There has been an increase in cases of drug addiction and prescription drug abuse worldwide. Recently, pregabalin abuse has been a focus for many healthcare agencies, as highlighted by epidemiological studies. We previously evaluated the possibility of pregabalin abuse using the conditioned place preference (CPP) paradigm. We observed that a 60 mg/kg dose could induce CPP in mice and that pregabalin-rewarding properties were mediated through glutamate neurotransmission. Notably, the dopaminergic reward circuitry is also known to play a crucial role in medication-seeking behavior. Therefore, this study aimed to explore the possible involvement of dopaminergic receptor-1 in pregabalin-induced CPP. Mice were randomly allocated to receive saline or the dopamine-1 receptor antagonist SKF-83566 . After 30 min, the mice received either saline or pregabalin (60 mg/kg) during the conditioning phase. Among the control groups that received saline or SKF-83566, the time spent in the two conditioning chambers was not significantly altered. However, among the pregabalin-treated group, there was a marked increase in the time spent in the drug-paired chamber compared to the time spent in the vehicle-paired chamber. Notably, blocking dopamine-1 receptors with SKF-83566 completely prevented pregabalin-induced place preference, thus demonstrating the engagement of the dopaminergic system in pregabalin-induced reward-related behavior. The intake of pregabalin in high doses or in combination with other drugs has a significant addiction risk3. Moreover, the European Medicines Agency and the European Monitoring Centre for Drugs and Drug Addiction have recently reported a list of several drugs with the potential for abuse, including pregabalin, carfentanil, phenibut, and zopiclone4. Pregabalin prescriptions have increased by 150% in the UK within the last 5 years5. Moreover, the growing black market, including the online availability of pregabalin without a prescription6, indicates the importance of understanding the neurochemical effects behind pregabalin addiction.Worldwide, the abuse of mind-altering prescription drugs has increased dramatically in recent years10. Pregabalin is recommended to treat neuropathic pain, partial epilepsy, and common anxiety disorders13. Moreover, pregabalin is routinely used off-label for many health conditions, including bipolar disorder, trigeminal neuralgia, restless legs syndrome, and alcohol withdrawal states17. We previously reported that 60 mg/kg pregabalin tends to cause conditioned place preference (CPP) in mice18.Pregabalin is a gamma-aminobutyric acid (GABA) analog that binds strongly to the auxiliary alpha-2 delta subunit of the presynaptic voltage-gated calcium channel receptor to reduce the activation of postsynaptic neurotransmitter release19. Specifically, most drugs of abuse can elevate the extracellular dopamine levels in the nucleus accumbens (NAc), which is involved in the reward circuitry, motivational drive, and learning facilitation19. For instance, studies determined that opiate, methamphetamine, and cocaine administration are correlated with an increase in dopamine efflux from key brain regions in animal models23. Notably, pregabalin could produce changes in dopamine level similar to other drugs of abuse. Previous findings indicate that the dopamine-1 (D1) receptor is an important factor in dopaminergic neurotransmission26. It is involved in strengthening cognitive performance30, response control31, and reward management34. Therefore, this study aimed to explore the potential causes behind pregabalin’s reinforcing effects, which we hypothesized could involve dopaminergic system activation through the D1 receptors.The reinforcing and rewarding properties of a variety of abused drugs are related to the neurotransmission of dopamine, a key driver of the neurobiological modifications in drug addictionWe obtained male BALB/c mice from King Fahad Medical Research Center  with a weight range of 25–35 g. Mice had access to food and water ad libitum at standard conditions, where the temperature and relative humidity were adjusted to 21 °C and 30%, respectively, with a 12-h light/dark cycle. Moreover, before the experiments began, the mice were habituated for seven days. All experiments were carried out in accordance with the Institutional Animal Care and Use Committee of the National Institutes of Health and were approved by the Research Ethics Committee at Taif University (42-0112). All methods are reported in accordance with ARRIVE guidelines.36. Moreover, cumulative evidence indicates that an SKF dose of 0.03 mg/kg is sufficient for blocking D1 receptors in rodents38. Finally, a dose of 0.03 mg/kg of SKF has been shown to effectively block amphetamine- and scopolamine-induced locomotor stereotypy and hyperlocomotion40.We dissolved pregabalin  and SKF-83566  in 0.9% saline. An SKF dose of 0.03 mg/kg was selected, as several studies have indicated that this dose is safe and effective to use in rodents18.Briefly, the apparatus was constructed using acrylic and had two identically sized conditioning chambers separated by a removable wall. These conditioning chambers differed in tactile and visual cues as previously reported18  30 min before the administration of saline (10 ml/kg) or pregabalin (PGB) . Each mouse was administered pregabalin or the vehicle four times every other day for eight conditioning days. Subsequently, we conducted the postconditioning test on day 12, with each mouse being placed in the CPP apparatus without restriction between the chambers for 30 min. Finally, the time spent in each chamber was assessed using the same method that was used to investigate the pretreatment behavior.During the conditioning phase (days 4–11), the mice received pretreatment intraperitoneal injection (i.p.) of saline or the D18. Group 4  mice received SKF  30 min before receiving pregabalin (60 mg/kg) for four sessions , with alternating saline sessions during the conditioning phase (n = 7). Subsequently, the place preference was assessed after all the conditioning sessions had been completed.We randomly assigned the mice to one of four groups, as shown in Fig. For all CPP behavioral studies, the time that each mouse spent in each chamber pretest and posttest was analyzed using two-way repeated-measures analysis of variance (RM ANOVA). We performed the Newman-Keuls multiple comparisons test using GraphPad Prism. A p-value of < 0.05 was the chosen level of significance.In group 1 , two-way RM ANOVA identified no significant effect on the phase  = 1.000, p = 0.3632) or chamber  = 0.07136, p = 0.8000), as well as no significant phase–chamber interactions  = 0.3981, p = 0.5558) , we observed a significant effect on the phase  = + infinity, p < 0.0001) and chamber  = 24.90, p = 0.0041), as well as a significant interaction between the phase and chamber  = 28.55, p = 0.0031). The post hoc analysis revealed a significant increase in the time spent in the pregabalin-paired chamber compared to that spent in the saline-paired chamber during the postconditioning test  and the NAc, is considered a critical neural region underlying reward and drug-seeking behavior58. Activation of D1 receptors, but not D2 receptors, in the NAc is essential for long-term potentiation and positive reinforcement behavior59. However, blocking D1 or D2 receptors has shown to impair locomotion and rearing effects associated with dopaminergic neuron inhibition in the NAc core and shell60. Several reports have investigated the role of substance abuse in the release of dopamine in rewarding circuits61. Importantly, repeated morphine treatment for four doses has been shown to induce CPP in mice62. The latter study suggested the development of receptor supersensitivity for postsynaptic dopamine in mice. In confirming this effect, apomorphine (a dopamine agonist) produced stimulated response in ambulatory activity. Moreover, administration of the NMDA antagonist, Mk-801 inhibited the morphine-induced CPP behavior and the development of postsynaptic dopamine receptor supersensitivity. In addition, this effect was observed with other drugs of abuse including cocaine and methamphetamine in mice64. This is consistent with our findings that blocking D1 receptors attenuated pregabalin-induced CPP. Therefore, pregabalin might induce rewarding effects through the activation of postsynaptic D1 receptors in the NAc. Further studies are needed to examine the effects of pregabalin on dopamine release in the NAc.Accumulating evidence indicates that dopamine transport is important for behavioral reward regulation in the NAc68. For example, cocaine-seeking behavior has been linked to the downregulation of GLT-1 expression69. Furthermore, the disturbance in the glutamatergic system is linked with the spillover of glutamate when the uptake of glutamate by a synapse is decreased, which in turn overactivates the postsynaptic receptors that mediate drug-seeking behavior70. Moreover, presynaptic glutamate receptors such as metabotropic glutamate receptors Type 2 (mGlu2/3) have been shown to regulate glutamate release in the NAc and prefrontal cortex and be involved in reward and drug-seeking behavior71.Additionally, the glutamatergic system is significantly involved in mediating the drug-seeking effects of several abuse drugs. Drug-seeking behavior has been linked to glutamatergic imbalance in the NAc and downregulation of the glial excitatory amino acid transporter (GLT-1), which is the main regulator of glutamatergic homeostasis in the brain1 receptors are located in the presynaptic glutamatergic terminal of VTA72. Activating D1 receptors facilitates the release of glutamate in the VTA52. Additionally, it has been mentioned that ethanol-induced spontaneous excitatory postsynaptic currents (sEPSCs) via glutamate alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors are suppressed by SKF in the VTA74. Several studies have shown that ceftriaxone, via the upregulation of GLT-1, can attenuate ethanol intake and relapse in rats76. The involvement of glutamatergic neurotransmission in pregabalin-induced CPP has been previously reported18 and could be due to the activation of presynaptic D1 receptors located within glutamatergic synapses. Where, activating the D1 dopamine receptor could augment AMPA receptor transmission as shown in the NAc cell cultures prepared from rat pups77. Of note, the interaction between dopamine and glutamate is complex in the NAc. The glutamatergic activation in the VTA has been shown to increase dopaminergic activity and release in the NAc79. Moreover, glutamate, at presynaptic level in the NAc, can facilitate dopamine release81. Dopamine can also modulate glutamatergic firings in the NAc that originate from the hippocampus amygdala82. Interestingly, this effect has been shown to be mediated through D1 receptors.Interestingly, these two systems of dopamine and glutamate have been shown to be interconnected and to influence each other in brain regions. It has been proposed that D85. Reportedly, dopaminergic neurotransmission is associated with glutamate release86. In fact, dopamine terminals within the NAc cross on single dendrites with glutamatergic terminals across several key brain regions such as the hippocampus, prefrontal cortex, and amygdala87. Furthermore, reports indicate a strong similarity between the activation of the glutamate receptor N-methyl-d-aspartate (NMDA) and the D1 receptor in drug reward paradigms89. Moreover, it has been shown that glutamate is released upon dopaminergic neurotransmission in the midbrain region in in vitro and in vivo models95. For example, in the NAc shell, dopaminergic terminals were found to release glutamate when activated with channelrhodopsin-296. In methamphetamine seeking, both the NAc and dorsomedial prefrontal cortex showed high levels of glutamate and dopamine when analyzed by microdialysis97, whereas systemic administration of a D1 receptor antagonist (SCH 23390), but not a dopamine receptor-2 antagonist (eticlopride), attenuated methamphetamine seeking98. Although standard receptor binding tests have revealed that pregabalin is not bound to the D1 receptor99, it might increase dopamine levels and glutamate release with the euphoric mental state being achieved. Therefore, the D1 receptor antagonist counteracted the D1 receptor effects in glutamate release regulation and inhibited the dopamine effects. This could lead to a decrease in glutamate and dopamine release100. This is in line with our earlier results which confirmed an association between glutamatergic neurotransmission and the rewarding effects of pregabalin18. These interconnections between dopamine and glutamate in key brain regions support our previous findings that ceftriaxone, a known regulator of glutamate homeostasis, attenuates pregabalin- induced CPP18. Blocking the D1 receptors with SKF in the current study also blocked pregabalin induced CPP. Together, dopaminergic and glutamatergic neurotransmissions in key brain regions might play a significant role in pregabalin-induced CPP. This is consistent with several known drugs of abuse where both dopaminergic and glutamatergic neurotransmissions have been shown to be involved in their rewarding effects68. Studies are warranted to investigate the neurochemical interactions between dopaminergic and glutamatergic systems in pregabalin induced reward.Several studies have suggested that repeated dopaminergic activation during behavioral conditioning performs an essential function in the cue stimuli, leading to drug-seeking behavior101. However, the study did not investigate other brain areas such as the NAc subregions (core and shell), prefrontal cortex, or the VTA. Thus, testing only a single area may not be enough to determine the effects of pregabalin on the dopaminergic system as a whole. Another limitation of this study was not assessing the impact of SKF on pregabalin-induced CPP in a dose-dependent manner. This should be considered for examination in future studies. Moreover, it may be worthwhile to assess whether the effect of SKF in blocking pregabalin-induced CPP may also affect other brain neurotransmitters.The fact that there are no biological studies to confirm these findings is one of the limitations of the present study. Measuring the level of dopamine and glutamate in key brain regions during the conditioning phases, as well as the posttest phase, could provide insights into the mechanisms of pregabalin rewarding properties. A recent study revealed that acute administration of pregabalin did not affect the dopamine level in the NAc102. However, acetylcholine-mediated activation of the D1 receptor by SKF-38393 in striatal cells has been shown105, and this activation was inhibited by SKF103. Similarly, SKF appears to partially act against serotonin receptor-1c106. Pregabalin interaction with the alpha-2 delta subunit of the calcium channel remains only partially understood. Previous findings regarding pregabalin indicate an essential role of GABAergic neurotransmission in the reward and dependence effects of drugs of abuse107. Since pregabalin is a GABA analog, its abuse liability might involve GABA-modulating properties. Specifically, pregabalin administration has been found to slightly increase the extracellular GABA levels in the brain110. Therefore, the weak GABAergic activity of pregabalin may induce GABA-mimetic activity that influences the euphoria and relaxation described by some patients and drug abusers. Thus, future investigations of the effects of SKF on several neurotransmitter systems are needed to understand the mechanistic effect of pregabalin-induced CPP.A previous in vitro study on human neocortical slices assessed different neurotransmitters and reported that pregabalin modulates acetylcholine, serotonin, and norepinephrine release without changing dopamine release1 receptors might play a crucial role in the ability of pregabalin to induce behavioral sensitization through the dopamine reward system. However, there is a need for further neurochemical studies to identify similarities in the abuse liability mechanism between pregabalin and other defined addictive drugs.In conclusion, findings after pretreatment with SKF indicated that the D


We study the vulnerabilityof single-molecule nanowires againsta temporary disconnection of the junction. To this end, we comparethe room and low-temperature junction formation trajectories alongthe opening and closing of gold–4,4′-bipyridine–goldsingle-molecule nanowires. In the low-temperature measurements, thecross-correlations between the opening and subsequent closing conductancetraces demonstrate a strong structural memory effect: around halfof the molecular opening traces exhibit similar, statistically dependentmolecular features as the junction is closed again. This means thatthe junction stays rigid and the molecule remains protruding fromone electrode even after the rupture of the junction, and therefore,the same single-molecule junction can be reestablished if the electrodesare closed again. In the room-temperature measurements, however, weakopening–closing correlations are found, indicating a significantrearrangement of the junction after the rupture and the related lossof structural memory effects. Upon further pulling, the moleculeslides to the apex and only the linkers couple to the electrodes,yielding a decreased junction conductance (LowG configuration). Here,we study the stability of these molecular arrangements investigatingthe two possible scenarios in T = 4.2 K).BP molecules canattach to gold electrodes in two different bindinggeometries, resulting in double-step molecular plateaus.6 the room-temperature opening traces yield a double-peakfeature −1 is the conductance quantum unit. Onthe other hand, the low-temperature measurements exhibit one dominantpeak at the LowG conductance and only a shoulder is observed aroundthe HighG conductance . The coexistence of the LowG peak in theopening and closing histogram indicates that the LowG configurationcan be recovered as the junction is closed again in a low-temperatureenvironment.The low-temperatureclosing histogram  confirm that molecular plateaus extendto longer displacement during the closing of the junction. Furthermore,these two-dimensional histograms reveal that at low temperature, asignificant number of traces are measured without molecular signatures;i.e., tunneling traces are mixed with molecular ones in both the openingand the closing direction. In contrast, the room-temperature measurementsdo not indicate significant number of tunneling traces in either direction,thus almost all opening and closing conductance traces exhibit molecularplateaus.The two-dimensionalconductance histograms .The above analysis shows that≈45% of the low-temperaturemolecular opening traces exhibit molecular plateaus along the closingprocess as well. In the following, we further analyze this subsetof the traces  is likely tobe reproduced on the subsequent closing trace. This reproducibilityis restricted to the molecular conductance range, and to the opening–closing trace pairs. Note that the strong correlations vanish abovethe HighG conductance range (see the vanishing correlations abovethe conductances of the enframed molecular region in r and the closing trace in the r + 1 opening/closingcycle (see the absence of strong correlations around the diagonalin At this point, it is important to emphasizethat opening/closing2D conductance histograms and average conductance traces 4A convey


Dopamine neurotransmission has been ascribed multiple functions with respect to both motivational and associative processes in reward-based learning, though these have proven difficult to tease apart. In order to better describe the role of dopamine in associative learning, this series of experiments examined the potential of dopamine D1- and D2-receptor antagonism (or combined antagonism) to influence the ability of rats to learn neutral valence stimulus-stimulus associations. Using a sensory preconditioning task, rats were first exposed to pairings of two neutral stimuli (S2-S1). Subsequently, S1 was paired with a mild foot-shock and resulting fear to both S1 (directly conditioned) and S2 (preconditioned) was examined. Initial experiments demonstrated the validity of the procedure in that measures of sensory preconditioning were shown to be contingent on pairings of the two sensory stimuli. Subsequent experiments indicated that systemic administration of dopamine D1- or D2-receptor antagonists attenuated learning when administered prior to S2-S1 pairings. However, the administration of a more generic D1R/D2R antagonist was without effect. These effects remained constant regardless of the affective valence of the conditioning environment and did not differ between male and female rats. The results are discussed in the context of recent suggestions that dopaminergic systems encode more than a simple reward prediction error, and provide potential avenues for future investigation. Dopamine neurotransmission has been shown to be critical for aspects of reward-related learning in many different preparations, making it of key interest in the study of disorders involving dysregulation of the reward system, such as addiction, depression, ADHD, and schizophrenia. However, it has proven difficult to pinpoint the precise function(s) of dopamine with respect to associative learning (the formation of connections between cues or actions and their associated outcomes) and motivational processes. Whilst there is strong background literature implicating dopamine in aspects of effort  of these processes, it is not straightforward to isolate the different neurochemical mechanisms, and their neuroanatomical loci, through which dopamine is able to perform these different functions. One reason for this is that the majority of work investigating these functions naturally takes place in the context of reward-based conditioning procedures, making it difficult to tease apart the separate processes involved and study them in isolation. Accordingly, in order to get a clear picture of dopamine’s role in learning, it may help to look beyond reward-learning procedures such as appetitive conditioning and reinforcement learning and explore dopamine’s involvement in other associative preparations. For example, there is evidence to suggest that dopamine may be important in neutral-valence, stimulus-stimulus learning  before these are ever paired with reward  but not D2-like receptors , between 12–16 weeks of age at the beginning of experimental procedures. Rats were housed in groups of four, in a temperature- and humidity-controlled environment (22°C) operating on a 12 h light/dark cycle (lights on at 07:00 h). Experimental procedures took place during the light cycle. For Experiments 1, 3, and 4 rats were placed on a restricted food schedule prior to behavioural training to induce appetitive motivation for food. Weights never reduced past 85% of free-feeding values and water was continuously available in home-cages. In Experiment 2 both food and water were continuously available. Animal procedures were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals , and were approved by the UNSW Animal Care and Ethics Committee.Behavioural training and testing took place in eight standard operant chambers , each housed in a light- and sound-attenuating compartment. The sidewalls of each chamber were constructed of aluminium, and the back wall, ceiling, and hinged front wall were made of clear Perspex. Floors consisted of 19 stainless-steel bars , aligned perpendicular to the back wall of the chamber. A constant current shock generator  was used to deliver a brief duration electric current to the grid floor of the chamber . Floors were cleaned after each experimental session.The auditory stimulus used was a 70 dB 1 kHz square-wave tone produced through a speaker located at the top back left corner of the chamber. The visual stimulus was a 28 W light located on the ceiling of the compartment, flashing at a rate of approximately 3 Hz. The physical identity of the stimuli was fully counterbalanced in each experiment. Chambers were also equipped with a recessed food magazine located at the bottom Centre of the right-hand wall, into which reward pellets could be delivered from a pellet dispenser . Head entries to the magazine were detected by breaks of an infrared beam across the opening of the magazine.via an infrared light source on the compartment ceiling. Cameras mounted on the back wall of each compartment recorded rats’ behaviour during training and test sessions. Recordings were stored on an external hard drive. Experimental events were controlled and recorded via a PC running Med-PC software.Each chamber was illuminated Dopamine receptor antagonists were dissolved in 0.9% saline (w/v) and injected subcutaneously at a volume of 1 ml/kg 15 min prior to sensory preconditioning (Stage 1 training session). For Experiments 2 and 3, the antagonists used were the selective D1R antagonist SCH39166 , administered at a dose of 0.0125 mg/kg (Low), 0.025 mg/kg (Mid), or 0.05 mg/kg (High), and the selective D2R antagonist eticlopride hydrochloride , administered at a dose of 0.003 mg/kg (Low), 0.0125 mg/kg (Mid), or 0.03 mg/kg (High). For Experiment 4, the antagonist used was the non-selective dopamine receptor antagonist α-flupenthixol , administered at a dose of 0.5 mg/kg. SCH39166 and eticlopride doses were determined on the basis of the range observed within our laboratory to be behaviourally effective in an appetitive conditioning context .A summary of the experimental timeline from context exposure onwards can be found in . ExperimStage 1 sensory preconditioning (SPC) occurred over a single session approximately 46 min in duration. The session involved eight presentations each of the visual and auditory stimuli, designated S1 or S2 . S2 was presented on a fixed time schedule with 5 min 10 s between each presentation, and each presentation lasting 30 s. In groups receiving paired (contingent) S2-S1 presentations, S1 began at the termination of S2. S1 presentations lasted 10 s. In groups receiving unpaired (non-contingent) S2 and S1 presentations, S1 was presented in the middle of the interval between S2 presentations. Rats remained in the conditioning chamber for 1 m following the final stimulus presentation. For experiments involving drug manipulations , animals were randomly assigned to receive an injection of either saline or dopamine antagonist prior to this session.Stage 2 first-order fear conditioning (FOC) took place the next day over a single session of approximately 22 min duration. The session involved four 10 s presentations of the stimulus previously designated S1  and four presentations of the US . S1 presentations were separated by an interval of 5 min. For groups receiving paired S1-US presentations, the shock was delivered in the final 0.5 s of each S1 presentation. For groups receiving unpaired S1 and US presentations, the shock was delivered in the middle of the interval between S1 presentations. Rats remained in the conditioning chamber for 1 min following the final stimulus presentation.On the day following FOC, rats received two 30-min sessions of context extinction in which they were placed in the conditioning chamber but no programmed events occurred. These sessions were separated by a minimum interval of 3 h. The purpose of these sessions was to extinguish any freezing elicited by the context, in order to better observe freezing specifically elicited by conditioned stimuli on subsequent tests.On test days rats first received an additional 10 min of context extinction, followed 2 h later by the test session. Test sessions involved 8 presentations of the S2 stimulus alone (Test 1) or S1 stimulus alone . In each test stimulus durations were as in training  and presentations were separated by 3-min intervals.post hoc pairwise comparisons (Scheffe correction for multiple comparisons) where relevant. Significance was set at α = 0.05.Freezing was the measure of conditioned fear, defined as the absence of all movement (except breathing) in an awake animal. For each rat, observations were made every 2 s during stimulus presentations and a baseline period (2 min at the start of session), where the rat was scored as either “freezing” or “not freezing.” Scoring was conducted by an observer blind to experimental condition. A proportion of observations (~10%) were cross-scored by a second independent observer  to ensure observer reliability. In all cases, there was a high degree of agreement between primary observer and cross-scorer (<90%). Overall freezing scores were expressed as a percentage of total observations and used to calculate a difference score (% freezing during stimulus presentations − % freezing during baseline) to be used in statistical analysis. Data were analysed using between-subjects univariate analysis of variance (ANOVA) with Experiment 1 comprised a control study based on Holmes et al. , designet-test; t(23) = 3.989, p = 0.001, where mean (±SD) for session 1 = 9.919 (±3.834) and session 2 = 12.892 (±4.629)]. For the Neutral condition, context exposure proceeded without any programmed events.Forty-eight rats were randomly allocated to receive training in either a neutral or appetitive context. For the Appetitive condition, rats were placed on a food restriction schedule during handling. Context exposure occurred on Days 1 and 2, and for the Appetitive condition Day 2 exposure involved the delivery of food rewards throughout both sessions in order to establish the training context as a positive environment. This manipulation was shown to be successful, as indicated by a significant increase in entries to the food magazine per min from the first to the second session of context exposure with food presentation , indicating that the manipulation was successful. Immediately prior to the SPC session, rats were randomly allocated to receive an injection of either saline (Group VEH), SCH39166 (Group SCH), or eticlopride (Group ETI). In this experiment, all rats received paired S2-S1 presentations during the SPC session on Day 3 and paired S1-shock presentations during the FOC session on Day 4. Context extinction was conducted on Day 5, and tests of S2 and S1 were carried out on Days 6 and 7, respectively. One rat did not receive a shock during the FOC session and was excluded from the analysis. Final group sizes (N = 35) were as follows: Group VEH, n = 11 ; Group SCH, n = 12 ; Group ETI, n = 12 .All rats were placed on a food restriction schedule during handling, and context exposure occurred on Days 1 and 2. Day 2 involved exposure to food rewards throughout both sessions in order to establish the training context as a positive environment. There was a significant increase in entries to the food magazine per min from the first to the second session of context exposure with food presentation , which is at least suggestive that the manipulation was successful overall. For the Neutral condition, context exposure proceeded without any programmed events.Fifty-six male rats were randomly allocated to receive training in either a neutral or appetitive context. For the appetitive condition, rats were placed on a food restriction schedule during handling and context exposure occurred on Days 1 and 2. Day 2 involved exposure to food rewards throughout both sessions. Magazine entry data was unfortunately recorded incorrectly for eight of 24 rats in the appetitive condition. In the remaining 16, there was a significant increase in entries to the food magazine per min from the first to the second session of context exposure with food presentation . There are also numerous examples  where this dose of α-flupenthixol has been shown to be capable of mimicking the impact of more selective dopamine D1R and D2R antagonists at the doses used in our earlier experiments  was the same as (or higher than) doses used in our lab and elsewhere, which have established impacts on learning and performance  could, for example, play a role if these proved to be differentially sensitive to selective and non-selective dopamine receptor antagonists. Similarly, given what we know about other neural substrates of sensory preconditioning (Ward-Robinson et al., Given the potential that functioning of D1R and D2R systems in attenuating sensory preconditioning appears to operate as a logical NAND gate, then another possibility to explain the differential impact of selective vs. non-selective dopamine antagonists is that it might be the relative balance of D1R and D2R activity that is critical. That is, it may be that blocking either D1R or D2R activity and disrupting the relative balance between the two results in impaired sensory preconditioning. In contrast, blocking both D1R and D2R together reduces dopamine signalling overall, but preserves the relative balance of D1R and D2R activity and allows sensory preconditioning to proceed unimpaired. This notion is not without precedent. For example, Furlong et al.  demonstrIn summary, both dopamine D1R and D2R activity appear to be independently important for sensory preconditioning in a manner which is also independent of the motivational status of the learning environment. This confirms and extends previous findings indicating a role for dopamine signalling beyond reward prediction error, and lays the groundwork for further investigation of the downstream mechanisms supporting this function in dopamine systems. Moreover, these findings underscore the utility of the sensory preconditioning procedure as a contrast to more typical reward-based learning procedures in further delineating the range of dopaminergic function in learning. However, the potential that combined dopamine D1R and D2R blockade does not impact sensory preconditioning further highlights the complexity of signalling in dopaminergic systems in relation to learning. In decoding this complexity, future studies are well-placed to employ neuroanatomically targeted approaches to identify the brain regions in which D1R and D2R activity exerts an influence in sensory preconditioning and undertake more direct and controlled manipulations, for example in the balance of activity in D1R- and D2R-expressing neural populations. Furthering our understanding of dopamine function in this manner has important clinical implications for our understanding of psychological disorders involving dysfunctional associative and motivational processes, such as in addiction, schizophrenia, depression, and ADHD.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by UNSW Animal Care and Ethics Committee.SR and SK conceived and designed the experiments. SR and AM collected and analysed the data. SR and SK interpreted the data and wrote the manuscript, with input from AM. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


E. coli.Antimicrobial resistance (AMR) is among the most pressing One Health issues. While interventions and policies with various targets and goals have been implemented, evidence about factors underpinning success and failure of interventions in different sectors is lacking. The objective of this study is to identify characteristics of AMR interventions that increase their capacity to impact AMR. This study focuses on AMR interventions targeting We used the AMR-Intervene framework to extract descriptions of the social and ecological systems of interventions to determine factors contributing to their success.E. coli AMR interventions. We mainly identified interventions implemented in high-income countries (36/42), at the national level (16/42), targeting primarily one sector of society (37/42) that was mainly the human sector (25/42). Interventions were primarily funded by governments (38/42). Most intervention targeted a low leverage point in the AMR system, (36/42), and aimed to change the epidemiology of AMR (14/42). Among all included publications, 55% (29/52) described at least one success factor or obstacle (29/52) and 19% (10/52) identified at least one success factor and one obstacle. Most reported success factors related to communication between the actors and stakeholders and the role of media, and stressed the importance of collaboration between disciplines and external partners. Described obstacles covered data quality, access to data and statistical analyses, and the validity of the results.We identified 52 scientific publications referring to 42 unique E. coli interventions were poorly described with limited evidence of the factors that contributed to the intervention success or failure. Design and reporting guidelines would help to improve reporting quality and provide a valuable tool for improving the science of AMR interventions.Overall, we identified a lack of diversity regarding interventions. In addition, most published The online version contains supplementary material available at 10.1186/s12879-021-06483-z. Antimicrobial resistance (AMR) is a pressing global issue in human and animal health and is frequently included as a priority in political agendas at national and international levels. AMR is a natural phenomenon that has been accelerated by the broad use of antimicrobials (AM) in modern medicine and the food producing system. Development of multi- and pan-resistant bacteria decreases our capacity to treat common infections, and increases the burden of such infections in terms of lives lost and financial costs. Over the past ten years, efforts to tackle AMR have increased worldwide. For example, the healthcare sector adopted tailored stewardship programmes and guidelines for antimicrobial use (AMU)  and streIn a previous publication, we defined an intervention as a coordinated action driven by a social group among a targeted population in a bio-ecological context that interferes with the outcome or course of a difficult situation or process in order to improve it or prevent it from getting worse . The terAMR issues are the result of complex interactions in tightly coupled social-ecological system  and should therefore be designed according to their context to maximise impact . While iThe need for systems thinking to support description, study, and evaluation of complex issues such as AMR has been acknowledged in previous publications , 17. A pEscherichia coli (E. coli). Resistance in gram-negative bacteria, such as E. coli, has been identified as one of the most pressing AMR issues [E. coli is a potential pathogen that is frequently identified in humans and animals and can survive for a long time in the environment. Also, E. coli is considered a good indicator of resistance levels in the community. This study is part of the AMResilience (https://amr-resilience.gtglab.net/) project, which aims to provide and validate a comprehensive multi-method assessment of resilience and transformability to limit AMR and AMU in national and regional One Health systems [The objective of this narrative review is to identify characteristics of AMR interventions that affect their implementation in social-ecological AMR systems, focusing on interventions targeting resistant R issues , 21. E.  systems . Here weWithin the scope of the AMResilience project, we set up a general database of published AMR interventions based on a scoping review of the literature. We used a Boolean query with search strings validated among the co-authors , referencing the text used for each coding. The first author then conducted a thematic analysis of the text extracts following the classification of the framework for advancing science Consolidated Framework for Implementation Research (CFIR) , but codE. coli AMR interventions , slaughterhouse (3/15) or slaughterhouse and farm (3/15), or no specific setting (6/15). Interventions targeting human health were mainly implemented in hospitals (21/28). Interventions targeting food mainly targeted supermarkets (2/3).Only 2 of the 42 interventions were implemented in more than one country. All others were implemented in a single country, most frequently the USA (5/42), Canada (4/42), the Netherlands (4/42), China 3/42) United Kingdom (3/42), and Denmark (3/42). Among the coded interventions, 36 targeted high-income countries, 4 middle high-income countries and 1 a low-income country. The interventions were implemented from 1982 to 2012 , but the maximum was seven AM classes . Most interventions had one specific trigger (31/38), usually the epidemiologic state of AMR (15/38) or the health impact of AMR such as the morbidity/mortality (12/38) , some in collaboration with private partners (2/23), followed by research-based organisations (6/42) and private organisations (3/42); also shown in Additional file /42, someAmong reported key results, we identified 101 positive results towards AMR . Key results can be identified as 104 outputs of the interventions and 33 outcomes. Among all key results, we identified 3 related to the drivers of AMR, 37 related to the pressure of AMU towards AMR, 59 related to the state of AMR, 5 related to the impact of AMR, and finally 32 directly related to the intervention and its delivery, implementation or success.Among all publications, 13% (7/52) mentioned the identification of positive and negative unintended consequences and six (6/7) described them. The identified unintended consequences can be organised in three categories. First, the intervention enabled hiring of more medical professionals as well as specialists in AM treatments in a hospital. Second, the intervention enhanced a shift in the use of AM from one class to another. Finally, issues related to the duration of an intervention’s effect suggests that the observed reduction of AMU might not be permanent.Among all coded documents, 55% of the publications described at least one success factor or obstacle to intervention implementation 29/52). Among them, 10 publications  explained at least one success factor and one obstacle. All factors are detailed in Table 9/52. AmoSuccess and failure factors included the intervention characteristics , the outer and inner settings of the intervention , and the implementation process . Details are provided in Table E. coli AMR interventions that affect their implementation in social-ecological AMR systems. We used the AMR-Intervene framework to describe the interventions in their diversity regarding the social-ecological factors of complex systems, as well as reporting quality in scientific publications. The study of 42 unique E. coli AMR interventions highlighted a lack of diversity of interventions and their action profile, a lack of reporting regarding implementation strategy, and a need for new, adaptive, flexible and complex interventions to tackle AMR.This study identified characteristics of . identified 17 types of interventions [The interventions coded in the database had limited diversity, mainly in their social system and governance. Table ventions .Since diversity of action is a key driver of resilience in social-ecological system, this might be an obstacle to building societal resilience to AMR. Moreover, the use of system thinking has highlighted the need for new forms of co-evolutionary governance to tackle AMR , 27. NumBased on the 52 publications reviewed, it seems that factors related to the implementation, sustainability and success of an intervention are part of a black box , i.e., tIn this narrative review, assessment of the interventions was mainly focused on quantitative indicators, which rationalize and simplify the description process. While this facilitates comparison of intervention results, it obscures the drivers of successes. Furthermore, many different indicators of success or failure were identified in this review. Units and definitions for indicators are not harmonized between evaluations. And still, the main indicators are quantitative, and authors did not explore qualitative methods to assess the interventions. Qualitative assessment of interventions would allow publication of unusual results or feedback about the intervention and things that may not typically be measured in quantitative assessments of interventions  but may be important to intervention success . It coulIn most publications we identified a lack of contextualization, as the intervention was not implemented and reported in a global system perspective and was usually seen as an individual action. All readers would benefit from knowing the context, the means, and other details of the intervention –38. ManyE. coli AMR interventions using the AMR-Intervene framework. Several limitations could have influenced the study results. First, coding bias has been largely prevented, but some variables of the framework still rely on subjective assessment by the coder. Therefore, we implemented a versioning system of AMR-Intervene that can be updated depending on research findings and needs of users. Second, the identification of interventions can also be affected by selection bias and affect our capacity to identify them via the online review process. Some interventions might not have clearly mentioned E. coli as a targeted pathogen. For example, interventions targeting many pathogens including E. coli among others, interventions based on clinical syndromes , as well as all interventions targeting ESBL (extended spectrum beta-lactamase) resistance. Finally, our study does not avoid the classical publication bias such as the year of publication, the publication of successful interventions only, publication habits and the dominance of high-income countries in the scientific literature. Details of unsuccessful interventions or even failure factors or difficulties that the implementers faced are rarely reported. Thus, this study should be complemented by a parallel survey to directly reach implementers and better capture success and failure factors, non-published or ongoing interventions that are not covered by this study.This study characterized 42 Despite these limitations, AMR-Intervene is the first framework built to characterize AMR interventions from a social-ecological perspective. While the framework helps make sense of the variety of interventions targeting AMR among a diversity of contexts worldwide, the present study suggests that the literature focuses on certain types of interventions. Ultimately, we hope that the application of the framework will help to improve how interventions are identified, conceptualized, described, reported, and assessed.One publication mentioned that activities to fight AMR might have diminishing returns and the reduction of AMU could not be further improved by current means  and we might need a shift to other more ambitious interventions and/or improve intervention implementation .Improving implementation of interventions is needed to increase our capacity to tackle the global issue of AMR. Indeed, there is a need for interventions that are better adapted to their social-ecological context, interventions that are diverse and flexible, with various angles of attack, at all levels of the social and bio-ecological system. However, if interventions are not detailed and reported in a more systematic manner, we might not have sufficient understanding of the current situation of AMR interventions. Many publications mentioned a lack of data quality and quantity, which was identified as a failure factor of the intervention. A planned and considered data management process would be an asset to any interventions, e.g., include data managers early in the process, identify with stakeholders and partners an appropriate way to collect, transcribe and share the data and information between them.A successful intervention should also be able to evolve by integrating feedback and adapting to change. Kruk et al.  mentione. [Future interventions are also in need of qualitative data about implementation, such as feedback about the experience, success and failure factors, and contextual factors that can impact the effectiveness or continuity of interventions. In this study we gathered a certain number of different success and failure factors, described in the publications. All factors were identified in previous literature but no publications reported factors about the characteristics of individuals involved, the political and economic climate, or the process of executing the intervention among other factors classified by Darmschroder et al. . Therefo. . While cE. coli AMR interventions identified several factors that can improve the success of interventions when it comes to tackling resistance in this ubiquitous colonizer and pathogen. The study highlighted the lack of diversity in the design and implementation of reported interventions. We clearly identified a lack of One Health interventions, a trend toward interventions with similar goals, governance, and components of response to AMR. It also pointed out that interventions are not designed using a complex system approach. To support adaptation and transformation [The study of ormation , the figAdditional file 1. Online search of E. coli AMR interventions included in the current study via a scoping review.Additional file 2. Supplementary results for the description of articles referring to E. coli AMR interventions, coded in AMR-Intervene.Additional file 3. Supplementary results for the description of E. coli AMR interventions.Additional file 4. Adherence to Reporting Guidelines, the checklist.


Regular evaluation of integrated surveillance for antimicrobial use (AMU) and resistance (AMR) in animals, humans, and the environment is needed to ensure system effectiveness, but the question is how. In this study, six different evaluation tools were assessed after being applied to AMU and AMR surveillance in eight countries: (1) ATLASS: the Assessment Tool for Laboratories and AMR Surveillance Systems developed by the Food and Agriculture Organization (FAO) of the United Nations, (2) ECoSur: Evaluation of Collaboration for Surveillance tool, (3) ISSEP: Integrated Surveillance System Evaluation Project, (4) NEOH: developed by the EU COST Action “Network for Evaluation of One Health,” (5) PMP-AMR: The Progressive Management Pathway tool on AMR developed by the FAO, and (6) SURVTOOLS: developed in the FP7-EU project “RISKSUR.” Each tool was scored using (i) 11 pre-defined functional aspects ; (ii) a strengths, weaknesses, opportunities, and threats (SWOT)-like approach of user experiences ; and (iii) eight predefined content themes related to scope . PMP-AMR, ATLASS, ECoSur, and NEOH are evaluation tools that provide a scoring system to obtain semi-quantitative results, whereas ISSEP and SURVTOOLS will result in a plan for how to conduct evaluation(s). ISSEP, ECoSur, NEOH, and SURVTOOLS allow for in-depth analyses and therefore require more complex data, information, and specific training of evaluator(s). PMP-AMR, ATLASS, and ISSEP were developed specifically for AMR-related activities—only ISSEP included production of a direct measure for “integration” and “impact on decision making.” NEOH and ISSEP were perceived as the best tools for evaluation of One Health (OH) aspects, and ECoSur as best for evaluation of the quality of collaboration. PMP-AMR and ATLASS seemed to be the most user-friendly tools, particularly designed for risk managers. ATLASS was the only tool focusing specifically on laboratory activities. Our experience is that adequate resources are needed to perform evaluation(s). In most cases, evaluation would require involvement of several assessors and/or stakeholders, taking from weeks to months to complete. This study can help direct future evaluators of integrated AMU and AMR surveillance toward the most adequate tool for their specific evaluation purpose. The importance of combatting antimicrobial resistance (AMR) was highlighted in the Global Action Plan (GAP) released by the World Health Organization (WHO) in 2015 ] During 2019–2020, an international network of scientists in the project “Co-Eval-AMR—Convergence in evaluation frameworks for integrated surveillance of AMR” . The objIn the following section, the six tools used are presented in brief.The Assessment Tool for Laboratories and AMR Surveillance Systems (ATLASS) is a tool designed by the FAO for assessing and defining targets to improve national AMR surveillance systems in the food and agriculture sectors . It is cThe Evaluation of Collaboration for Surveillance (ECoSur) tool aims at evaluating the organization, functioning, and functionalities of collaboration taking place in a multi-sectoral surveillance system . The finThe AMR integrated surveillance system evaluation project (ISSEP) tool is a conceptual tool developed in Canada with the aim to structure an evaluation of the added value of integrated surveillance systems for AMR . It compThe Network for Evaluation of One Health (NEOH) tool is part of a framework resulting from the EU COST Action “Network for Evaluation of One Health” to provide science-based guidance for the evaluation of One Health and other integrated approaches to health , 20, 21.The Progressive Management Pathway tool for AMR (PMP-AMR) tool is a self-assessment tool designed by the FAO to provide guidance to countries for implementation of their National Action Plans (NAP) for AMU and AMR , 21. It SURVTOOLS was developed as a part of the EU FP7-funded project RISKSUR: risk-based animal health surveillance systems. The evaluation tool (EVA tool) is a support tool for the evaluation of animal health surveillance systems, developed to provide guidance for evaluation of animal health surveillance including economic evaluation , 21. WheThe details of the scoring scheme for functional aspects, the SWOT-like approach, and the scoring scheme for the themes describing the scope of the tools are presented below.A total of eight country-based case studies of AMU and AMR surveillance systems were included in the study . Each coThe assessors met regularly, and initially, there was an assessment methodology developed in collaboration with selected members of the Co-Eval-AMR network group. The methodology included two standardized scoring schemes; a strengths, weaknesses, opportunities, and threats (SWOT)-like analysis scheme; and templates for reporting and instructions. The evaluation tools were applied on the country-based case studies using one or more tools on each case. Overall, the outcome was the users' experience regarding applicability of the tool. Each tool was assessed between one and four times.A scoring scheme aiming at assessing 11 functional aspects was developed, and answers were scored numerically, where 1 = not covered, 2 = not well-covered, 3 = more or less covered, and 4 = well-covered. With each score, a comment was requested explaining the score. The 11 aspects were as follows: (1) user friendliness, (2) compliance with evaluation objectives, (3) efficiency , (4) use of a step-wise approach to the evaluation, (5) overall appearance, (6) generation of actionable evaluation outputs, (7) evaluation of OH aspects, (8) workability in terms of required data, (9) workability in terms of required people to include, (10) workability in terms of analysis to be done, and (11) time taken for application of the tool.The combined scores for each tool were presented in a heat map. In the case one assessor/assessor group scored over a range of numbers, averaging was used followed by rounding up if necessary to obtain a whole number for the total score. A crude summary score for each tool was calculated and presented in heat maps. The scores should only be interpreted relatively within this study material. The justification for each score, provided by the individual assessors, was condensed by the first author and checked for correctness by the other authors, and the “condensed results” were then presented.A SWOT-like scheme was developed asking the assessors to answer four questions: (1) things that I liked or that the tool covered well; (2) things that I struggled with when using this tool; (3) things people should be aware of when using this tool; and (4) things that this tool covers insufficiently. A qualitative synthesis of the result was done in two steps. First, all individual phrases were captured. In a second step, phrases with the similar meaning were reduced into one, implying that a phrase was simplified or made into one word, if possible. It also implied that no phrase or word was repeated for each of the SWOT analyses and tools. The first synthesis was carried out by the assessors for the tools they had applied. The second synthesis was condensed by two of the authors, and the condensed results were checked for correctness by the other authors and subsequently presented.A second scoring scheme consisted of eight themes to describe the scope of the tool: developed specifically for AMU and AMR, collaboration, resources, output and use of information, integration, governance, adaptivity, and technical operations. Seven of the themes included in the scheme were developed in the Co-Eval-AMR project . Additiohttps://coevalamr.fp7-risksur.eu/) and in Nielsen et al. . ISSEP, ECoSur, NEOH, and SURVTOOLS allow for in-depth analyses and, therefore, require more complex data, information, and specific training of the evaluator(s). PMP-AMR and ATLASS seemed to be the most user-friendly tools, particularly designed for food safety authorities managing the surveillance system.The results of the SWOT-like approach applied to assess the tools are shown in The ECoSur was liked because it allowed evaluation of collaboration in detail; however, the level of abstraction in the language in the existing version of the tool was a struggle. ISSEP was liked because it described the relationship between the integrated surveillance activities for AMU and AMR, OH outputs produced, and the different expected outcomes very well.NEOH was liked for being comprehensive, multi-faceted, and fit for a transversal analysis of OH initiatives. The main struggle related to NEOH was that it was cumbersome and time-consuming to use. Similarly, SURVTOOLS was liked because information for evaluation of all aspects of a surveillance system including the epidemiological part is provided as scientific references. Furthermore, an epidemiological calculator is provided. However, SURVTOOLS is one of the tools that only provide an evaluation plan.The results from the scoring of each tool for the eight themes describing the scope of the tool in relation to surveillance are shown in PMP-AMR, ATLASS, and ISSEP have been developed specifically for AMR-related activities. NEOH and ISSEP were perceived as the best tools for evaluation of all OH aspects, and ECoSur and ISSEP for evaluation of the quality of collaboration. ATLASS is the only tool evaluating laboratory activities specifically. Only ISSEP produced a direct measure of the “integration” and “impact on decision making.” SURVTOOLS has an epi-sample size calculator and is, hence, the only tool providing a quantitative assessment of the technical operations in surveillance.Only PMP-AMR, ATLASS, and ISSEP have been developed especially for evaluating AMU and AMR surveillance. Generally speaking, ISSEP was the only tool assessed that addressed AMU and integration aspects. The strengths of PMP-AMR and ATLASS are governance and, hence, strategic implementation of NAPs. PMP-AMR addresses neither evaluation of design of surveillance nor integration or collaboration. ATLASS is structured in such a way that detailed information about the sectors involved and the laboratories in the surveillance system can be captured. Thereby, it addresses the gaps in a laboratory's capacity to implement surveillance activities. A quantitative evaluation of the epidemiological designs is impossible in ATLASS. Moreover, ATLASS does not provide an output of the level of integration—but all data collated could provide the evaluator with an impression of the level of integration in the system evaluated.However, the other evaluation tools were also considered suitable for evaluation of AMU and AMR surveillance programs. In fact, several of the tools showed a high degree of flexibility and were applicable to different surveillance evaluation objectives. Still, the most accurate evaluations originated from the tools that match the specific evaluation questions. Generally speaking, an evaluation of integrated AMU and AMR surveillance systems will benefit from using tools developed specifically for evaluating AMR surveillance and OH aspects since specific characteristics are encountered.The PMP-AMR and ATLASS tools are to a high extent self-instructive and the questions were, therefore, easy to answer. The structure of PMP-AMR was very easy to understand, whereas ATLASS was more complicated to fill in, since it comprises many questions at all levels of organization. The handbook/guidance/surveillance evaluation wiki to SURVTOOLS was perceived by some of the assessors as very clear and easy to read. It also provides advice on how to cover many of the required aspects of evaluation. The online evaluation tool itself looks very aesthetic but covers less information than the handbook and is not fully self-instructive for all evaluation objectives. NEOH requires knowledge of both the relevant context (in the NEOH framework denoted “the underlying system and its system boundaries”) and the integrated surveillance activities  in question, because the assessor must define all components that form part of the underlying system (the context) included in or affected by the surveillance. NEOH allows the assessor to identify and assess expected outcomes based on the ToC of the initiative. ToC is a specific type of methodology for planning, participation, and evaluation that is used in companies, philanthropy, and not-for-profit and government sectors to promote social change. Further, it defines long-term goals and then maps backward in time to identify the necessary preconditions and actions to be taken. The ToC focus will lead to learning and perhaps a better understanding of the surveillance and its potential societal impacts. It is easy to get lost in the extensive handbook published to assist in using NEOH, and a quick guide is currently missing. The many detailed questions about integration such as OH implementation including systemic organization and level of sharing (infrastructure aspects) and learning  allow for nuances in the answers and, thereby, a better quality of the results. However, the evaluator should be aware that applying this tool requires time investment and training, including specific training in “systems thinking.”ISSEP, ECoSur, and SURVTOOLS also allow for an in-depth analysis requiring collection of more complex data and information. For SURVTOOLS, a specific training in design of epidemiological studies and a wide spectrum of analytical methods are needed before a full exploitation of the tool can be expected. Many of the tools could also be used to guide the design of AMU and AMR surveillance systems in addition to evaluation of existing systems.Many of the tools, especially ATLASS, produce intermediate outputs of how well the different parts of the program are integrated and how well the partners collaborate. In contrast, the interpretation of evaluation results of ECoSur supports the identification of strengths and weaknesses of collaboration and the formulation of recommendations. Among the six tools investigated, this tool allows for addressing collaboration in most detail and in different dimensions.It became clear during this study that adequate resources are needed to perform a full evaluation, sometimes requiring involvement of many assessors and/or stakeholders, and it might take weeks to months to finalize. For all tools, training and instructions would be required to understand the tools sufficiently well to work effectively. Furthermore, the assessor should preferably have a moderate level of understanding of surveillance processes. Moreover, it is important to balance the degree of complexity of the evaluation tool with the available resources in terms of number of people, data, and time.https://www.jpiamr.eu/project/coeval-amr-phase-2/).The ISSEP and partly SURVTOOLS approaches provide a conceptual basis for structuring the evaluation of different surveillance outcomes, from the level of integration to the evaluation of the decisions as well as economic efficiency. The outputs of an evaluation may consist of first-level outputs, such as epidemiological performance measures, as well as intermediate outputs, such as how well the system is integrated. For successful AMU and AMR surveillance, the final impact would be that there are antibiotics available to treat future generations of humans and animals against infections. PMP-AMR and ATLASS only produce intermediate outputs through the theme collaboration. It remains unknown whether this and similar themes really reflect what is necessary to implement to reach the final desired impact in the AMU and AMR surveillance. ATLASS and PMP-AMR are contributing to this final impact by providing evaluation of the governance, strategic support, and budgets for surveillance. An evaluation of the impact of surveillance will be further addressed in a phase 2 of the Co-Eval-AMR, just initiated as a follow-up project funded by Joint Programming Initiative for AMR (JPIAMR)  of the tool. For NEOH, ECoSur, ISSEP, and PMP-AMR, co-developers of the tools were involved in the assessment, but the tools were also assessed by other case study groups. The assessments were done by different persons, and the scores were perceived as crude and subjective. The assessors had varying levels of understanding of the evaluation tools; some were involved in the development of one of the tools, whereas others were trained in using a specific tool. The first group of assessors may have had greater insights into the tool(s) that they assessed and may have been biased in some aspects of the assessment, e.g., user friendliness. During the assessment process, there was some convergence in the scoring done by the assessors due to the development of a common understanding of the words and sentences used in the tools. Therefore, the results of the scoring of the functional criteria had a higher variation than the results of the scoring of the attributes that was done later in the process. The qualitative assessments are probably more informative for the pros and cons of each tool than the actual scores. The remaining tools were assessed by “non-developers.”https://coevalamr.fp7-risksur.eu/). Most of the participants in the case study groups were veterinarians or professionals working within veterinary public health. Persons in human health only participated indirectly when being interviewed, and there was no focus on the environment. In phase 2 of the project, collaboration among others and with social scientists will broaden the scope and the way of looking at surveillance and evaluations.Monitoring and stewardship of AMU as part of AMR surveillance were not addressed in the assessment. In the second phase of the Co-Eval-AMR, additional assessments using other tools are planned. Moreover, focus will be on how to assess the impact of integrated surveillance systems for AMU and AMR as well as on how to evaluate governance. The online assessment system made by the Co-Eval-AMR project group can also be used by other scientists for doing similar comparisons and hence more experiences will be collected . We encourage users of the tools to provide their inputs and expect that over time a growing collection of experiences will help users in choosing more easily among the existing tools.The developed reporting template enables other assessors to report their experiences using the tools in a comparable way. The template consists of four sections; (1) general information, (2) scoring of 10 functional aspects, (3) SWOT-like approach, and (4) scoring of eight themes describing the scope of the tool. The idea was to develop a kind of user experience scoring overview similar to many internet applications such as TripAdvisor and Google reviews providing the readers with quick, yet detailed, insights of the tools. The template is placed in an online platform on the homepage of Co-Eval-AMR (Evaluation of integrated surveillance is needed at regular intervals using robust tools. It is important to choose a tool that adequately addresses the specific evaluation objectives. We provided a portfolio of the experiences of 20 users representing eight country-based case studies in which six different tools were applied, to highlight their attributes, pros and cons, and requirements.Only PMP-AMR, ATLASS, and ISSEP have been developed especially for evaluating AMU and AMR surveillance—with ISSEP being the only tool providing a semi-quantitative score of AMU and AMR integration. All six tools demonstrate a high degree of complementarity. Depending on the evaluation questions selected, assessors may choose among the different tools to conduct the evaluation as such, namely, ECoSur for addressing collaboration, NEOH for the OH-ness and the relationship between ToC and expected outcomes of the surveillance, ATLASS for the laboratory capacities, and SURVTOOL for epidemiological and economic performance.https://guidance.fp7-risksur.eu/welcome/decision-support/. Furthermore, this platform could help further extend general user experience of AMU and AMR surveillance evaluation tools.An online platform for reporting of users' experiences will help users interested in conducting an evaluation of AMU and AMR surveillance in choosing the most adequate tools for their specific evaluation needs: The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.MS, AH, MB, and LA were substantially involved, and took the lead, in all steps of the study from the conception to the design of the work. AH and UB designed the layout of MB was involved in the development of ECoSur and LN was involved in the development of NEOH. The remaining 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.


N = 890) included in the study were tested for BTB at least once using the comparative intradermal tuberculin test (CIDT). Fertility, mortality, and offtake were assessed in 21 dairy farms where herd follow-ups over 3 years were performed. In addition, an independent abattoir survey was conducted to assess carcass weight and visible TB-like lesions upon meat inspection. Animal movements (purchasing and offtakes) were documented for each farm. The impact of BTB status on the intervals been birth, service, and calving times and the intercalving intervals was analyzed using a Cox proportional hazards model. The hazard ratio associated with BTB-positive animals was smaller than 1 for all fertility parameters, suggesting that BTB status increases the time between events; however, the effect was small and only statistically significant (95% level) for the time between calving and service. Offtakes included a higher percentage of reactor animals (58%) as compared with non-reactor animals (42%) (p = 0.0001). Overall, reactors were eliminated from the farms within 238.6 days after receiving test results, which was 54.9 days faster than for negative animals. The majority of owners purchased animals within their town or its surrounding. Nearly a quarter of reactors were sold directly to other farms. Animals were also sold further away, including other regions, raising the question of disease spread and the need for an animal tracing mechanism. In the abattoir survey, a total of 349 carcasses were weighed, of which 8% showed visible TB-like lesions and 53.6% had fasciolosis. Negative adult bull carcasses were 7.5 kg heavier than reactor bulls.Bovine tuberculosis (BTB) has substantial impact on fertility, milk, and meat productivity in cattle. However, these assumptions are based on outdated data. Recent global studies on the impact of BTB on cattle productivity are scarce and show sometimes inconclusive and/or contradicting results. This pilot study is the first longitudinal study performed in urban upgraded dairy cattle in Ethiopia that are kept under intensive husbandry. We assessed whether BTB has an impact on various animal productivity parameters and animal movement. Animals ( Mycobacterium bovis, with cattle being the primary host. However, a wide-range of domesticated and wild mammal species as well as humans can become infected with BTB through direct contact with a positive host or consumption of raw animal products (Bovine tuberculosis (BTB) is a chronic bacterial zoonotic disease caused by products , 2.Besides being an animal and public health concern, BTB has globally substantial direct and indirect economic implications related to direct impacts on animal health and productivity, surveillance-control and eradication programs, market losses, and animal movement restrictions.Costs of BTB control and interventions have been quantified in some contexts –6. The eM. bovis.More recent small-scale studies showed diverging or sometimes inconclusive results. Boland et al.  showed aCattle productivity analyses have rarely been performed. Such analysis require large animal numbers, longitudinal studies, or very well kept detailed records on productivity parameters over years. Studies in the scale of the Meisinger study have never been replicated in recent times. In Africa, only very rare longitudinal studies were performed to estimate fertility and mortality of local zebu cattle under traditional husbandry management –19.BTB is endemic in Ethiopia , 21. OveThis present study is the first longitudinal study performed in urban dairy cattle in Ethiopia that are being kept under intensive husbandry. We aimed in this pilot study to assess whether BTB has an impact on animal movement and on various animal productivity parameters and, through establishing biological effect sizes, inform the necessary sampling scale for future larger studies.The methods used are similar to the ones described in Tschopp et al.  and are This study was carried out in Central Ethiopia within the greater milk production belt, namely, Addis Ababa , as well as Debre Zeit and Sendafa, 50 and 40 km away from the capital, respectively. Data were collected between November 2015 and September 2018. The area is conducive to dairy farming and has high production potential. It benefits from a temperate climate and an abundant rainfall  with good animal fodder potential and holds the largest high-yielding dairy cow numbers in the country. The milk shed of the study areas has access to big markets including Addis Ababa .Intensive urban dairy farms were selected based on the willingness of the owners to collaborate on a 3-year longitudinal study. Animals were high milk-yielding Holstein–Frisian cross-breed cattle. All farm sizes were represented and categorized into small farms , medium farms , and large farms . Except for one government farm, all farms were privately owned. Husbandry was similar within farm size categories.Animals were categorized into sex and age class. Young stock were animals younger than 12 months, replacement stock were animals from 12 months to around 3 years, and breeders were from 3 years onwards.An initial registration of all animals was performed in each farm at the start of the study. A herd-book was prepared capturing parameters such as any new animal entry , animal exit , detailed data related to selling and purchasing of animals , morbidities including mastitis, mortalities, and fertility [artificial insemination (AI) dates and calving dates]. The farms were visited twice a month by the same investigators. Herd-book information was updated on hard copies during each visit, and data were entered in a Microsoft Access table. All animals were dewormed once a year with albendazole.Animals were tested early in the study (spring 2016) using the comparative intradermal tuberculin test (CIDT). They were tested again toward the end of the study (2018) by a trained veterinarian. Two sites of 12–15 cm apart in the middle of the neck were shaved and cleaned. Skinfold thickness was measured with a digital caliper at both sites. Animals were then injected intradermally with 0.1 ml  avian PPD and 0.1 ml  bovine PPD  using insulin syringes. The injections sites were examined, and the skin thicknesses measured 72 h post-injection. The difference in the increase of skin thickness at the bovine and avian sites before and after inoculation was measured and recorded by the same veterinarian. A reaction was considered positive if the increase in skin thickness at the bovine site of injection was 4 mm greater than at the reaction shown at the site of the avian injection. The reaction was inconclusive if the increase was between 1 and 4 mm  and negaFasciola hepatica infection and graded as + low infection, ++ moderate infection, and +++ heavy infection, leading to organ confiscation. Carcasses were also assessed for visible TB lesions, and samples from these lesions were collected and cultured in duplicate on glycerol and 0.4% pyruvate LJ medium  and incubated at 37°C. Cultures were observed daily for growing colonies during the first week and then weekly from the second week onwards. Cultures with no growth at week 8 were considered negative.An abattoir study was carried out independently from the above longitudinal study . Carcasses from cattle slaughtered in two abattoirs in the study area (Addis Ababa and Sululta) were weighed by quarters. Liver was examined for Raw log book data from farms within the study were entered in Microsoft Access tables. Each fertility parameter can be considered as a time interval, measuring the time to either calving or service from a given reference time . As such, each of these measures is potentially right censored either through the removal of the animal from the herd before the end of the study or by the end of the study itself. Survival analyses specifically account for this censoring and are therefore the most appropriate way to assess the impact of BTB status, or other explanatory factors, on each of these parameters . To thisWe performed an initial exploratory analysis by plotting the Kaplan–Meier survival curves for each fertility measure in turn, stratified by farm size, farm identification, and TB test status using the survival package in R . These rAs the Cox model only provides the hazard ratio with respect to BTB status, we also present summary statistics for each fertility parameter in terms of the median value and 95% quantiles .This research study was approved by the Institutional Review Board (IRB) of Aklilu Lemma Institute of Pathobiology, Addis Ababa University (reference number IRB/ALIPB/2018) and the IRB of AHRI (AAERC) (reference number PO46/14) and supported by the Ethiopian Ministry of Livestock and Fisheries. Skin testing of cattle was based on the international standards . Verbal N = 356) and 31.7% after adjusting by herd size (95% CI: 19.5–47%). Overall, 53.2% (N = 474) were negative and 6.8% (N = 60) were inconclusive. Three-hundred twenty-seven animals were tested twice over a period of 3 years, of which 237 (72.5%) kept the same BTB status, whereas 42 animals (12.8%) converted from inconclusive or negative to being reactors. An additional eight animals (2.4%) converted from negative into inconclusive. The remaining 40 animals that were initially positive either became inconclusive or negative.A total of 890 animals were tested at least once for BTB in 21 farms. Sixteen farms (76.2%) had at least one reactor animal. Overall, animal crude apparent prevalence for BTB was 40%  but were forced into the model to adjust for this important source of variation identified by the exploratory analysis. The hazard ratio associated with BTB-positive animals was smaller than 1 for all fertility parameters, suggesting that BTB status increases the time between events. However, the estimated effect was small and only significant (at the 95% level) for the calving to service time see . Table 2Among the BTB tested animals, 34 animals died of a natural cause during the study period. Seventeen were reactors and 17 were BTB negative. The numbers were too small to justify further analyses.Overall, at the end of the study, 245 animals among the 830 tested animals had been eliminated (selling or slaughtering); among them, 103 (42%) were BTB negative and 142 (58%) were BTB positive. Among all 356 BTB-positive animals, 142 were eliminated (39.9%) and 214 (60.1%) were still in the farms at the end of the study. Among the 474 BTB-negative animals, 103 (21.7%) were eliminated and 371 (78.3%) were kept.N = 236), 96 (40.7%) were BTB negative and 140 (59.3%) BTB positive. Overall, for the 236 animals that were eliminated, median time between test result and elimination of the animal was 191.5 days (95% CI: 158–254) regardless of TB status. Overall, reactors were eliminated faster than BTB-negative animals (mean difference was 54.9 days). This was particularly true in medium farms (p = 0.02)  see .N = 60; 73.2%) mainly in Addis Ababa, whereas a quarter were sold directly to other farms , and one animal was sold to a broker.Detailed information on where these positive animals went was available for 82 animals. The great majority was sent to abattoirs  and 209 (59.9%) carcasses were from Addis Ababa and Sululta abattoir, respectively. One hundred ten were male (41.2%) and 157 female (58.8). Replacements accounted for 48 animals (13.7%) and breeders for 299 animals (85.7%).Fasciola prevalence was 53.6% (N = 187). Half of the animals had moderate Fasciola infestation , whereas 32.1% (N = 60) had severe and 17.1% (N = 32) had mild infestation. Twenty-eight (8%) animals had visible TB-like lesions upon meat inspection examination. Culture was done on 18 lesions, and 13/18 (72.2%) were culture positive for Mycobacteria.Overall BTB-negative breeding bull carcasses were overall 7.5 kg heavier than BTB-positive animals; however, there was no statistical difference at the 95% level see .Overall, 102 animals were purchased. Among them, 53 were included in the original stock during registration, and 49 were further purchased during the study time for 48 of which detailed information was collected. The majority of animals were purchased directly from another farm (93.7%), while 6.2% were purchased from local markets. No brokers were involved. The majority of farmers bought their animals within their town or its surrounding. Addis Ababa farmers purchased 89.6% of the total purchased animals in the study.N = 275; 51.1% of all animals sold) followed by male calves that were eliminated within the first 2 weeks of life . The majority of sold animals originated in the Addis farms  and the least in Sendafa . Reasons for selling were given for 467 animals, as follows: immediate need for cash (18.6%), underperforming animals (14.6%), depopulation (10.3%), diseased animals (6.8%), old animals (6%), and animals suffering a trauma such as broken legs (2.4%). The rest were young male calves eliminated within their first 2 weeks of life. TB was never given as a direct reason. Most animals were sold directly to abattoirs . Nearly a quarter  were sold directly to another farms, whereas 15 animals (4%) were sold via brokers. None were sold via a market system; 66.6% of all animals were sold to Addis Ababa, where large abattoirs are found. Overall, 538 animals were sold during the study period. The majority were heifers and cows . This result highlights potential substantial hidden economic impact of BTB on farms that cannot be easily measured. There is an indication that farmers are selling more reactors than non-reactors and that they are selling their reactors faster than they would in average. Therefore, they might lose on optimal market prices, affecting both farm dynamic and business through, for instance, economic and logistic challenge of animal sourcing for restocking; loss of markets; and a possible additional factor (among many others) of observed farm depopulation. In our study, nine out of the 24 study farm (37.5%) had their animal number decrease over the last 3 years including 80% of all large farms . Overall, reactors were eliminated faster than non-reactors  and the African buffalo (Syncerus caffer), where lower pregnancy probability was described , a common liver fluke found in cattle. A third (32.1%) showed even severe infestation of the liver. F. hepatica infections were shown to be linked with altered immune responsiveness to PPD, hence having implications with BTB diagnosis and disease progression  were infested with gression –45. The This study is the first one in Ethiopia to assess the potential impact on BTB on dairy cattle productivity parameters in a holistic way . Due to the lack of accurate record keeping of milk yield, we could not calculate the effect of BTB on milk productivity. Although monetary translation of productivity loss was difficult to perform at this stage, and although our sample size was too small for definitive conclusions, our pilot study indicated that BTB has likely an impact on productivity (lower fertility and weight loss) and has likely further invisible costs and indirect economic impacts such as forgoing access to better markets, time, and change in farm business. Besides a regular BTB testing follow-up of each animal over time, a much larger sample size including >5,000 animals is warranted in future follow-up studies to corroborate these findings and enable to control other factors influencing productivity such as husbandry factors or comorbidities. With the growing intensification of cattle production in Ethiopia, analysis of productivity and impact of diseases in productivity are important. The results of this study will provide important information to conduct further cost–benefit analysis of different control strategies for BTB.The datasets presented in this article are not readily available because restrictions linked to project (EThicobots) regulations.This research study was approved by the Institutional Review Board (IRB) of Aklilu Lemma Institute of Pathobiology, Addis Ababa University (reference number IRB/ALIPB/2018), the Institutional Review Board of AHRI (AAERC) (reference number PO46/14) and supported by the Ethiopian Ministry of Livestock and Fisheries. Written informed consent for participation was not obtained from the owners because Verbal informed consent was obtained by owners.RT designed the research, performed the data analysis, and drafted the manuscript. GG and GA collected field data and contributed to drafting the manuscript. AC and JH contributed to the data analysis. JW and JZ provided critical comments to the manuscript. All authors have contributed to the manuscript and have approved the submitted version.The members of the Ethiopia Control of Bovine Tuberculosis Strategies (ETHICOBOTS) consortium are as follows: Abraham Aseffa, Adane Mihret, Bamlak Tessema, Bizuneh Belachew, Eshcolewyene Fekadu, Fantanesh Melese, Gizachew Gemechu, Hawult Taye, Rea Tschopp, Shewit Haile, Sosina Ayalew, and Tsegaye Hailu, all from Armauer Hansen Research Institute, Ethiopia; Rea Tschopp from Swiss Tropical and Public Health Institute, Switzerland; Adam Bekele, Chilot Yirga, Mulualem Ambaw, Tadele Mamo, and Tesfaye Solomon, all from Ethiopian Institute of Agricultural Research, Ethiopia; Tilaye Teklewold from Amhara Regional Agricultural Research Institute, Ethiopia; Solomon Gebre, Getachew Gari, Mesfin Sahle, Abde Aliy, Abebe Olani, Asegedech Sirak, Gizat Almaw, Getnet Mekonnen, Mekdes Tamiru, and Sintayehu Guta, all from National Animal Health Diagnostic and Investigation Center, Ethiopia; James Wood, Andrew Conlan, and Alan Clarke, all from Cambridge University, United Kingdom; Henrietta L. Moore and Catherine Hodge, both from University College London, United Kingdom; Constance Smith at the University of Manchester, United Kingdom; R. Glyn Hewinson, Stefan Berg, Martin Vordermeier, and Javier Nunez-Garcia, all from Animal and Plant Health Agency, United Kingdom; Gobena Ameni, Berecha Bayissa, Aboma Zewude, Adane Worku, Lemma Terfassa, Mahlet Chanyalew, Temesgen Mohammed, and Miserach Zeleke, all from Addis Ababa University, Ethiopia.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.