sealed cassette at −80°C and the autoradiography images were captured and analysed using imagej .Female SD rats weighing 225‐275 g maintained on a LD 14:10 hour daily photoperiod and fed regular rodent chow were sacrificed by decapitation, and their brains were quickly removed and frozen in liquid isopentane (−30°C) for 30 s. Coronal sections (16 μm) including the peri‐SCN and SCN regions were cut on a cryostat at −20°C and thaw‐mounted onto cold, gelatin‐coated glass slides. After drying at room temperature using an air blower, the slides were stored at −80°C in slide boxes with desiccant. Prior to using the tissue, the section mounted slides were gradually brought to room temperature. For dopamine D2 receptor binding determination, the slides were first equilibrated in Tris‐ions assay buffer (15 min at room temperature) containing 50 m2.15icycler iq (Bio‐Rad). The dopamine D1 and D2 receptor qPCR assays were repeated using a different set of primers, reagents, PCR instrument and software. RNA was isolated with an Absolutely RNA Miniprep Kit (Agilent) and cDNA was synthesised with an AffinityScript cDNA Synthesis Kit (Agilent). Amplification was conducted with a Brilliant III SYBR green qPCR kit (Agilent) using an AriaMX instrument and analyzed with ariamx software (Agilent).Female SD rats weighing 225‐275 g , maintained on a 14:10 hour photoperiod and fed a RC diet were sacrificed by decapitation and the brains were quickly collected, frozen on dry ice and stored at −80°C. On the day of the experiment, one coronal slice (150 μm) through the medial preoptic area (mPOA) and three consecutive coronal slices (150 μm × 3) through the peri‐SCN/SCN region were cut on a cryostat at −16°C. The mPOA, combined peri‐SCN/SCN and SCN tissues were dissected with pre‐chilled brain punches  under a dissecting microscope and immediately homogenised in ice‐cold lysis buffer. For quantification of dopamine D1 and D2 receptor levels, RNA isolation  and cDNA synthesis  were carried out in accordance with the respective manufacturer's instructions. qPCR was performed with a Bio‐Rad SsoAdvanced Universal Probes Supermix using the Bio‐Rad Dopamine D1 receptor (DRD1) assay qRnoCEP0027016, Bio‐Rad Dopamine D2 receptor (DRD2) assay qRnoCIP0023714 and Bio‐Rad GAPDH assay qRnoCIP0050838 in accordance with the manufacturer's instructions. Matching Bio‐Rad PrimePCR templates were used to quantify the absolute amount of DRD1, DRD2 and GAPDH mRNA using Primers:DRD1‐F 5′‐GCGTGATCAGCGTGGACAGGDRD1‐R 5′‐AGGGCCATGTGGGCTTTGCCDRD2‐F 5′‐GGTGGCCACACTGGTAATGCCDRD2‐R 5′‐CAGTAACTCGGCGCTTGGAGCTGAPDH‐F 5′‐AACTTTGGCATTGTGGAAGGGAPDH‐R 5′‐ACACATTGGGGGTAGGAACA2.16t test for two group comparisons or one‐way anova for more than two group comparisons, or two‐way repeated measures anova for comparisons of treatment groups undergoing repeated measurements at different time points, as appropriate. When the overall anova result was statistically significant, a post‐hoc Dunnett's test was carried out to highlight where these differences occur. A statistical value of P < .05 (2‐tailed) was considered statistically significant.All data are expressed as the mean ± SEM. Statistical analysis were performed using an unpaired Student's 33.1l‐DOPA administration in the regulation of seasonal metabolism.Our previous studies suggested a role for circadian responsiveness to systemic 125]‐SCH23982 and [125I]‐iodosulpride autoradiography, as well as reverse transcriptase (RT)‐PCR of D1 and D2 receptor mRNA expression. Autoradiographic analysis revealed moderate dopamine D2 receptor binding density within the peri‐SCN region and much lower D2 receptor binding density within the SCN itself  at the SCN and its perimeter followed by double‐immunohistochemical staining with fluorogold and TH (the rate‐limiting enzyme in dopamine synthesis) antibodies to trace the origin of primary dopaminergic neurones that project to the peri‐SCN /SCN region. These studies revealed a predominant collection of TH positive (+), primary neurones within the SuMN that project to the peri‐SCN/SCN region Figure A‐D. Someanova with repeated measures on HVA indicates a time effect  and also a time and treatment interaction effect . SCN DOPAC was increased in AMPA treated vs vehicle groups . There is also a time effect  and a time and treatment interaction effect .In a separate study, administration of AMPA receptor agonist directly to the SuMN resulted in robust dopamine release at the SCN area , confirming that this anatomical apparatus was functional in providing dopaminergic input to the SCN area Figure F,G. Two‐3.2l‐DOPAP < .0001, Student's t test) . The area under the glucose and insulin GTT curve in the HFD fed group increased by 23% and 57%, respectively, compared to the RC fed group . HFD feeding reduced the insulin sensitivity . Extracellular microdialysis samples collected at 2 hour intervals over a 24 hour period from the peri‐SCN region of free living lean rats fed either RC or HFD and maintained on daily 14 hour photoperiods were subsequently analysed for dopamine metabolites. Such analysis revealed a robust circadian rhythm of dopamine release (measured as changes in extracellular levels of HVA and DOPAC) with a peak  at the onset of locomotor activity  among rats fed regular chow that was completely abolished in rats fed a HFD . There is also a time and group interaction effect , which indicates circadian difference of SCN dopamine activity between HFD and RC fed groups. Two‐way anova with repeated measures on DOPAC reveals a time of day effect  and a time and group interaction effect . Furthermore, immunohistochemical staining of SuMN neurones dual positive for cFOS and tyrosine hydroxylase  in lean animals fed low‐fat chow similarly demonstrated a time of day dependent variation in activated SuMN dopamine neurones with a peak activity at the onset of locomotor activity. The number of activated dopamine neurones at the SuMN/adjacent posterior hypothalamus in the brains from RC lean rats was 46% higher at ZT16 than at ZT4  and this daily peak is abolished in the brains from HFD fed obese rats  a circadian rhythm of responsiveness to systemically administered ) Figure A, as wel) Figure B and ins) Figure C during 3.3m induced a marked potentiation of the SCN firing rate equally at both ZT5 and ZT14  peri‐SCN glutamate‐evoked action potentials from the SCN following administration of 5 mm dopamine directly to this peri‐SCN/SCN region demonstrated a marked inhibition in SCN firing rate in response to such dopamine administration. Moreover, in rats held on a regular chow diet, there was a robust (approximately 5‐fold) increase in sensitivity to peri‐SCN dopamine inhibition of such glutamate‐evoked SCN neuronal firing rate in response to its administration at ZT14 (a time coinciding with the daily peak in dopamine release at this area) relative to its administration at ZT5 . The half maximal effective concentration of dopamine inhibition to the SCN neuronal response at ZT5 (EC50 = 14.5 ± 1.3 mm) was 4.8 times greater than at ZT14 (EC50 = 3.0 ± 0.5 mm), (P < .05). By contrast, among animals made obese by HFD feeding, the daily peak in SCN electrochemical responsiveness to peri‐SCN dopamine administration (at ZT14) was reduced 50% relative to RC fed controls   and little activity during the dark phase ZT14), as established previously,, as esta50) plus or minus dopamine D1 or D2 receptor antagonists, as well as with a selective presynaptic dopamine D2 receptor antagonist (AJ76), revealed that postsynaptic dopamine D2 receptors on peri‐SCN neuronal dendrites were responsible for mediating the SCN response  without any change in daily food consumption (of the HFD)   in animals made obese/insulin resistant by feeding of a HFD at the time of day of circadian peak dopaminergic neuronal activity at this site in healthy lean animals  resulted in a substantial reduction in epididimal fat pad (29%), retroperitoneal fat pad (38%), liver triglyceride content (39%), hyperleptinaemia 55%) and hyperinsulinaemia (42%), whereas there was elevated plasma NE (36%) and improved insulin sensitivity indices   , as did repeated measures anova on MHPG . Repeated measures anova on 5‐HIAA also revealed a group effect . Following 2 weeks of infusion of dopamine or vehicle into the peri‐SCN/SCN region of obese/insulin‐resistant rats at either ZT13 or ZT19 and subsequent to death, brain punches of frozen sections of the hypothalamus were obtained for the analysis of VMH and PVN NE and its metabolites within these study groups. It was found that daily dopamine infusion at the peri‐SCN area at ZT13 (the daily peak in dopaminergic activity at this site) but not at ZT19 significantly reduced NE activity   Figure . Repeate4These studies demonstrate the functionality of a unique, potent and previously unrecognised role for the circadian organisation of dopaminergic activity at the peri‐SCN/SCN neuronal area in the regulation of peripheral metabolism and of the responsiveness to the deleterious metabolic effects of a HFD. This circadian physiological activity is characterised by a temporal interaction of a circadian rhythm of dopamine release at this peri‐SCN/SCN site from SuMN neurones with a circadian rhythm of SCN neuronal electrophysiological responsiveness to such dopamine release. The SuMN is a heterogeneous neuronal locus involved in regulation of reward/primary reinforcement, hippocampal theta rhythm and arousal response; however, the present study is the first report of SuMN dopaminergic neurones projecting directly to the peri‐SCN/SCN area. The SuMN dopamine neurones also project to the lateral septum and MPOA,Receptor activation competition studies indicate that the peri‐SCN/SCN dopamine electrophysiological response system is mediated by a post‐synaptic dopamine D2 receptor in the peri‐SCN/ SCN region. Moreover, in this peri‐SCN/SCN region, qPCR identified dopamine D2 receptor mRNA and in situ autoradiographic binding studies identified functional dopamine D2 receptors both with predominance in the peri‐SCN region. Furthermore, ample TH+ fibres from the SuMN  were identifiable in this peri‐SCN region and their sparse presence was even identifiable in the SCN itself. Some peri‐SCN dopaminergic neurones were found to originate from the posterior hypothalamus as well, and it cannot be ruled out that some peri‐SCN TH+ neurones represent short interneurones at the SCN perimeter with the employed technique. Such observations are in agreement with and also add site of origin information to the previous identification of TH+ dopaminergic neurones in this peri‐SCN/SCN region of adult rodents.Subsequent to our initial postulate that temporal interactions of circadian neuroendocrine input activity rhythms to the biological clock modulate its output signals that regulate peripheral metabolism,The present studies suggest that a contributing mediator of this circadian peri‐SCN dopaminergic effect on metabolism likely includes SCN communication with the VMH and PVN, where elevated levels of NE  Figure  are reduob/ob mice, db/db mice, Ay/J mice, offspring of malnourished mother rats and offspring of insulin‐treated mother rats.In seasonally obese/insulin‐resistant animals, the diminution of the circadian peak of dopaminergic activity at the peri‐SCN is coupled with substantial increases in NE release at the VMH.Moreover, infusion of NE into the VMH of lean/insulin‐sensitive inbred laboratory rats held on a low‐fat diet to raise the extracellular NE levels induces a rapid and sustained (chronic) simultaneous increase in sympathetic nervous system (SNS) tone, plasma NE, blood pressure, insulin, glucagon and leptin levels, as well as adipocyte lipogenic activity and adipocyte responsiveness to the lipolytic effects of noradrenergic stimulation  interval manifests this physiological response. It is equally instructive that the impact of the HFD feeding to reduce peri‐SCN/SCN dopamine activity was restricted to the circadian peak dopaminergic activity period of the day at this site and showed no such effect during the long low trough activity period of the day Figure , implicaImportant unanswered questions of the present study include: (i) what is the genesis of these circadian dopamine stimulus and response rhythms at the clock  and (ii) how does HFD feeding reduce the concurrent circadian peaks in SuMN‐SCN tyrosine hydroxylase activity and dopamine release, as well as SCN responsiveness to peri‐SCN dopamine stimulation? However, the finding of reduced dopaminergic activity is generally consistent with a multitude of studies indicating a reduction of dopamine and/or dopamine receptor levels in other brain areas, particularly the striatal‐mesolimbic system, following chronic HFD feeding, although no specific investigation of such feeding on circadian aspects of dopaminergic activity at these brain sites has been investigated.Although this circadian dopamine input signalling system to peri‐SCN/SCN neurones to regulate clock functions controlling peripheral fuel metabolism is a unique finding, it mirrors the similar circadian dopamine regulation of striatal clock gene expression.Neuroanatomical studies indicate that the major source of the peri‐SCN dopamine derives from the SuMN. The SuMN is a hypothalamic nucleus with connections to widespread regions of the hippocampus, forebrain areas, raphe nuclei and limbic system areas.In conclusion, the present study has identified a previously unrecognised circadian dopaminergic neuronal input modulator system to the biological clock comprised of a circadian rhythm of dopamine release at the peri‐SCN area from SuMN neurones and a circadian rhythm of SCN neurone electrophysiological responsiveness to such dopamine release (at the dopamine D2 receptor) for which temporal interaction is coupled with the regulation of peripheral fuel metabolism without inducing a change in daily food consumption. In lean animals, the circadian peaks of these dopaminergic stimulus and response rhythms (are programmed to) coincide, whereas, in animals made obese/insulin resistant by HFD feeding, the daily peaks in these circadian rhythms of both dopamine release and electrophysiological response to dopamine at the SCN are markedly attenuated. The reversal of this attenuated circadian peak in dopamine signal at the peri‐SCN/SCN of HFD‐induced obese/insulin‐resistant animals by the appropriately circadian timed daily 1‐minute pulse infusion of dopamine directly to the peri‐SCN area reverses the obese, insulin‐resistant, glucose intolerant state, without altering food consumption, whereas dopamine treatment outside this circadian temporal window is ineffectual with respect to producing this metabolic response. This circadian‐time dependent effect of dopamine input activity at the SCN to improve the obese/insulin‐resistant condition involves a reduction in noradrenergic activity at the VMH and PVN of the hypothalamus, comprising areas where increased noradrenergic activity has been shown to induce the obese insulin‐resistant state even in animals fed a low‐fat diet. Normal activation of this circadian dopaminergic clock regulatory circuit appears to set the responsiveness to the adverse effects of a HFD because its activation maintains the lean/insulin‐sensitive state even in the presence of a prolonged exposure to the HFD without any alteration of feeding. Modulation of a disrupted circadian dopaminergic neuroendocrine regulation of metabolism may be an effective means of treating a host of metabolic disorders.All authors are current or former employees of VeroScience LLC.


Laeverin is a placenta-specific membrane-bound aminopeptidase. In this study we wanted to: 1) serially measure plasma levels of laeverin in healthy women during the second half of pregnancy and postpartum, 2) determine whether laeverin is differently expressed at 22–24 weeks in women who later develop preeclampsia compared to controls, 3) compare laeverin protein expression in placenta and umbilical vein serum in healthy and preeclamptic pregnancies at birth.+0 to 39+6 weeks of gestation, and at 22–24 weeks from 15 women who later developed preeclampsia. Enzyme-linked immunosorbent assay was used to measure laeverin protein concentration. Serum from healthy non-pregnant premenopausal women (n = 10), menopausal women (n = 10) and men (n = 11) were used as negative controls. Protein extracts from placental tissue were obtained after birth from healthy- (n = 11) and preeclamptic women (n = 13). Paired umbilical artery and vein serum samples from the neonates (n = 10) of healthy mothers were also analyzed. Multilevel modeling was used to determine the reference centiles. Differences between groups were analyzed using Student’s t-test.Plasma was obtained serially, approximately every 4-weeks, from 53 healthy women with uncomplicated pregnancies during 22p < 0.0001) compared to men (mean 0.18 ± 0.31 ng/mL), non-pregnant premenopausal women (mean 0.77 ± 0.26 ng/mL) and postmenopausal women (mean 0.57 ± 0.40 ng/mL). Maternal plasma laeverin levels decreased with advancing gestation, from 6.96 ± 0.32 ng/mL at 22–24 weeks to 4.95 ± 0.32 ng/mL at term (p < 0.0001) in uncomplicated pregnancies. Half of the women who developed preeclampsia had plasma laeverin levels below the 5th percentile at 22–24 weeks gestation. However, laeverin levels were 1.6 fold higher in preeclamptic compared to healthy placentas (p = 0.0071). Umbilical venous samples of healthy neonates (n = 38) had higher (p = 0.001) mean levels of laeverin (16.63 ± 0.73 ng/mL), compared to neonates of preeclamptic (n = 14) mothers (12.02 ± 1.00 ng/mL). Postpartum plasma levels of laeverin decreased in healthy and preeclamptic women with a half-life of 3 and 5 days, respectively.Healthy pregnant women at term (37–40 weeks) had significantly higher plasma levels of laeverin (mean 4.95 ± 0.32 ng/mL; Maternal plasma levels of laeverin decrease with advancing gestation during the second half of normal pregnancy and lower levels measured at 22–24 weeks might be associated with the development of preeclampsia later in gestation. Laeverin (aminopeptidase Q) is a protein that is expressed by human villous trophoblasts . More spWe hypothesized that plasma laeverin levels vary with gestational age during the second half of pregnancy and laeverin is differentially expressed in normal and preeclamptic pregnancies.In this study we aimed to 1) serially measure plasma levels of laeverin in healthy women during the second half of pregnancy and postpartum to establish longitudinal reference ranges, 2) determine whether laeverin plasma levels measured at 22–24 weeks are different in women who later develop preeclampsia compared to controls, and 3) compare laeverin protein expression in placenta and umbilical vein serum in healthy and preeclamptic pregnancies at birth.The study was approved by the Regional Committee for Medical and Health Research Ethics-North Norway (REK Nord ref. number 5.2005.1386 (approved 27.09.05) and 2010/2058–4 (approved 13.01.05)) and informed written consent was obtained from all the participants.Fifty-three healthy pregnant women were recruited to the longitudinal arm of the study . Blood sAmong a total of 641 pregnant women recruited to the cross-sectional arm of the study , 29  analysis was performed according to the manufacturer’s specifications . ELISA is a specific and sensitive method for detection of small amounts of protein in serum , 6. MeasFor quantification studies of the total placental protein samples from healthy and preeclamptic women 50 μg total protein was added to each well. Laeverin concentration was then derived from standard curves.Total protein, extracted from placental samples, was used as positive controls of laeverin expression for validation of the kit. The lowest standard concentration of the assay was 2 ng/mL and the minimum detectable amount of the kit was 0.312 ng/mL (according to the manufacturer). The kit has sensitivity to detect concentrations down to 0.156 ng/mL. Intra- and inter-assay coefficients of variation (CV) calculated from duplicate patient samples in the concentration range 0.312–20 ng/mL were <6%.t-test for parametric variables and Mann-Whitney U test for non-parametric variables. Linear mixed model ANOVA was used to evaluate the differences in laeverin concentrations between different gestational weeks antenatally and different days post-partum. A P-value <0.05 was considered statistically significant.Analysis of the longitudinal data was performed using SAS v.9.4 . Multilevel modeling was used to estimate the reference percentiles . SPSS soThe phenotype of the study population is presented in Table Three (5.6%) women had cesarean section; two due to dystocia and one because of breech presentation. Six (11.2%) women had vacuum delivery; four due to failure to progress and two because of fetal distress in the second stage of labor.n = 15) had higher BMI, higher mean uterine artery pulsatility index, and were delivered at an earlier gestation (32 weeks (n = 1), 36 weeks (n = 1), 39 weeks (n = 3), 40 weeks (n = 3) 41 (n = 6) weeks and 42 (n = 1) weeks). Two (13%) women had cesarean section, three (20%) women were delivered by vacuum extraction and ten (67%) had normal vaginal delivery.Pregnant women participating in the cross-sectional arm of the study who developed preeclampsia (n = 11) and preeclamptic women (n = 13) whose placentas were used for placental laeverin quantification are presented in Table The baseline clinical characteristics and pregnancy outcome of the healthy (n = 10) of healthy mothers whose umbilical cord blood samples were used to determine arterio-venous gradient of laeverin concentration were all delivered at term and had a mean birth weight of 3317 ± 200 g, placental weight 566 ± 38 g and normal umbilical artery pH (7.26 ± 0.04) and base excess (−1.05 ± 1.25 mmol/l).The neonates , non-pregnant premenopausal women (mean 0.77 ± 0.26 ng/mL) and postmenopausal women (mean 0.57 ± 0.40 ng/mL) had very low levels of laeverin (mean 0.50 ± 0.19 ng/mL).p < 0.0001). We found significantly lower laeverin levels on postpartum day 2 (p = 0.0454) and 3 (p = 0.01) compared to the antepartum laeverin levels, but the difference was not statistically significant on the first postpartum day. Laeverin levels decreased further after delivery from a median value of 4.98 ng/mL on 1st day postpartum to 1.31 ng/mL on 6th day postpartum  in laeverin levels between different postpartum days. In the postpartum period, in healthy women, laeverin protein half-life (t1/2) was 3 days.Gestational age specific longitudinal references ranges for maternal plasma laeverin concentration during the second half of pregnancy are presented in Table p = 0.006). In four women (27%), plasma laeverin levels were between 50-75th percentile, three women (20%) had laeverin levels below the 50th percentile and eight (53%) had below the 5th percentile. In women developing preeclampsia, the plasma laeverin concentration decreased postpartum from 4.27 ng/mL on 2nd day to undetectable level on the 6th day after delivery. The half-life of postpartum laeverin was 5 days in preeclamptic women. Among the 15 preeclamptic women, only one had severe  early-onset  disease. The serum laeverin levels of this woman were not significantly different compared to the rest of the preeclamptic women (p = 0.069).Plasma laeverin concentrations of 15 women at 22–24 weeks of gestation, who later developed preeclampsia, plotted against the normal longitudinal reference curves, are shown in Fig. With a cut-off value for laeverin concentration set at ≤5 ng/mL, the sensitivity, specificity, positive predictive value and negative predictive value for the prediction of preeclampsia were 0.60, 0.79, 0.82 and 0.88, respectively.p = 0.0071).Placental tissue of 83 mg gave 5 μg total protein, of which 0.26 ng was laeverin protein. Laeverin protein concentration was 1.6 fold higher in preeclamptic placentas (mean 8.29 ± 0.73 ng/mL) compared to healthy placentas (mean 5.18 ± 0.75 ng/mL) (n = 22) and boys (n = 16) . In the neonates of women who had preeclampsia (n = 14), the mean laeverin level in the umbilical vein was significantly lower (12.02 ± 1.00 ng/mL) compared to healthy pregnancies (p = 0.001).The neonates of the healthy mothers had a mean laeverin concentration of 16.63 ± 0.73 ng/mL in the umbilical vein serum samples with no significant difference between girls  and vein  was not statistically significant.This is the first study to investigate gestational age-associated longitudinal changes in plasma laeverin levels in healthy women with uncomplicated pregnancies. We found that plasma laeverin levels gradually decrease with advancing gestation during the second half of pregnancy and are undetectable few days after delivery. The reasons for decreasing levels of laeverin are unclear. As laeverin is a placenta-specific protein, it is likely to have an important role in the process of placentation during early pregnancy, but less might be required after this process is complete. Indeed, we showed previously that laeverin effects migration and invasion of first trimester trophoblast cells . AlternaFurthermore, we found lower plasma levels of laeverin at 22–24 weeks in women who later developed preeclampsia compared to healthy controls. These women most likely had shallow placentation. We have previously shown that laeverin is overexpressed in preeclamptic placenta both at mRNA as well as protein level . We havePreeclampsia is associated with increased apoptosis or cell death of cytotrophoblasts. The exact molecular mechanisms behind these phenomena are not known. It is suggested that endoplasmatic reticulum stress can lead to cell apoptosis and protein degradation , 13. TheAnother possible explanation of the reduced laeverin protein expression in the maternal circulation might be that laeverin is trapped in microvesicles in the fetal capillaries in the placenta of preeclamptic patients . HoweverOur study has some limitations. Samples were stored for up to 5 years at −70°C and some samples were thawed once. However minimal differences in laeverin levels were found when thawed and non-thawed samples were compared (data not shown). In a study by Rana et al. minimal effect on plasma and serum quality was seen when samples were thawed several times . FurtherWe used a commercial kit to measure laeverin concentration, which has not been previously validated for use in the context of pregnancies and placenta samples. Therefore, we performed extensive validation prior to analysis. Healthy men, non-pregnant premenopausal women and menopausal women had a very low expression of laeverin indicating that laeverin is a placenta-specific protein and the aminopeptidase Q ELISA kit appeared to be a versatile test for the quantitation of laeverin in serum-, plasma- and placental tissue protein samples.In conclusion, our longitudinal study showed that the maternal plasma laeverin concentration gradually decreases during the second half of normal pregnancy. Women who later developed preeclampsia had lower levels of laeverin compared to healthy controls at 22–24 weeks of gestation in our cohort. Further research is needed to investigate the potential role of laeverin as a predictive biomarker of preeclampsia.


Although the engagement of stakeholders is a key factor for the eradication programme’s success, social factors have been often ignored in the control programmes of animal diseases, especially in developed countries. In this study, we used a qualitative approach to investigate perceptions, opinions, attitudes, and beliefs of farmers, and veterinarians who may influence the effectiveness of the Spanish bTB eradication programme. The study was carried out in two phases. First, 13 key representatives of different groups involved in the programme were interviewed through exploratory interviews to identify most relevant themes circulating in the population. Interviews focused on strong and weak points of the programme; reasons for failure to achieve eradication; benefits of being disease free; future perspectives, and proposed changes to the programme. Based on these results, a thematic guide was developed and detailed information was gained through face-to-face in-depth interviews conducted on a purposive sample of 39 farmers and veterinarians. Data were analysed following an ethnographic methodology. Main results suggested that the bTB programme is perceived as a law enforcement duty without an adequate motivation of some stakeholders and a general feeling of distrust arose. The complexity of bTB epidemiology combined with gaps in knowledge and weak communication throughout stakeholders contributed to causing disbeliefs, which in turn generated different kinds of guesses and interpretations. Low reliability in the routine skin test for bTB screening was expressed and the level of confidence on test results interpretation was linked with skills and experience of public and private veterinarians in the field. Lack of training for farmers and pressure faced by veterinarians during field activities also emerged. Few benefits of being bTB free were perceived and comparative grievances referred to wildlife and other domestic reservoirs, sector-specific legislation for bullfighting farms, and the absence of specific health legislation for game hunting farms were reported. Understanding reasons for demotivation and scepticism may help institutions to ensure stakeholders’ collaboration and increase the acceptability of control measures leading to an earlier achievement of eradication.The effectiveness of health interventions against bovine tuberculosis (bTB) is influenced by several “ The influence of social factors on public health interventions is well known in human medicine and several studies taking these aspects into account have been done –3; howevknowledge for action” and a “bottom up approach” in contrast to conventional research, which is more “top-down”  in Europe represents a significant obstacle to the sustainability of the livestock sector and since 1964 many efforts have been made to eradicate it . Even thIn Spain, several aspects of bTB epidemiology have been investigated. In particular, research has been conducted on: spatial and spatiotemporal dynamics of the disease –19; riskIn spite of all these studies, no major decrease in the bTB herd prevalence has been observed in Spain over the last decade (1.8% in 2004 and 1.7% in 2014) and, in 2015, the bTB prevalence has increased to 2.8% . This coIn this study, we aim to investigate farmers and veterinarians’ perceptions, opinions, attitudes, and beliefs about the Spanish bTB eradication programme by using a qualitative approach in order to assess the influence that these aspects may have on the effectiveness of the programme.The study was carried out in two Autonomous Communities of Spain, Andalusia and Catalonia, as representatives of high- and low-prevalence areas, respectively Figure .In Spain, Regional Veterinary Services (RVS) has been set up in each Autonomous Community under the coordination of the Spanish Ministry of Agriculture and Fisheries, Food and Environment. Moreover, each administrative county has a Local Veterinary Service (LVS) attached to the RVS. Besides, there are accredited veterinarians working in the field (private sector) that collaborate in carrying out disease prevention programmes. Often, they are also responsible for hygiene, productivity, and treatment programmes of the same farms.Catalonia is located on the north-eastern extremity of the Iberian Peninsula; it consists of 4 provinces and 42 counties. The Autonomous Community can count on 47 official veterinarians working on bTB at the LVS and 113 specialised private veterinarians supporting the routine screening tests for bTB in about 1,900 beef herds, 700 dairy herds, and a few bullfighting herds. Since 2008, the bTB herd prevalence at regional level remained lower than 1%, decreasing to 0.04% in 2013, but in 2015 bTB herd prevalence slightly increased to 0.32% .Andalusia is located in southern Spain and it is divided into 8 provinces and 62 counties. There are 63 official veterinarians, operating at the LVS, directly engaged with the bTB eradication programme. These are assisted by about 270 specialised private veterinarians for the implementation of routine screening in about 5,300 beef farms, 800 dairy farms, and 400 bullfighting farms. Over last 10 years, herd prevalence for bTB in this region has persisted above 4% and in the last 2 years has dramatically increased to 11% in 2014 and 17% in 2015 .The present study was carried out in two phases, first, exploratory interviews followed by qualitative in-depth interviews, and it was conducted by a team of veterinarians, sociologists, and anthropologists. In both phases, people to be interviewed were selected through a purposive sampling.In accordance with the national and institutional guidelines, ethical approval was not required for this study as it did not include samples or experiments on people but only their expression of opinions in relation to a specific topic.With regard to the informed consent of participants: as the interviews were anonymous, the data were analysed anonymously and the decision to participate in the study was solely up to each contacted person, we did not consider it necessary to obtain a written consent. We orally informed all participants of the elements of consent and permission was obtained verbally before starting the interview.At the beginning of each interview: interviewers introduced themselves and the contacted person was informed on the study design and its objectives.It was explained that the participation was voluntary and completely anonymous  and that they could stop the interview at any time.It was explained that there were no expected risks and no expected personal benefits associated with participation in the study. We also asked their approval for using information collected through the interview and for using direct quotes from them and these would only be cited as from a “farmer” or “veterinarian,” keeping the anonymity.stakeholder sampling strategy , three veterinarians of the public sector , two private veterinarians (operating in two different counties), and three farmers, covering the main livestock production types: beef, dairy, and bullfighting farms were interviewed. In the low-prevalence area , two veterinarians of the public sector (RVS and LVS), one private veterinarian, and two farmers (beef and dairy farmers) were also interviewed.The interviews were conducted face-to-face following a general script in order to allow, as much as possible, open and informal conversations in which key aspects on the bTB management could emerge.Each interview lasted between 50 and 120 min and focused on the following six points: (i) strong points of the bTB eradication program; (ii) weak points of the bTB eradication program; (iii) reasons for the failure of bTB eradication; (iv) future perspectives; (v) proposed changes to the bTB eradication program; and (vi) benefits of being bTB free.Two of the researchers, taking handwritten notes, were present at each interview. After the interview, notes from both researchers were compared in order to transcribe the main arguments expressed. The review of the transcription of the different exploratory interviews was done in different steps. In a first step, the transcription of the exploratory interviews was send to all the team members (paper’s authors) and then we organised a group meeting where all team members discussed together the results from those interviews. After that, the team of sociologist prepared a first draft of the interview guide for the qualitative in-depth interviews and they send it to all the authors of this paper for the final discussion and agreement.Interviews in Andalusia were conducted at the beginning of December 2014 (from 1/12 to 11/12), whereas in Catalonia they were performed in two rounds: middle July 2015 (from 17/07 to 22/07) and middle September 2015 (from 15/09 to 21/09).This study phase was aimed at gaining detailed information on the themes that emerged from the exploratory interviews in order to understand perceptions of farmers and veterinarians and their interpretation of problems related to the eradication of the disease in Spain.A “thematic guide” was developed based on previous results and it provided an orienting framework of the different stakeholder groups.maximum variation sampling strategy in order to identify as many different “speeches” as possible . Interviews were tape-recorded and transcribed by the team of sociologist and anthropologists.Prior to the interview, a formal letter (headed by the university logo and signed by the research team) was hand delivered to each interviewee and permission was secured at all levels. Participants were informed about: (a) the purpose of the study; (b) the research team members and their university department ; (c) the freedom to accept or not to do the interview and to withdraw from it at any time; and (d) the explicit guarantee of anonymity and confidentiality of their personal opinions. Interviews only took place after they were read, and verbal consent was obtained from each participant.In order to make respondents as comfortable as possible during the interview and encourage them to talk extensively and “freely ramble on,” all in-depth interviews started with a few general questions, which respondents could answer easily. These questions were related to their professional career, type of livestock farm, daily working activities , and variation in their workday across the year. As the interview progressed, the interviewer gradually introduced new elements in the conversation directing it to more specific and targeted topics.Interviews in Andalusia were conducted and transcribed between March and October 2015, whereas in Catalonia they were conducted and transcribed between January and June 2016.To ensure the protection of sensitive data, recordings and transcripts were stored by the research team, and access to them is reserved exclusively for members linked to this research, who have undertaken to maintain the confidentiality and anonymity specified in the mentioned letter. All the real names of individuals and companies, entities, or institutions were eliminated in order to ensure anonymity. Instead, an alphanumeric code that identifies each sample was assigned to each interviewed person. Each interviewee was warned that if any of the phrases pronounced during the interview were used to illustrate results in some public document, and that in no case would the person’s name be mentioned, but replaced by the mentioned code or attributed to the sample as a whole.An ethnographic methodology was used in this study. Interview transcriptions were analysed through a method inspired on the grounded theory approach, based on the constant comparisons between data of the whole dataset  and on the use of a repeated coding, which provided a scheme of the main perceptions, opinions and beliefs circulating in the discourses of the study population . The recn}, where “S” means “sentence” and the “n” is an integer number whose value represents the unique identifier of the sentence.For each theme that emerged, the most representative sentences were transcribed in their original language  and included in the Supplementary Material. From here onwards in the text, we will refer to each sentence as {SFollowing the general script previously described, the exploratory interviews allowed us to identify the following themes to be further investigated in the second study phase.In general, the programme was perceived as technically correct. The increased implication of veterinary services, the systematic use of the interferon-γ assay (IFN-γ), and the implementation of mandatory training courses for veterinarians (public and private) organised by the Spanish Ministry of Agriculture and Fisheries, Food and Environment were perceived as major improvements of the programme in the last years Figure .Main weak points were related to the communication flow, organisational issues and the suitability of the human and economic resources currently assigned to the programme Figure .Concerns were expressed in relation to the coordination with the labs, the experience of official veterinarians who supervise private veterinarians in performing the single intradermal test (SIT), the lack of homogeneity in the implementation criteria of the bTB eradication programme and the lack of human resources. Interviewees also mentioned that some of the implemented control measures were too restrictive or infeasible.Some stakeholders reported the comparative grievance that is generated due to the special legislation that is in place for bullfighting herds, as in herds with cattle that is older than 24 months bTB testing is not performed. Moreover, the presence of wildlife and other domestic bTB reservoirs not included in the eradication programme was perceived as a comparative grievance by farmers and contributed to generate uncertainty on the achievement of bTB eradication.Arguments that emerged in this section were related to the lack of confidence in the results of the diagnostic tests, the heterogeneity in the bTB detection capacity among the different slaughterhouses, the relationships among stakeholders and pressures faced by private veterinarians when interpreting the skin test Figure .The importance of the level of implication of the different actors in the bTB eradication programme  and the lack of trust between farmers and official veterinarians were also mentioned.Moreover, the reason for certain sanitary measures was somewhat unclear or not well understood and the presence of infected wildlife animals was perceived as a major obstacle for the bTB eradication, especially in the south of Spain.In this section, very different views were expressed Figure : some peThe different stakeholders considered that improvements to the bTB programme should focus on training  and communication. It was also mentioned that measures related to movement restrictions should be relaxed Figure .With the exception of some awareness on the potential zoonotic risk of bTB reported from some people, few benefits of being bTB free were perceived Figure . The per(i)bTB detection and control .(ii)Training, information, and communication .(iii)Role of wildlife and other domestic reservoirs .(iv)Perception of social aspects .(v)Risk perception on bTB and benefits of eradication .(vi)Future perspective on the progress of bTB and proposed changes to the programme.Based on these results, we developed a thematic guide to be used in the qualitative in-depth interviews Table  which ouMain results obtained from the ethnographic reports of both areas are described below. Since we did not observe major differences in attitude and opinions between the two study areas, results are presented together, highlighting differences when these were identified.A generalised lack of confidence in the bTB diagnostic tests clearly emerged during the in-depth interviews. Both farmers and veterinarians expressed strong uncertainties on the reliability of test results, although this perception was widespread especially among farmers; so much that some people used the term “lottery” when explaining their perception about test results {S1}. Actually, farmers expressed that they do not want to have any bTB-infected animal in their herd, but that they want to be sure that the test-positive animal is truly infected {S2}.Uncertainties were mostly associated with the SIT and mainly attributed to the lack of confirmation of positive results and they asked for the application of complementary tests for the verification of the final results {S3}. Reasons provided were the absence of visible lesions in slaughtered animals {S4}, discordance of results between the SIT and the IFN-γ {S5} and the use, as screening test, of the SIT instead of the single intradermal comparative cervical test (SICCT), as it could give cross-reactions with paratuberculosis or other environmental mycobacteria {S6}.Concerns with the existence of false-negative results were also mentioned but mainly by the official veterinarians and related to bad practices in the field and erroneous execution of the SIT. This group, more than others, disagreed on the systematic use of the SICCT and defended the use of SIT as the screening test. Even though, they admitted a certain degree of subjectivity in the interpretation of the SIT results and a great influence of the level of experience of the veterinarian in question {S7} emphasising and warning about the importance of the professional training of veterinarians {S8}.Private veterinarians also highlighted that a correct application of the SIT is not always easy as some cattle are difficult to manage and farms do not always have the necessary infrastructure. The importance of having good infrastructure was highlighted by several interviewed, not only to correctly perform the SIT but also to prevent veterinarians from risk of injuries and lesions. The lack of support from the official veterinary services to ensure the existence of adequate infrastructures for bTB testing {S8b} was also mentioned.On the other hand, the IFN-γ was generally perceived as a better diagnostic test than the SIT; thus, its introduction and systematic use was perceived as positive by most of the participants in the study {S9}. Especially, veterinarians highlighted that the IFN-γ is a valid and helpful tool to dispel doubts on diagnostic results {S10} and that it reduces pressure on veterinarians during field activities as it is performed in labs {S11}. However, some concerns were expressed on the IFN-γ regarding the possible existence of false-positive animals {S12} and the high cost of this diagnostic test that makes its systematic use not always feasible {S13, S14}. Furthermore, the difficulties in sending blood samples to the laboratory on time from remote areas and the lack of support from the labs {S15} were also reported. Finally, another issue mainly expressed by private veterinarians and farmers was the over-saturation of some laboratories and the consecutive delay in receiving the results due to the lack of coordination {S16}; on their side, official veterinarians acknowledged that organisational problems have happened in some occasions due to the lack of enough personnel in the lab. Lack of enough human resources for bTB activities was also related to a deficient post-mortem inspection in the slaughterhouses or field activities supervision {S17, S18}.Another important issue that emerged in relation to the perception of the diagnostic techniques as unreliable was the lack of understanding of test results . Both farmers and private veterinarians mentioned experiences with doubtful results that nobody has been able to explain and clarify {S19, S20}, and they asked for further investigation and more efficient dissemination of information {S21}.In the last few years, official veterinarians were in charge of supervising the performance of the skin test done by the private veterinarians. This has generated some conflicts as some private veterinarians consider that the official veterinarians who have to supervise them do not always have sufficient experience {S22}. Furthermore, the eradication programme in areas of high prevalence (as is the case of the south of Spain) has established a stricter lecture of the SIT in infected farms by which doubtful results are considered as positive. This measure has not been well accepted by the interviewed farmers and private veterinarians who would wish to verify positive results {S23}, whereas official veterinarians do think that it is a good change that will benefit the eradication programme.The screening intervals set by the bTB eradication programme for routine testing were considered functional and adequate by official veterinarians and most of private veterinarians and farmers, albeit they asked for more coordination among different sanitary controls to avoid generating stress in animals and workers {S24}.Only in certain rural areas of Andalusia, the implementation of two screening round per year was perceived as excessive, especially by farmers, due to the difficult management of beef cattle in extensive farming systems. In addition, farmers expressed the management difficulties that they face during the bTB testing, especially in those farms with extensive managements or in bullfighting farms {S25}. Direct loses due to abortions, work hours, injured animals, and decrease in milk production were mentioned as a major issue related with bTB testing, especially in those infected herds subjected to a high frequency of tests.Some criticisms were reported in Andalusia with regard to the sector-specific legislation for bullfighting cattle farms (bTB screening exemption for cattle older than 24 months), although different points of views were expressed {S26–S28}. Some interviewees considered that no exceptions should be allowed with bullfighting animals, while others justified this measure and evaluated it as reasonable on the basis of their difficult management, the risk of injuries in animals of high value or changes in their behaviour making them unfit for bullfighting {S29}. However, even within the group of farmers that agree with the exemption of bTB testing, not everyone agreed with the argument of difficult management as still these animals are subjected to other health measures (such as vaccination or deworming). The high genealogical value of bullfighting animals and the economic difficulties that the sector is going through were considered as more relevant for these persons.In relation to the control measures provided by the programme, the huge economic consequences derived from movement restrictions was mentioned, especially for those farms without infrastructures for fattening animals. This measure was perceived as too restrictive and as the origin of fraudulent activities. Nevertheless, in the last few years, farmers have been allowed to send these animals to specific fattening units; a measure that has been positively received, despite that calves are sold at a lower price {S30}.An improvement in the application of the bTB programme in the last few years was highlighted and mainly attributed to the organisation of mandatory training courses. Both official and private veterinarians acknowledged that some bad practices in the field were largely caused by a lack of knowledge and training among veterinarians {S31}.Official and private veterinarians also expressed the importance of organising such activities also for farmers, ensuring that they could have access to all the available information by increasing awareness and knowledge on the diseases as well as on its impact to the farm {S32, S33}. Some of the interviewees also emphasised the importance of training for farmers in order to improve the understanding of sanitary measures provided for the bTB eradication programme and increase its acceptability {S34}.Among farmers, the lack of understanding of test results and control measures gave rise to some disbelief and to different guesses, for example, that a high mutability rate of the Mycobacterium invalidates the diagnostic tests and that bTB is just an excuse to reduce the cattle population in Southern Countries {S35}.It was not clear which should be a more efficient way to deliver such training as some people expressed concerns due to the high number of courses that are already organised for farmers {S36} and a lack of motivation in relation to animal health by some of them {S37}. Among the different stakeholders, private veterinarians were identified as one of the more adequate professionals to inform farmers and raise their awareness on the disease, as they are the ones that usually inform farmers on other matters {S38}.In relation to the effectiveness of communication between stakeholders, different opinions were reported. On the one hand, some farmers expressed the lack of meeting places to exchange information and to express doubts and concerns on the disease and its control. As a matter of fact, most times they have learnt about the bTB eradication programme and changes in the regulation by talking to other farmers in the bars {S39, S40}.On the other hand, some other farmers expressed that the communication through their private Vetrinarian group (ADGS) was good enough and they were informed of any changes through them {S41}. Most of the farmers also reported that they would prefer attending informative days about specific issues rather than formal courses and that it would be preferred to organise these meetings during animals’ markets.Regarding the communication of bTB test results, differences emerged between the two study areas. In Catalonia, it was described by farmers and veterinarians as adequate {S42, S43}; while in Andalusia a general perception of low feedbacks on test results was reported and both farmers and private veterinarians demanded easier and more flexible procedures to get all needed information on lab results {S44, S45}, results of the post-mortem inspections and the cultures {S46}.The existence of bTB wildlife reservoirs was mentioned as a major obstacle for bTB eradication in Andalusia and Catalonia, but was especially highlighted in those areas with high prevalence and extensive herd management in Andalusia. Different opinions on the role of wildlife reservoirs arose; some people attributed a secondary role in the maintenance of the disease to these species while others were of the opinion that wildlife reservoirs could represent a primary source of infection for cattle {S47–S48}.In general, controlling bTB in these animals was perceived as a very difficult task and several people expressed the hope of having a vaccine in the future to control the disease in these animals. The development of biosecurity plans to reduce the risk of transmission from wildlife to cattle was also mentioned. However, different views were expressed and some people considered it possible, whereas others considered it impossible to prevent cattle and wildlife interaction {S49}.Other factors that in the opinion of some people increased the risk of bTB transmission was related to hunting activities and the lack of biosecurity, as different groups of dogs, vehicles, people, etc., interacted with infected wild animals and could spread the disease to other places {S50}. In this regard, farmers and veterinarians agreed on asking for more controls in wildlife, especially in hunting farms as they are managed as livestock farms {S51–S52}.Several interviewees negatively perceived the supplementary feeding for hunting purposes, as it was linked to an increase of wildlife population and as a consequence an increased risk of infection for cattle herds. Moreover, the economic benefits provided by hunting activities was suggested to lead to the establishment of several mixed farms (wildlife and cattle), therefore, increasing the risk of bTB transmission. In this sense, the importance of the coordination between the different governmental statements responsible to manage animal health and the environment was highlighted {S53}.In relation to other bTB domestic reservoirs not subjected to any control programme, the potential role of goats, sheep, and extensively reared pigs  was mentioned. The interviewees reported that sharing pasture by cattle and these other domestic reservoirs poses another risk of infection for cattle and complained about the lack of specific legislation for this matter.Although the relationship between farmers was considered to be good, bTB was described as a sensitive issue that is normally avoided in their talks. In some occasions, conflicts between neighbouring farmers were generated to the perception that the adjacent farm was responsible for the bTB infection of the herd as the neighbouring farmer has not complied with the eradication programme and has been the source of the outbreak {S54, S55}.The relationship between farmers and private veterinarians was described as good as in general, it is an enduring relationship and farmers tend to have a very high confidence on them {S56}. However, the existence of a “patronage relationship” between some farmers and private veterinarians was also mentioned, because private veterinarians conduct in the farm other duties than only the bTB testing that are paid by farmers. This fact could generate pressure on private veterinarians, which might not always act with professionalism as could be strongly influenced by the consequences for farmers due to the bTB control measures and for the fear of losing “customers” {S57}. In this regard, some of the interviewees also mentioned that sometimes the pressure faced by veterinarians generated conflicts, as the most rigorous veterinarians were not well accepted by all farmers {S58, S59}. In this sense, to have a greater support from the official veterinary services was perceived as a way to reduce pressure to private veterinarians {S60}.There were different opinions about the relationship between official veterinarians from LVSs and private veterinarians and farmers. Some people reported to have a close and effective relationships and a good coordination with them, despite official veterinarians have the role to control and inspect them {S61, S62}. Others described the relationship as tense and of mutual mistrust. Main reason for this difficult relation was due to the perception of fraudulent activities with bTB testing.The existence of fraudulent practices was acknowledged by some farmers, however, they also argued that, even though not all farmers act the same, they are all treated the same way, and they perceive that the official veterinary services are treating all of them as “delinquents” {S63, S64}.Concerning the fraudulent practices, the missed communication of animals with doubtful test results and the non-rigorous reading of the SIT were the most reported by both farmers and veterinarians {S65, S66}. These behaviours contributed to generate demotivation especially among farmers but also among veterinarians {S67, S68}.Some differences arose between groups on the perceived burden of the bTB. Official and private veterinarians acknowledged both the health and the economic impact of the disease. They emphasised that animal health is the base of the development of the livestock sector and it is fundamental to an efficient animal production and, therefore, to food security and human health {S69}. The group of veterinarians expressed the need to eradicate bTB also because it represents a public health problem, not only because of the obvious trade benefits but also because of the positive repercussions on animal health {S70}.On farmer’s point of view, bTB is not seen as an important animal health problem. Most of the farmers perceived that benefits of eradication were mainly commercial, as bTB was not considered having an impact on public health neither a disease causing production losses. The fact that the meat from infected animals can be passed as “fit for human consumption” after the removal of the affected tissue  generated doubts about the public health implications of bTB {S71–S73}. Moreover, they strongly disagreed that veterinary services focus so much on bTB instead of controlling other diseases that they consider more severe for human health {S74}.Generally, farmers did not perceive any production losses due directly to bTB and some of them referred that bTB does not affect animal at all. Only few farmers perceived a direct relationship in the long term between the productivity of animals and the presence of the disease {S75, S76}. In this sense, veterinarians admitted that due to the early detection of the disease, most infected animals do not develop lesions and, in this context, it is difficult to make farmers aware on the impact of the disease {S77}. Thus, farmers mainly perceived the control of bTB as an imposition rather than a necessary activity to protect their animals {S78, S79}. They also mentioned that few studies have been done so far to quantify production losses due to bTB in the current epidemiological context and asked for updated scientific evidence on it. Nevertheless, the economic impact of the disease was strongly underlined by all interviewed groups and the commercial consequences of being bTB positive were perceived as worrisome {S80}. It was reported that some farmer abandoned the sector due to economic cost faced for the control of bTB. This is because, despite the fact that the central veterinary service provides the diagnostic tests and current law provides for indemnity for slaughtered cattle, farmers assume the rest of the costs, mainly due to restrictions on trade and animal movements and field activities for the routine screening  {S81}.With regard to the amount of the indemnification, veterinarians generally opined that it is adequate and that increasing indemnity payments would mean rewarding the maintenance of the disease; they also reported that no significant complaints have been received from farmers {S82, S83}.Most of the interviewees were sceptics on the possibility of eradication mainly due to the presence of wildlife and other domestic reservoirs. The possibility of maintaining the disease at low levels was seen as the more realistic option but it was conditioned to the existence of a stable regulation {S84}.Some farmers also doubted about the need of so restrictive measures  taking into account the possibility of developing a vaccine for cattle {S85}. Others would prefer to live together with the disease rather than applying such restrictive measures that, on their opinion, will end up penalising the cattle industry in the country {S86}.Suggestions and changes proposed to the programme were related to the main problems highlighted, for example, more investigation on diagnostic test, to improve the control on fraudulent activities, to increase the personnel of the LVSs and the implementation of controls plan also on other reservoirs and wildlife.non-biological” context, as it might influence the effectiveness of the eradication plan Autonomous community level  with responsibilities in the coordination of the programme in their autonomous community. These veterinarians, together with official veterinarians of other autonomous communities, also participate in the technical meetings organised at national level to review and discuss the bTB programme;(ii)County level: with responsibilities in the coordination of the programme in their area.Considering both study phases, the main stakeholders involved in the Spanish bTB eradication programme were included in our study. We interviewed cattle farmers ; Researchers with experience on bTB; Veterinarians working in the diagnostic labs: with responsibilities in the performance of the tests  that are performed in the bTB eradication programme; Private veterinarians who conduct bTB testing; and Official veterinarians working at different levels:Although it is true that some stakeholder profiles are missing, for example, we did not included veterinarians working in the slaughterhouses, trading partners, or consumers; however, we have included representatives from the groups most involved in the implementation of the National bTB eradication programme. Therefore, we believe that the results of this study may have a wide applicability as we have gained information on the main discourses.Overall, 52 people were interviewed (13 people for exploratory and 39 for in-depth interviews), among those there were 22 veterinarians and 30 farmers. The selected number of participants relied on previous studies based on grounded theory and wanted to maintain a balanced emphasis between the homogeneity  and the heterogeneity (requiring larger size) of the sampling target , 46. In In recent years, the application of ethnographic methods has been extended to the description and analysis of social relations within any group of people: social, professional, or conceptual , making One of the main results of this study was an apparent lack of motivation of some stakeholders and a general feeling of distrust in control measures and disbelief in test results. The complexity of the disease combined with gaps in knowledge and the lack of an efficient communication about the interpretation of diagnostic test results and control interventions seems to be important causes of disbeliefs, which in turn might generate different kinds of guesses and interpretations. Good communication and coordination between the different stakeholders have been previously described as having paramount importance in any health programme, since it might be a critical factor for the success of bTB control interventions , 40. TheSimilar to our findings, Calba et al. , in a stpost-mortem inspection have been already described as possible barriers toward bTB eradication in previous studies, as they might reduce the engagement of farmers in preventive health interventions  and that they could stop the interview at any time. It was explained that there were no expected risks and no expected personal benefits associated with participation in the study. We also asked their approval for using information collected through the interview and for using direct quotes from them and these would only be cited as from a “farmer” or “veterinarians,” keeping the anonymity.Conceived and designed the study: GC, AA, and JE. Performed and transcribed qualitative exploratory interviews: GC, AA, SN, and JC. Performed and transcribed qualitative in-depth interviews: PI, EC, and SL. Analysed collected data: GC, AA, PI, EC, and SL. Wrote the paper: GC and AA. Revised the paper: AA and JE.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Dopamine neurons of the substantia nigra compacta (SNc) and ventral tegmental area (VTA) are critical components of the neuronal machinery to control emotion and movement in mammals. The slow and gradual death of these neurons as seen in Parkinson's disease has triggered a large investment in research toward unraveling the molecular determinants that are used to generate these neurons and to get an insight in their apparent selective vulnerability. Here, I set out to summarize the current view on the molecular distinctions that exist within this mesodiencephalic dopamine (mdDA) system and elaborate on the molecular programming that is responsible for creating such diversity. Regulation of behavior and more specific emotion is critically regulated by dopaminergic neurons located in the mesodiencephalon. These dopaminergic (DA) neurons can be sub-classified within their own groups by specific molecular features  to follow the developmental trajectory  enriched in the SNc of the mdDA population, were it was proposed to have a major function in local retinoic acid synthesis and was suggested to play a role in disulfiram (Antabuse) induced neurotoxicity leading to parkinsonism and catatonia  , also known as paternal expressed gene 1(Peg1), is a is a member of the divergent alpha-beta hydrolase protein family. In the liver, Mest inhibits Lrp6 glycosylation through enhancing ubiquitination of beta-catenin and therefore is able to regulate balancing of canonical and non-canonical Wnt signaling (Jung et al., Based on initial gene expression comparison studies performed on human PD patients and controls, NeuroD6 was identified to be enriched in VTA neurons (Chung et al., The first data that hinted toward subset specific molecular coding involved in the generation of mdDA neuronal subsets was published at least a decade ago (Smidt et al., The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Vibrio vulnificus is a Gram negative, rod shaped bacterium that belongs to the family Vibrionaceae. It is a deadly, opportunistic human pathogen which is responsible for the majority of seafood-associated deaths worldwide. V. vulnificus infection can be fatal as it may cause severe wound infections potentially requiring amputation or lead to sepsis in susceptible individuals. Treatment is increasingly challenging as V. vulnificus has begun to develop resistance against certain antibiotics due to their indiscriminate use. This article aims to provide insight into the antibiotic resistance of V. vulnificus in different parts of the world as well as an overall review of its clinical manifestations, treatment, and prevention. Understanding the organism's antibiotic resistance profile is vital in order to select appropriate treatment and initiate appropriate prevention measures to treat and control V. vulnificus infections, which should eventually help lower the mortality rate associated with this pathogen worldwide. The emergence of multidrug resistant strains of bacteria has become an international health crisis as illustrated by statements issued by the World Health Organization (WHO) highlighting antimicrobial resistance as a significant threat to human well-being WHO, . This crVibrio species including Vibrio cholerae, Vibrio parahemolyticus, and Vibrio vulnificus are among the common causes of foodborne infections in humans as a result of consumption of contaminated food, particularly seafood. Aside from this, these infections of aquatic livestock by organisms have also been responsible for large-scale losses in the aquaculture industry leading to prophylactic as well as therapeutic use of antimicrobials  , followed by Mid-Atlantic, North Atlantic, and the Pacific  rather than at harvest  presented with septicaemia and the mortality rate was 75%. The prognosis of primary septicaemia was found to be worse than that of wound infection  accounted for a large proportion of the total cost of seafood-related illness treatment costs (US$306 million), followed by medical care (US$25 million), hospitalization (US$6 million), and loss of productivity (US$15 million), and these inevitably become a great healthcare burden to the U.S. , tetracycline (6.06%), aztreonam (24.24%), streptomycin (45.45%), gentamycin (93.94%), tobramycin (100%), and cefazolin (100%). Fortunately, all the isolates obtained were sensitive to ampicillin, ampicillin-sulbactam, piperacillin-tazobactam, cephalothin, ceftriaxone, cefetaxime, ceftazidime, imipenem, ciprofloxacin, levofloxacin, nalidixic acid, trimethoprim-sulfamethoxazole, chloramphenicol, and nitrofurantoin  and an aminoglycoside while doxycycline and fluoroquinolones are contraindicated  in Italy  in Italy and resistant profile (8/8) in South Africa. While aminoglycosides are also safe to use in children with V. vulnificus infection, it has an intermediate resistant and resistant profile in Italy, Germany, and China; and resistant profile alone in Hong Kong and India. With all the evidence, it is clearly vital for clinicians and microbiologists to work with policy makers to review updated resistance profile in that particular country in order to facilitate usage of the most appropriate antibiotics to maximize the treatment efficacy.However, the first-line antimicrobial agents suggested above may no longer be applicable to all countries with Other than conventional medication mentioned above, it is essential for patients with severe soft tissue infection to undergo surgery  when handling raw shellfish, and wear protective footwear (wading shoes) when wading in warm salt or brackish water  compared to at harvest  were immunized with a recombinant bivalent expressed outer membrane protein (OMP) of V. vulnificus and Aeromonas hydrophila and the immunogenicity of this novel vaccine antigen was evaluated  protein and found that it successfully induced a protective immune response against V. vulnificus  has been proposed to be one of the effective ways to attenuate virulence of organisms (Defoirdt et al., Vibrios and thus do not pose any selective pressure for the development of resistance. The fact that the probability of resistance development is smaller compare to the usage of antibiotics makes QS an attractive biocontrol strategy (Defoirdt et al., QS disrupting compounds is also known as antipathogenic compounds because of its characteristics in attenuating virulence without affecting the growth of the bacteria. The QS disrupting compounds mentioned above were shown to have no effect toward the growth of the V. vulnificus is a potentially fatal infection that represents a great health care burden in certain countries. The emergence of multidrug resistant V. vulnificus with highly variable regional resistance profiles is a significant healthcare concern. It is essential to gain a more thorough understanding of the antibiotic resistance profile in different countries which is key to drawing up appropriate clinical practice guidelines for treatment and prevention of this potentially lethal pathogen. Efforts to create an effective vaccine are currently underway but are still in early stages, and identifying effective medical therapy seems to be the more immediate goal. Besides the effort of the researchers, both patients and clinicians also have to be educated in terms of effective prevention measures and antimicrobial resistance profile for an effective therapy in order to lower the fatality rate more convincingly. Ultimately, it is also key to find ways to limit antibiotic use in order to prevent increasing emergence of drug resistant microorganisms.SPH and VL performed the literature search and writing of the manuscript. CYD, NSAM, TMK, LHC, KGC, BHG, PP, and LHL provided critical review and insight to improve the writing. The research project was conceptualize by LHL.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.