The present study aimed to explore the consequences of a single exposure to a social defeat on dopamine release in the rat nucleus accumbens measured with a fast-scan cyclic voltammetry. We found that 24 h after a social defeat, accumbal dopamine responses, evoked by a high frequency electrical stimulation of the ventral tegmental area, were more profound in socially defeated rats in comparison with non-defeated control animals. The enhanced dopamine release was associated with the prolonged immobility time in the forced swim test. The use of the dopamine depletion protocol revealed no alteration in the reduction and recovery of the amplitude of dopamine release following social defeat stress. However, administration of dopamine D2 receptor antagonist, raclopride , resulted in significant increase of the electrically evoked dopamine release in both groups of animals, nevertheless exhibiting less manifested effect in the defeated rats comparing to control animals. Taken together, our data demonstrated profound alterations in the dopamine transmission in the association with depressive-like behavior following a single exposure to stressful environment. These voltammetric findings pointed to a promising path for the identification of neurobiological mechanisms underlying stress-promoted behavioral abnormalities. Stressful events provoke a chain of biochemical and neurochemical responses in the body and brain ,4,5,6,7.A synapses on VTA dopamine neurons was blocked 24 h following acute exposure to stress [To reproduce the phenotype of stress-related psychopathologies, repeated social defeat was predominantly employing ,28,33,34o stress . Since do stress ,39, streo stress . In agreo stress . It was Therefore, the present study was designed to explore the consequences of a single social defeat on accumbal dopamine release evoked by an electrical stimulation of the VTA measured by a fast-scan cyclic voltammetry (FSCV). Based on the results of previous studies ,40, neurMale Sprague Dawley (300–350 g) and Long Evans (450–500 g) rats were housed on a 12 h light/dark cycle with food and water available ad libitum. All animal procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All experiments conformed within international guidelines on the ethical use of animals. For social defeat procedures, a Sprague Dawley rat was placed into the home cage of the resident (Long Evans) in order to create a robust stressful environment for the intruder ,41,42. DEach 20 min social defeat session and control group interactions were video recorded. Behavioral patterns were recognized as follows: defensive, freezing (rat does not move), explorative , running, submissive (rat lying on its back) and other . Duration of each behavioral element was assessed in both socially defeated (SD) and control rats. Every effort was made to minimize suffering and reduce the number of animals in this study. Rats (including control) were tested in the standard battery of behavioral tests 24 h following social defeat and social interaction paradigms.Sucrose preference test was performed to reveal whether an anhedonic condition could be triggered by short-term stress exposure. A two-bottle choice design was used where the rats had access to water and a 10% sucrose solution 18 h/day for 3 days/week over 2 weeks. At the beginning and end of the drinking period the bottles were weighed and a fluid intake was calculated. No previous food or water deprivation was applied before the test. The intake of water and 10% sucrose was measured during the dark circadian phase . Final mAn open field test was used to explore short-term consequences of a single bout of social defeat on locomotion and stereotypical behaviors. Rats were acclimated to the testing room and then individually placed at the center of the plastic arena (40 × 40 × 40 cm). Their spontaneous activity was registered) for 6 min with a video-recording system . Between subjects, the arena was cleaned with 3% hydrogen in order to eliminate olfactory cues. The duration of behavioral patterns  was counted manually by analyzing the recorded videos .Finally, the forced swim test procedure ,46 was pElectrically evoked dopamine release was measured in the nucleus accumbens (NAc) of anesthetized rats 24 h after the social defeat procedure. Rats were anesthetized by using a single intraperitoneal (i.p.) injection of urethane 1.5 g/kg) and secured in a stereotaxic frame. Holes were drilled to allow for the implantation of electrodes into the brain. A stimulating electrode was inserted into the VTA , a carbon fiber working microelectrode  was placed into the NAc  and an Ag/AgCl reference electrode into the brain tissue of the contralateral hemisphere. The electrodes were connected to the voltametric amplifier interfaced with a computer running the specialized software. To explore the difference in frequency-dependence of dopamine release between SD and control rats, 1-s electrical stimulations (330 µA) of the VTA were made at different frequencies  every 10 min. For the pharmacological study, following acquisition of a stable baseline dopamine signal , raclopride (dopamine D2 receptor antagonist) at 2 mg/kg (i.p.) dose was injected. This raclopride dose robustly affects dopamine transmission at the level of presynaptic terminals in a way that allows for reliable detection of increased electrically evoked dopamine release with FSCV  g/kg and. ElectriThe depletion protocol included three consequent stimulations , which were made with 1–2 s intervals. Then, a regular stimulation  was employed with 1 min (14 stimulations), 5 min (3) and 10 min (5) intervals to allow dopamine signal recovery.Extracellular dopamine was detected at the carbon fiber electrode every 100 ms by applying a triangular waveform . The dopamine signal was verified by a background-subtracted cyclic voltammogram characterized by oxidation and reduction peaks occurring at +0.6 and −0.2 V, respectively ,48,49.To verify the placement of the detecting electrode animals were overdosed with chloral hydrate (1000 mg/kg) and perfused intracardially with 0.9% saline. The brains were rapidly removed, and brain samples were fixed in a buffered 10% formalin solution for 24 h, then were washed in water for 20 min and kept at +4 °C in 30% sucrose in 0.1 M phosphate buffer for 48–96 h as previously described . Next, bt-tests, otherwise nonparametric Mann–Whitney U test was performed. All analysis was carrying out using GraphPad Prism . The data are expressed as a mean ± SEM with a criterion for significance set at p ≤ 0.05.Data for each behavioral element was accounted as a percent of total observation time. Voltammetry measurements  were converted into a concentration of released dopamine and expressed in molar (M) or as % of preceding background values. Statistical analysis of voltammetric data was performed by applying a repeated-measures two-way ANOVA. For behavioral data D’Agostino and Pearson omnibus normality test was used for counting whether the values followed Gaussian distribution and then in case of normal distribution we used unpaired two-tailed p = 0.0006) with a decrease in other behaviors, such as walking and standing , with no significant difference in explorative behavior such a rearing and sniffing  in defeated rats when compared to control rats.The present study revealed evident behavioral alterations in the “intruder” rats during SD exposure. In the paradigm , the avep = 0.8048, Mann–Whitney U test) in two-bottle choice test , time spent in the center of arena , rearing , grooming  and locomotor activity . However, socially defeated rats showed a significant increase in the immobility time during the forced swim test  (p = 0.0201). Sidak’s multiple comparisons test revealed a higher dopamine release in defeated rats vs. controls at higher frequencies  but not at lower frequencies   = 15.59,  0.0001) . However > 0.05) .p < 0.0001), while a greater effect was observed in controls compared to defeated rats  = 5.321; p < 0.05)  resulted in significant increase of electrically evoked dopamine release in both groups  = 30.26;  < 0.05) .p < 0.0001) with no difference between groups  = 1.141; p > 0.05)  = 14.76;  > 0.05) .The present study was mainly focused on the short-term consequences of a single social defeat on accumbal dopamine dynamics, while behavioral assessments were also achieved. Expectedly, robust stress-associated behaviors were evident in intruder rats during their confrontation with an aggressive resident. However, the majority of performed tests did not reveal significant alterations in behaviors of defeated rats 24 h following the interaction. Thus, a sucrose intake, locomotor activity, rearing and grooming were identical to those in control animals. Nevertheless, stressed animals demonstrated a substantial increase in the immobility time during the forced swim test. This social defeat consequence, which indicates a deficiency in natural escape behavior, was paralleled with changes in dopamine release evoked by an electrical stimulation of the VTA. Remarkably, the increased dopamine efflux was observed following the VTA activation at high frequencies , while lowest frequencies  did not result in the differences in accumbal dopamine response between defeated and control rats. Furthermore, the administration of dopamine D2 receptor antagonist, raclopride  caused a significantly weaker effect on electrically evoked dopamine release in stressed subjects compared to control. The use of the dopamine depletion protocol revealed no alteration of the reduction or recovery of the amplitude of dopamine release following stress triggered by a social defeat.During social defeat in rodents, acute physiological adaptations, which include an activation of cardiovascular and endocrine systems ,52 alongNevertheless, the immobility time in the Porsolt forced swim test (FST), which is considered an indicator of a depression-like state ,46, was In the current study this behavioral consequence of social defeat was paralleled with changes in the accumbal dopamine release evoked by an electrical stimulation of the VTA. Noticeably, dopamine release was enhanced under the condition, when the stimulations were performed at high frequencies, which trigger phasic pattern of dopamine neurotransmission ,57. The A synapses on VTA dopamine neurons [A single stress promotes several neuroadaptations, which can be responsible for observed dopamine changes. Thus, acute stress-induced blockade of the LTP induction at GABA neurons ,59 may r neurons . These f neurons .Remarkably, the consequences of social defeat on dopamine dynamics can be evident in an ex vivo preparation, where the release is evoked by electrical stimulation of accumbal slices, which have no connectivity from the dopamine cell body region (VTA) . In factOn other hand, corticotropin-releasing factor (CRF) can be responsible for the observed dopamine alterations. This neuropeptide releases in response to acute stress promoting adaptive and maladaptive behaviors ,66 throuAltered dopamine efflux and consequent changes in its extracellular concentrations potentially may lead to presynaptic neuroadaptations, which are directed to compensate nonphysiologocal changes. This may impact the processes of synthesis, autoreceptor regulation of neurotransmitter release and reuptake. For example, the acceleration in dopamine uptake presumably due to the persistently elevated dopamine concentration in dopamine terminals was found using different models of drug addiction ,15.To find whether the consequence of single social defeat on dopamine release is capable of affecting dopamine dynamics at the presynaptic level, we applied the dopamine depletion protocol that allows to reveal changes in synthesis, reuptake and autoreceptor regulation of dopamine ,71,72. TNevertheless, single social defeat resulted in a weaker effect of raclopride on electrically evoked efflux compared to the control that might suggest alteration in D2 receptor regulation of dopamine release. Interestingly, there is an evidence that acute exposure to aggression increases D2 receptor density in the nucleus accumbens of the opponent rat . HoweverOur data demonstrated marked changes in the accumbal dopamine neurotransmission together with the indication of depression-like condition determined 24 h following the exposure to a social defeat. Remarkably, these neurochemical and behavioral changes occurred as consequences of only a single exposure to stressful event. Collectively, these findings pointed in a promising direction for the identification of the neuronal circuitry and neurochemical mechanism underlying stress-triggered development of psychopathologies such as PTSD and depression.


Optimizing the properties of electrospun polymer-perovskite nanofibers is considered essential for improving the performance of flexible optoelectronic devices. Here, the influence of electrospinning setup parameters , rotation speed, as well as process time) on the properties  of electrospun polyvinylpyrrolidone nanofibers modified with cesium lead iodide nanocrystals has been studied. The results have shown that the structure of nanofibers is related to the electrical voltage, collector rotation speed, and process duration. Perovskite crystallinity and light absorption have improved by increasing the electrical voltage or/and the process time. The polymer’s glass transition temperature is affected by the embedded perovskite and the collector’s rotation speed. The shrinkage ratio and mechanical properties of nanofibers have been controlled by the rotation speed and the electrical voltage. The shrinkage is caused by the stress created in the nanofibers during the electrospinning process. The best mechanical properties can be noticed with the rotary collector at a rotational speed of 500–750 rpm. Nanofibers have shown good long-term stability and high thermal stability. The long-term stability is inversely proportional to the value of the electrical voltage. They found that an increase in the polymer concentration from 2% to 25% has led to a decrease in the perovskite crystallinity. The light absorption of the fibrillar structure was three times better than that of the nanolayer analogue. In this method, the nanocrystals are distributed in the center and near the surface of nanofibers [2 solution [3:PAN nanofibers, a decrease in the thickness of the polymeric shell has led to the formation of smaller crystals in the center of nanofibers due to the fast evaporation of the solvent, increasing the crystallization rate [g) of the polymer [3: polyvinylbutyral (PVB) nanofibers annealed at 400 °C for 1 h. Inspired by the previous results, herein we study the influence of electrospinning setup parameters on the properties of PVP:CsPbI3 nanofibers. The latter has been fabricated by the one-stage electrospinning method. Parameters that result in a strong change in the structure of nanofibers, e.g., the electrical voltage, the collector type, and the process time, were chosen to study their influence on the outer and inner structure of nanofibers, optical properties, mechanical properties, thermal behavior, and long-term stability.Electrospun nanofibers have attracted a lot of attention in scientific and industrial communities due to their unique properties, such as light weight, flexibility, controllable properties, and low cost, which have allowed them to be widely applied in many fields, e.g., energy generation and storage, sensors, drug delivery, filtration, etc. ,5,6,7,8.aterials ,29,30,31aterials ,33,34. Paterials ,33,34,35aterials . PVP is aterials ,33,34,36aterials ,29,30,31aterials  have stunofibers ,28, the nofibers , the consolution ,28. For  polymer  discover2, 99.5%) were purchased from Chemcraft, Kaliningrad, Russia. Glass substrates were purchased from Medtechnika 7, Saint-Petersburg, Russia.Polyvinylpyridine  and anhydrous dimethylformamide  were purchased from Sigma-Aldrich, Germany. Cesium iodide  and lead (II) iodide  were dissolved in the as-prepared polymer solution. The ratio of PVP to perovskite was 15%. The mixed solution was stirred for 60 min at 600 rpm to acquire a homogeneous solution. Solutions were made at a temperature of 27 °C and humidity of 21%. First, a 10% PVP solution was prepared by dissolving the polymer in DMF and stirring magnetically for 5 min at a speed of 300 rpm. Perovskite precursors , Chemat Technology Inc., at 6000 rpm for 60 s. Glass substrates measuring 25 mm × 25 mm × 1.2 mm (L × W × H) in size were used.Substrates were cleaned in an ultrasonic bath UZV7/100-TH  in water, acetone, and isopropanol for 25 min. each, then dried in an oven for 2 h at 200 °C . The appearance, average diameter, and orientation of nanofibers were studied using images from a scanning electron microscope (SEM) (MIRA3 TESCAN) and a microscope (Olympus STM6). The appearance of nanofibers was described using SEM images. The average diameter was calculated as the arithmetic mean of 400 nanofibers from 8 spots . The avePerovskite crystallinity was described by X-ray diffraction (XRD) patterns, which were recorded by a DRON-8 X-ray setup in a slit configuration with a BSV-29 sharp-focus tube with a copper anode, a NaI (Tl) scintillation detector, and a β-filter (Ni).The optical properties of nanofibers have been studied by recording light absorption and photoluminescence (PL) spectra in three different points of every sample. Light absorption spectra were obtained by a setup spectrometer consisting of a photonic multichannel analyzer PMA-12 (Hamamatsu) and an integrating sphere Everfine, 0.5 m, with a multi-photometer Photo-2000Z. PL spectra were recorded on an Agilent Cary Eclipse spectrofluorometer.2 with a flow rate of 50 mL/min. The temperature range was 25–475 °C at a growth rate of 10 K/min. DSC was carried out by DSC 204, NETZSCH in N2 with a flow rate of 50 mL/min. The temperature range was 25–350 °C at a growth rate of 10 K/min. For each sample, the average results of three tests were taken.The thermal behavior of the polymer and perovskite has been studied by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). TGA was carried out by TG 209 F1 Libra, NETZSCH in NIn this case, 10 samples of 10 × 1 cm (L × W) in size were cut in both the longitudinal (LD) and transverse (TD) directions of the drum. The shrinkage ratio was calculated as ΔL/L0 (%), where ΔL the change in the length after annealing, L0 is the initial length before annealing. Next, the average value was determined.The mechanical properties were evaluated using an Instron 5943 machine. The samples were conditioned in the laboratory environment for 24 h before being tested under the same conditions. The samples were 10 cm × 1 cm (L × W) in size. The speed test was 1 mm/min. The average value was calculated for five samples in each direction.The properties of electrospun nanofibers are known to be structure dependent. Without changing the concentration of the electrospinning solution, the structure of nanofibers can be manipulated by electrospinning setup parameters  and the process time. Electrospinning setup parameters lead to a change in the average diameter of nanofibers and their orientation. The process time results in an increase in the thickness of nanofibers on the substrate. Here, the relationship between, on the one hand, the previous parameters and the structure of nanofibers, and, on the other hand, between the structure of nanofibers and its properties will be studied.3 perovskite nanocrystals was developed  after electrospinning, which, after annealing, turns into polymer-perovskite nanocrystals composite . XRD hasrovskite ,33,34. Trovskite ,35,38,39rovskite . Under trovskite ,41,42,43rovskite . In the As the perovskite crystallinity is only affected by the electrical voltage and the thickness of nanofibers, the influence of the previous parameters on the optical properties of nanofibers has been studied by recording light absorption and PL spectra. The influence of electrospinning setup parameters on the thermal behavior of nanofibers has been studied by TGA and DSC. g of PVP in composite nanofibers is lower than that of the pure polymer. The distribution of perovskite into polymer nanofibers leads to an increase in the free volume not occupied by polymer chains. Thus, PVP chains can slide and move more easily at lower temperatures. In addition, a decrease in Tg can be noticed at a rotational speed of 500–750 rpm. This may be due to a change in the perovskite distribution into the polymer nanofibers or/and to a change in the interaction between the perovskite and the polymer, resulting in an increase in the free volume. Endothermic peaks observed around 323 °C confirm the formation of high-symmetry α-CsPbI3 [3 can be observed by manipulating the electrical voltage or the rotation speed of the collector. By comparing the results of XRD, light absorption, PL, and DSC, we can conclude that with small changes in the crystallinity of perovskite caused by manipulations in the structure of nanofibers, DSC may not be effective enough to accurately determine these changes. This could be attributed to various reasons:-DSC requires a specific preparation process for the samples to be placed in the crucible, which can potentially alter the structure of the tested nanofibers and affect the accuracy of the results. In contrast, XRD, light absorption, and PL methods do not require special preparation of the samples or even handling them. They are tested directly on the substrate. Therefore, the structure of the nanofibers is well preserved. So, DSC will be more useful for detecting changes in the perovskite crystallinity when the content of polymer or perovskite is altered; -2 (as in DSC) or air  and the annealing program  or a gradual increase in the temperature from room temperature (as in DSC technique)) [It should also be noted that the crystallinity of perovskite can be affected by various factors such as the conditions under which it is annealed ) ,33,34,45α-CsPbI3 ,47,48,49g of the polymer results in a higher increase in shrinkage due to the increased mobility of polymer chains. Thus, a decrease in Tg leads to greater shrinkage [g of the polymer) could lead to the formation of fiber–fiber fusions between overlapped fibers. After cooling, these fusions could form fiber–fiber bonding, increasing the mechanical properties of nanofibers [Nanofibers have been fabricated using planar and rotary collectors to study their mechanical properties. The effect of the electrical voltage on mechanical properties was studied by fabricating nanofibers with a rotary collector in order to be able to evaluate the mechanical parameters in both directions LD and TD. The sample is numbered 7  has resulted in an increase in perovskite crystallinity and light absorption properties. This can be attributed to the presence of mutual effects between nanofibers and possibly the formation of some crystals on the combined surfaces between nanofibers. The bandgap value is strongly related to the porous structure between nanofibers. The thermal analysis has shown that perovskite nanocrystals are formed at a temperature of 127–129 °C, and their size gradually increases with increasing temperature up to 322–324 °C. The glass transition of the polymer happens at a temperature of 172–175 °C. Composite nanofibers showed a weight loss of 1–2% at a temperature of 200 °C. Increasing the rotation speed of the collector to 750 rpm has led to an increase in the average placement angle of nanofibers from 76° to 90°, resulting in an increase in the mechanical properties and shrinkage ratio. The main mechanism that determines the shrinkage during annealing has been revealed. The relationship between the rotation speed of the drum, the glass transition temperature of the polymer, and the shrinkage of nanofibers was described. An increase in the rotation speed in the range of 500–750 rpm led to an increase in the stress in the nanofibers and a decrease in the glass transition temperature of the polymer, resulting in a higher shrinkage ratio. However, the highest mechanical properties have been registered at a rotational speed of 500–750 rpm. Mechanical properties provide the opportunity to analyze the behavior and performance of flexible optoelectronic devices under mechanical stress. The results of this work contribute to the optimal use of electrospun nanofibers for flexible optoelectronic devices. In addition, this paper provides some illustrations that may help to better understand the relationship between the polymer and perovskite in their composites and their behavior during processing and application.


Mycobacterium bovis. This study investigated farm management factors associated with recurrent bTB herd breakdowns (n = 2935) disclosed in the period 23 May 2016 to 21 May 2018 and is a follow-up to our 2020 paper which looked at long duration bTB herd breakdowns. A case control study design was used to construct an explanatory set of farm-level management factors associated with recurrent bTB herd breakdowns. In Northern Ireland, a Department of Agriculture Environment and Rural Affairs (DAERA) Veterinarian investigates bTB herd breakdowns using standardised guidelines to allocate a disease source. In this study, source was strongly linked to carryover of infection, suggesting that the diagnostic tests had failed to clear herd infection during the breakdown period. Other results from this study associated with recurrent bTB herd breakdowns were herd size and type (dairy herds 43% of cases), with both these variables intrinsically linked. Other associated risk factors were time of application of slurry, badger access to silage clamps, badger setts in the locality, cattle grazing silage fields immediately post-harvest, number of parcels of land the farmer associated with bTB, number of land parcels used for grazing and region of the country.Bovine tuberculosis (bTB) is a chronic, infectious and zoonotic disease of domestic and wild animals caused mainly by  Mycobacterium bovis is a zoonotic disease primarily affecting animals. Although cattle are the main hosts, the disease has been reported in many other farmed and wild animals [Bovine tuberculosis (bTB) caused by  animals . A key e animals  publishe animals , 4 and a animals , 4 on th animals –8.et al. (2016) used data from a national database APHIS  to inform definitions for prolonged and recurrent bTB breakdowns in chronic herds [Doyle ic herds , 9. The ic herds . It alsoic herds . This prBreakdown recurrence in Northern Ireland at the 6 monthly and yearly post-outbreak herd tests measured as a percentage of the tests carried out in the previous year was 11% and 9%, respectively, which mimics that for the herd tests carried out at derestriction , at 11% (DAERA unpublished data); its percentage only being surpassed by herd tests carried out at the beginning and during bTB herd breakdown events. Disclosures of bTB infection at the 6-month post-outbreak herd tests are demoralising for farmers and veterinarians, which are interpreted as a previously wasted effort and are viewed as simply returning the farm business to the beginning of a protracted disease control process. This also crucially erodes confidence in the integrity of the whole bTB eradication programme. In Ireland, bTB recurrence occurs when there are further bTB restrictions in the same herd within a specific time-frame resulting from either residual undetected infection in cattle, residual infection due to contamination of fomites (such as slurry), contiguous inter-herd spread or reinfection from local wildlife, however of the sources listed residual infection is thought to lead to recurrence more quickly  or bTB hThe objective of this study was to use a case control study design to identify farm-level management factors associated with recurrent bTB herd breakdowns, using data collected during on-farm epidemiological investigations.A case control study was conducted on a study population consisting of all bTB herd breakdown investigations during the period 23 May 2016 to 21 May 2018. The methodology and model framework applied to this study were as previously applied , 18 but Cases were bTB herd breakdowns of less than a year in duration followed by at least two further bTB herd breakdowns within the following 2 years . A disea1 and Stata/SE 15P ≤ 0.05 level.Microsoft Access™  and R Version 3.4.0P ≤ 0.25 were then analysed using a multivariable logistic regression model. The resultant model was further refined to produce a reduced multivariable logistic regression model, which utilised variables with P ≤ 0.05 from the first multivariable model. Following the fit of the reduced multivariable model, its estimated coefficients were compared to those in the initial multivariable model to determine if there was a magnitude change of >20%. This magnitude change known as Initial analysis was by univariable logistic regression. Any variables containing low numbers (<10) of cases at any factor level, which could not be logically merged with another factor level were removed after univariable analysis. Remaining variables with he model . VariablP > 0.25 in the initial univariable analysis were also individually added back in, to determine if they contributed to the multivariable model thus producing the preliminary main effects model. The only continuous variable included in the preliminary main effects model was herd size. Fractional polynomial analysis [P  ≤ 0.05); however, only those with probable biological significance were accepted as potential candidates for the model. Interaction terms accepted into the final model had an odds ratio (OR) calculated as a linear combination with their associated main effects (β0 + β1 + β2 + β1β2). The finalised model was then subjected to the Hosmer–Lemeshow goodness-of-fit test (decile sub-grouped) to determine its fit to the data. Furthermore, variables with analysis  was appln = 1924) or one breakdown (n = 819). The results from each stage of the model building process from univariable analysis through to the final multivariable model are detailed in A total of 2935 bTB herd breakdown investigations (192 cases and 2743 controls) were completed during the 2-year study period . These 2935 investigations were carried out in a total of 2730 individual Northern Ireland cattle herds as some herds had more than one investigation during the study period due to repeat herd breakdowns. Cases included in this work had a median duration of 164.5 days (interquartile range (IQR): 137.25–213), disclosed median number of SICCT reactors of 3 (IQR: 1–5) and a median time to previous herd breakdown of 169 days (IQR: 135.5–212). Controls were confirmed bTB breakdowns where there was no breakdown in the previous 2 years (P  ≤ 0.05 level. In terms of farm enterprise type dairy enterprises (reference category) formed 43% of cases. The potential for badgers to access silage clamps was found to be statistically significant  1.137–2.775) while the application of slurry/manure to farmland mostly in the spring time as opposed to continuously over the grazing season was protective . The ORs of a case in relation to geographical/other factors  are shown in Along with the main effects, addition of two interaction terms, ‘finding a dead badger on your farm in the past three years’ × ‘herd size transformed’ and ‘total parcels of land grazed’ × ‘herd size transformed’ improved the model. A third interaction term, ‘finding a dead badger on your farm in the past three years’ × ‘slurry/manure application mostly in the spring rather than continuously over the grazing season’, was not included as it was deemed biologically implausible. The results of the linear combination of the two interaction terms included in the model and their associated main effects . In terms of recurrent cases, 43.2% were dairy farm enterprises which is a large representation given that approximately 10% of Northern Ireland farming enterprises are defined as dairy [vs. 15 cows, respectively [In contrast to the DAERA Veterinarians who pointed to an unambiguous source of infection for recurrent breakdowns, farmers' responses returned associations to a much wider spectrum of management factors. Type of farming enterprise was a significant variable throughout the model building process. Dairy farming enterprise was taken as the baseline category and relative to it, none of the other levels showed a significant difference . Howeveectively , 27.vs. non-dairy) [In addition to the direct risk from herd size there has been a move towards intensification in Britain and Ireland with a trend towards larger farms and decline in absolute numbers , probabln-dairy) .M. bovis in faecal samples from bTB infected cattle was extremely low [As already noted  the recumely low .In terms of contiguous spread, recent upgrading of boundary fences was significant. However, the interpretation is unclear as it suggested that partial upgrade of boundary fences leads to an increase in recurrence while full upgrade was not a statistically significant factor level. One explanation might be that farmers suffering recurrent breakdowns were upgrading boundary fences as a reactive biosecurity measure, which was ineffective as, in many cases, the bTB source was carry-over of infection in their own herd. Another significant variable which could be related to both boundary fencing and wildlife in the locality was a farmer highlighting specific land parcels they associated with bTB in their cattle. This variable however has limitations in terms of its interpretation as such land parcels could be adjacent to herds at high risk of bTB breakdowns or favourable to wildlife/badger habitats, which if infected could act as a source for recurrent bTB herd breakdowns.Two other significant variables related to badgers were access to silage clamps and the presence of badger setts in the locality but not on the investigation farm. However, similar to the previous study , resultsGrazing cattle on fields after silage removal was also significantly related to the cases. This variable could be a proxy for farms with more intensive production, also allowing more exposure to contiguous herds . HoweverAs with our previous study , this stP < 0.05 for variable selection, there is a possibility that at least one spurious variable has been included. It is also possible with a study design where DAERA Veterinarian's select a breakdown source of infection there is potential for a degree of subjectivity. However, the DAERA Veterinarian with their local knowledge, training and standardised guidance is best placed to make these assessments.In this study the final multivariable model contained 13 variables and given the use of a statistical cut-off level of One of the most important tasks a DAERA Veterinarian performs is investigating bTB herd breakdowns where, after careful consideration using standardised guidelines, they allocate a disease source. In the situation where there was a high-frequency bTB herd breakdown pattern , this study showed that DAERA Veterinarians strongly associated the infection source to be carry-over of infection, indicating that the diagnostic tests have failed to clear herd infection in the time allocated to the restricted period for these cases. In terms of management factors associated with recurrent bTB herd breakdowns, the size of the herd and location in north-eastern Northern Ireland were significant, along with indicators of increased production intensity such as extended slurry spreading on grassland and after-grazing of silage fields. Herd type was a very strong contender variable in the final model, with dairy herds making up 43% of cases; however, factor level results did not show them to be statistically more at risk than other herd types. Even though badgers were not highlighted as a significant source of infection in this study, it cannot be ignored that so little effort is applied to create a biosecurity barrier between their setts/latrines and grazing cattle.


Molecular “cartoons,” such as pathway diagrams, provide a visual summary of biomedical research results and hypotheses. Their ubiquitous appearance within the literature indicates their universal application in mechanistic communication. A recent survey of pathway diagrams identified 64,643 pathway figures published between 1995 and 2019 with 1,112,551 mentions of 13,464 unique human genes participating in a wide variety of biological processes. Researchers generally create these diagrams using generic diagram editing software that does not itself embody any biomedical knowledge. Biomedical knowledge graphs (KGs) integrate and represent knowledge in a semantically consistent way, systematically capturing biomedical knowledge similar to that in molecular cartoons. KGs have the potential to provide context and precise details useful in drawing such figures. However, KGs cannot generally be translated directly into figures. They include substantial material irrelevant to the scientific point of a given figure and are often more detailed than is appropriate. How could KGs be used to facilitate the creation of molecular diagrams? Here we present a new approach towards cartoon image creation that utilizes the semantic structure of knowledge graphs to aid the production of molecular diagrams. We introduce a set of “semantic graphical actions” that select and transform the relational information between heterogeneous entities  in a KG to produce diagram schematics that meet the scientific communication needs of the user. These semantic actions search, select, filter, transform, group, arrange, connect and extract relevant subgraphs from KGs based on meaning in biological terms, e.g., a protein upstream of a target in a pathway. To demonstrate the utility of this approach, we show how semantic graphical actions on KGs could have been used to produce three existing pathway diagrams in diverse biomedical domains: Down Syndrome, COVID-19, and neuroinflammation. Our focus is on recapitulating the semantic content of the figures, not the layout, glyphs, or other aesthetic aspects. Our results suggest that the use of KGs and semantic graphical actions to produce biomedical diagrams will reduce the effort required and improve the quality of this visual form of scientific communication. Pathway diagrams and other molecular “cartoons” are widely used to summarize and communicate biomedical research results and hypotheses. Their nearly universal appearance in molecular biomedical publications clearly indicates the critical role they play in contemporary science. A recent survey of pathway diagrams (a subset of molecular cartoons) identified 64,643 pathway figures published between 1995 and 2019 with 1,112,551 mentions (denotations) of 13,464 unique human genes participating in a wide variety of biological processes . While mKnowledge graphs (KGs) are structured data models that computationally represent knowledge in the form of interlinked entities—objects, events, situations or abstract concepts, and the relationships between them. All entities and relationships in a knowledge graph are grounded to a formal meaning representation, generally a computational ontology such as the Gene Ontology . The GooIn principle, the rich information available in biomedical KGs could be used to support the creation of molecular cartoons. However, the communicative intent of a molecular cartoon differs significantly from the more detailed and exhaustive design required for computationally effective KGs. Direct visualization of the contents of such knowledge-bases is of modest utility; the information contained is both too detailed and too generic for the communication needs that molecular diagrams serve. Here, we characterize the differences between molecular cartoons and KGs, and present several novel computational approaches for using knowledge graphs to both identify appropriate information within a KG, and transform it into depictions that are effective visualizations. We demonstrate the utility of these techniques through a set of case studies reverse engineering multiple, diverse, peer-reviewed molecular cartoons using KGs. The techniques we describe form the basis of a new paradigm for producing molecular diagrams, making scientific communication faster and easier to produce, and potentially of higher quality.semantic graphical actions, that allow for the complex and abundant representation of information in a knowledge graph to be transformed into a useful diagram over a given domain. These semantic graphical actions search, select, filter, transform, group, arrange, connect, extract, and augment relevant subgraphs from KGs. Our focus is on the semantic content of the figures, not the layout, glyphs, or aesthetic aspects. Our case studies are rooted in three molecular cartoons from published pathway figures pertaining to Down Syndrome, COVID-19, and microbiome-mediated gut-brain connections (Our methodology introduces the idea of nections .indexing into the graph). Next, shortest paths are found between each source and target node pair using a breadth-first search algorithm with unweighted edges; note that there are generally many paths with the same length between a pair of nodes. These paths are then filtered or prioritized by various potential semantic graphical actions to produce a schematic of a final figure.We developed a pipeline to systematically index, search with semantic constraints, and visualize a given molecular pathway using both the PheKnowLator and the KG-COVID19 knowledge graphs . The purWe describe four semantic graphical actions which can be used to prioritize among paths between a given pair of nodes. The first is maximization of cosine similarity between intermediate nodes and the target (referred to as CS). The score for each path is calculated by summing the cosine similarities between the target node vector embedding and the vector of each other node along the path. The higher this score, the closer all nodes on the path are to the target. Vector embeddings are generated using Node2Vec    Eq. 1),,Eq. 1), For example, noting that a node has relation “only_in_taxon” to “Homo sapiens” is unlikely to be interesting, “Homo sapien” is a very higher degree node (connected to many other nodes). This action can therefore draw upon paths that describe molecular interactions as opposed to simple, known facts.Another semantic action, used in combination with the first two, are explicit requirements or prohibitions of edge classes on a path. As required or excluded edges may eliminate all paths between a pair of nodes in some cases, it can also be implemented as a soft constraint that is ignored when no paths satisfying it can be found. This action is paired with either the CS or PDP ranking algorithm for subgraph construction (referred to as EE).The final semantic action is to augment this KG with a semantically constrained nearest neighbor search. This search identifies immediate neighbors of extracted nodes and filters to only include those of specified ontological category . The result introduces information contained in the KG beyond the subgraph originally indexed, adding other relevant concepts and relationships. For example, the identification of drugs that affect proteins in a molecular pathway.The inputs to the pipeline are a file describing the desired nodes and some target connections for the cartoon and the KG . An interactive indexing interaction helps a user index to the specific intended nodes in the KG . Another challenge is integrating new findings into the resulting cartoons. For example, identifying the nearest neighbors of drug ontologies in the COVID-19 subgraphs added 113 potential treatments of which only 4 were included in the published figure. Those 4, along with the others in the published figure, have shown efficacy in clinical trials and this information has not yet been included in KG-COVID19.This work also illustrated some open challenges in generating molecular cartoons from KGs. For example, how to manage directionality in KGs and cartoons. Cartoons often indicate directionality and positive or negative influences indexing was the differential mappings for multiprotein families like AKT between the cartoon and KG. Using standardized entity linking resources . In thiNot all open challenges indicated by this work are semantic. The performance of the algorithms may be improved by filtering the neighbor list during the shortest path search, rather than generating all shortest paths and later prioritizing paths. The current visualization uses Cytoscape, which is a software that creates graphical representations of a network object consisting of nodes and edges. There is an extensive ecosystem of plugins in Cytoscape to curate an affordance for scientific communication. Our approach builds upon and adds to this ecosystem through the application of the semantic schema in KGs to the creation of figures. Future versions of the method will employ glyphs to facilitate the communication of biological concepts.These case studies demonstrate the potential of KGs and semantic graphical actions to provide innovative new functionality for molecular cartooning. Knowledge-based and semantic tools for cartoon construction could reduce the difficulty of creating high quality scientific communication, and are ripe for future development.


Control of bovine tuberculosis (bTB) is a priority for animal health, biosecurity, and human health authorities in Fiji as evident from the long-term funding of the Bovine Brucellosis and Tuberculosis Eradication and Control program (BTEC) and notable improvements to the program described in this paper. To evaluate the performance of the Fiji BTEC program from 2015 to 2020, all available bTB data for cattle were analyzed. Data sources included BTEC bTB testing records, abattoir records and laboratory records. We integrated all information to quantify the bTB tests applied, bTB positive farms and animals, meat inspection and laboratory findings. Test coverage was highest among dairy cattle in Central Division (~73%), where bTB was highly prevalent with 7.8% of dairy cattle and 61.7% of dairy farms found to be positive between 2015 and 2020. There was no visible downward trend in the apparent prevalence of bTB over the 6-year period. During 2019 and 2020, only 21.3% (51/239) of the tested dairy farms maintained their clear status, another 8.4% (20/239) reverted to infected status after 1 year or more of being bTB clear, and most farms remained infected during these 2 years. Factors observed to be contributing to this situation were persistent infections, related in part to the significant number of untested animals, uncontrolled animal movements, and larger farm size. Similar to other developing countries, bTB remains a serious concern and further strengthening of the program targeting the main contributors to bTB persistence, along with maintenance of a comprehensive reporting and traceability system, industry awareness and government support are needed. Control of bTB in Fiji is a long-term objective that must have multiple stakeholder engagement and regular review to measure success. Mycobacterium bovis and is a chronic disease that constitutes a significant economic burden to cattle production industries (https://www.cabi.org/isc/datasheet/91739). It is transmitted between cattle and people, and cattle and wildlife making control a substantial challenge for public health and animal health systems (M. bovis) has been achieved by few countries, notably Australia  is primarily caused by dustries , 2. bTB  systems . Program systems . In highustralia . In term and USA , 11. In  and USA –14.Globally there is a renewed focus on control of bTB because it is linked to the World Health Organization (WHO) strategy to end the human tuberculosis (TB) epidemic with targets for large reductions in TB incidence, TB-related deaths and household costs by 2030. People at risk of zoonotic TB are often members of neglected populations deserving greater attention. Thus, a Roadmap for Zoonotic TB was endorsed by WHO's Strategic and Technical Advisory Group for TB in 2016 .In Fiji, the need to consider zoonotic TB in the national TB program is reflected in the designation of bTB as a One Health challenge by the Fiji Ministry of Agriculture (MOA) and the Ministry of Health and Medical Services (MHMS) . The recIn fact, control of bTB has been a priority for the Government of Fiji for more than two decades, evidenced by the consistent annual funding of the Bovine Brucellosis and Tuberculosis Eradication and Control program (BTEC). Cattle are highly valued in this Pacific-island nation, which despite limited farmland  raises a total of 119,691 cattle  under extensive grazing management according to the 2020 Fiji Agricultural Census (2020 FAC) . This isThe key activities of the Fiji BTEC program over time have been mandatory on-farm bTB testing using the single intradermal test (SID) with unique individual identification of tested animals, data recording and analysis; removal of infected cattle from farms with compensation; laboratory and abattoir surveillance; and cattle movement control to reduce and prevent infection spread from infected farms. However, a retrospective investigation of BTEC bTB data drawn from hard-copy records for the 16 years from 1999 to 2014 demonstrated that despite sustained funding of the BTEC program, disease reduction and containment was not being achieved  and led to concern about the limited replacement stock being available in Fiji. Furthermore, as the prevalence of animals with generalized and gross bTB lesions in infected farms decreased, a greater proportion of culled reactors had no-visible lesions (NVL) on post-mortem inspection at the abattoir. All this raised questions about the sustainability of using the SID testing protocol . As a reBovibase, to record farm details on location, production, livestock, farm testing and cattle movement, with the intention of data sharing using Bovibase as a national recording system for the cattle industry. Bovibase was launched in 2019 during a 2nd BTEC forum, enabling the recording of on-farm testing, infectious status, and meat inspection results for bTB reactor animals. Functionally, BTEC staff were able to access information about farms that were due for bTB testing, thereby aligning testing dates and reading dates, and prioritizing infected farms. Furthermore, data for animals with an incomplete bTB test and missing animals were recorded.Under the new strategy, BTEC information was intentionally incorporated into the design of a new cloud data platform This paper presents a detailed collation and analysis of BTEC bTB records from 2015–2020, providing estimates of the coverage of the BTEC program and of the level of bTB infection in cattle herds in Fiji. Furthermore, it provides evidence of factors that are contributing to the transmission and maintenance of bTB in dairy cattle and guidance for updating the BTEC strategy based on the progress that has been made to date. These findings extend beyond the previous retrospective study for 1999–2014 and demonstrate the need for continued investment and collaboration between the government and the cattle industry in Fiji to achieve sustained control of bTB.M. bovis (PPD-B) injected intradermally into the caudal skin fold of the tail (CFT), with the result read 3 days after administration. Over the 6-years, it was deemed to be a positive test if the animal had developed a wheal of any size or redness at the injection site, following the OIE recommendation for detection of reactors in known infected farms  to conduct data analysis was received in March 2020. Data preparation and analysis were conducted from November 2020 to October 2021. The diagnostic test used throughout the 6-year period was the SID test using purified protein derivative tuberculin antigen for The determination of clear or infected status was based on the following criteria: farms with reactors determined by the SID were classified as “Infected”; and for an infected farm to be determined as “Clear” from bTB, it was necessary for it to have 3 consecutive negative SID tests at a minimum of 3-month intervals to obtain “Restricted', “Provisionally clear,” and then “Clear” statuses, respectively. Upgrading to a Clear status also relied on the compliance of the farmer to present all cattle older than 3 months of age for complete bTB testing  .Several types of data were collated and used in this study . Due to ® Excel® for Microsoft 365 MSO (16.0.14326.20908)) and R (version 4.0.4) to remove duplicate records and correct or delete errors after comparison against hard copies. Verification was performed by cross-checking against the unique identity farm (see below).Soft copies of BTEC bTB test data from 2015 to 2017 encoded in Microsoft Excel were provided by the MOA. For 2018, a soft copy of some data was provided, and the remainder was subsequently encoded in MS Excel by BTEC personnel. The data from 2015 to 2018 were systematically checked using MS Excel  and included an additional search of BTEC hard copy records and of soft copy files to exhaust all possible data sources, but no other files were found. Therefore, it was assumed that some 2018 test data records were permanently missing.Bovibase. The data provided by Bovibase were verified by cross-checking against the hard copies and confirmed to be correct.For 2019 and 2020, complete data were downloaded from the BTEC program section of the cloud data platform Bovibase ID) for each farm visited, and for farms that had also been visited previously during 2015 to 2018, the Bovibase ID was added to the earlier records.For farms visited only during 2015 to 2018, farm identification was based on unique farm names linked to the MOA dairy registration number or village, settlement, or town supply descriptions. For farms visited during 2019–2020, those data consistently contained a unique farm identification code  was used to validate location details recorded for each farm.Bovibase ID or farm name. There were 1,280 unique identity farms .In the results section, the term “Unique identity farm” was used to indicate the count of unique All the data compiled and used for further analysis are described in As a national program, BTEC is responsible for investigation of cattle in Central, Western, Northern and Eastern divisions of Fiji . BTEC prFarms of individual farmers, school farms, villages/settlements, government stations, cattle traders and impoundments were classified by type of operation . Dairy refers to an establishment that produced milk for a bulk supplier, town supply or for personal consumption, and/or traded dairy cattle. Beef refers to an establishment that only raised and/or traded cattle for meat production. Holding facilities refers to a site designated for holding impounded straying cattle with and without ear tags for short periods of time, ”.Dairy farms that had registered and paid a license fee to the MOA to officially sell milk to Fiji Dairy Limited (FDL) are referred as licensed dairy farms. These licensed farms must comply with additional regulations related to hygiene and sanitary standards to be authorized to sell milk. BTEC prioritizes the testing of these farms under the BTEC program with the aim to improve national dairy production and the safety of their dairy products. Among these licensed farms, the commercial farms that usually have higher milk production are called bulk suppliers, and Fiji Dairy (PTE) Limited collects the milk from the farm using the company-owned bulk tankers to transport it to the milk processing factory at Nabua, Fiji. For the smaller licensed dairy farms, the collection of milk from the farm in aluminum milk cans is coordinated by the Fiji Cooperative Dairy Company Limited (FCDCL) which operates chilling centers where the milk is stored for cooperative members until collection by Fiji Dairy Limited.Farms were classified based on herd size as commercial, semi-commercial or subsistence. A commercial farm was a farm with more than 40 cattle, a semi-commercial farm was a farm with 16 to 40 cattle and a subsistence farm was a farm with 1 to 15 cattle. Herd size was based on the median number of cattle bTB tested across the visits to a farm. If a farm was tested during 2015 to 2018 only, the median was calculated based on the total cattle tested per test visit during that period. If a farm was tested during 2019 to 2020, when untested animals were not counted, then the median was calculated based on the total cattle tested per test visit over these 2 years.Individual animals were identified by metal ear tags each with a unique number provided by BTEC. In the event that a tag was lost a new tag was provided by the BTEC Program. In the results section, the term “Unique identity animal” was used to indicate the unique metal ear tag animal identification listed on BTEC records.Bovibase, as well as the unique identity animal number of the animals that were not presented on the reading date (recorded as “Not Presented” or “NPD4”). The unique identity animal for animals that were presented by the farmers on Day 4 but that were not presented for testing on Day 1 were also recorded from 2019 (recorded as “Not Tested)”. The term “Untested animal” was used to describe both “Not Presented” and “Not Tested” animals.From 2015 to 2017, bTB SID tests were performed on animals older than 6 months and from 2018 onwards the tests were performed on animals older than 3 months as part of the enhancement of the standard operating procedures (SOP). For 2015 to 2018, it was assumed that all bTB SID test administered on Day 1 were read 3 days after the day of PPD-B tuberculin administration (Day 4). From 2019 the actual reading date was recorded in Bovibase was enhanced to classify unique identity animal numbers into a category called “missing animals” when not presented on Day 1 and Day 4 during a BTEC visit, without record of death, slaughter, or sale. When an animal was recorded as missing during 3 consecutive BTEC tests on a farm it was subsequently removed from the farm total stock.Furthermore, during 2019 and 2020, Bovibase ID, animals not presented on Day 4 for bTB test reading (NPD4), and animals not tested (Not Tested), date of reading, batch number and next test date.The dataset compiled from 2015 to 2018 included: date of test, animal identification number , animal type , animal gender , farm registration number, farm name, farm location  and bTB SID results . From 2019 to 2020, the dataset also included: farm Complete data for the post-mortem inspection of all reactors, issued by the meat inspectors of the MOA regulatory unit, were available only for the FMIB Nasinu abattoir. However, these records were not available for 2015 and 2017, thus Meat Compensation records issued by BTEC were used instead . These rFrom 2017 to 2020, records of the reactors' post-mortem inspection obtained from the FMIB Nasinu abattoir were, the “TB reactors record” from 2017 to 2018, and the “Meat Certificates” from 2018 to 2020 . TB reacThere were two major categories of outcome for bTB reactors recorded by meat inspectors at the abattoir: no visible lesions of bTB (NVL) or visible lesions of bTB  observed at post-mortem inspection. The type of bTB lesion was defined as: focal or generalized. Focal lesions were found in one area either in lungs, head, or lymph nodes such as prescapular lymph node, popliteal lymph node, internal iliac lymph node, mesenteric lymph node or superficial inguinal lymph node. Generalized lesions were recorded when there were very extensive bTB visible lesions in one region of the carcass or visible lesions in multiple locations. Visible lesions in the liver were also classified as generalized bTB due to the high risk of a bTB systemic infection.Granulomatous lesions found in any cattle during post-mortem inspection at the FMIB Nasinu abattoir were sampled for submission to the Fiji Veterinary Pathology Lab (FVPL). If there were multiple masses, up to three masses were sampled. For bTB reactors with no visible bTB lesions, a minimum of three samples from lymph nodes were collected: for example, a sample of the lymph nodes from the head, lung and liver, or any other enlarged lymph node.M. bovis, during 2019 and 2020, only samples from reactor animals with NVL were cultured to properly allocate the use of limited laboratory reagents and consumables, and samples from bTB reactor and non-reactor animals with gross lesions were not cultured. Samples of visible bTB lesions and NVLs were cultured in Löwenstein-Jensen pyruvate agar and Löwenstein-Jensen glycerol agar. Suspensions from culture plate colonies were prepared on glass slides, Ziehl-Neelsen staining was carried out and acid-fast bacilli were confirmed by light microscopy, enabling a presumptively culture positive classification. The FVPL dataset included: bTB culture line number, date of culture, animal identification number, laboratory case number, farm name, abattoir sample type , Löwenstein-Jensen pyruvate agar result, Löwenstein-Jensen glycerol agar result, Ziehl-Neelsen staining result and date read.Records for sampled individual bTB reactors and non-reactors in 2018 and sampled bTB reactors from 2019 and 2020 were obtained from the FVPL. The laboratory records did not distinguish between gross lesions from a bTB reactor or a non-reactor, and it was not possible to verify against any other data source in this study. Since the laboratory was carrying out confirmatory tests for For 2019–2020, animal movements were identified based on a unique identity animal being present on different farms. The following types of movements were studied: animal movements from non-clear farms to a farm in a different province; animal movements from non-clear farms to a farm in the same province; animal movements from clear farms to a farm in a different province; and animal movements from clear farms to a farm in the same province.All data analysis was performed using R (version 4.0.4).Budget allocation for each year of the study was calculated as the sum of monthly estimates of the BTEC program operations that included the bovine tuberculosis and brucellosis testing performed during the same farm visit, from January to December.The number of bTB tests conducted was the sum of the number of tests done each year.BTEC program coverage for 2019 and 2020 was calculated from the number of unique identity animals with a complete bTB test as numerator and the Fiji Agricultural Census 2020 animal count as denominator. Coverage was tabulated by division and by type of farm operation.Annual and 6-year figures on the proportions of infected unique identity farms and animals were calculated at national, divisional and provincial levels, and by type of farm operation. Unique identity farms were used to identify previously and newly enrolled farms in the BTEC program and to calculate their bTB apparent prevalence.Further analysis of dairy farms was done including the detection of bTB by size of dairy farm operation, geographical regions, and the number of bTB test visits. The proportion of licensed dairy farms was investigated using Dairy Inspection department records.The proportion of incomplete on-farm testing outcomes in 2019 and 2020 was calculated according to the number of farms with untested animals across all the farms tested by BTEC. The proportion of incomplete on-farm testing was tabulated by division and by dairy and beef farm operation types.For farms tested in 2019 and 2020, the proportion of missing animals was calculated based on the median number of animals tested and missing per BTEC testing visit during each year.From 2015 to 2017, the unique identity animal number from BTEC Meat Compensation records was used to count the number of reactor animals sent to the abattoir; all duplicated unique identity animal numbers were excluded . From 20The proportion of positive bTB cultures was calculated for reactors with NVL.During 2019 and 2020, unique identity animals that were present on more than one farm were used to calculate the proportion of animal movements from bTB clear and non-clear farms.Bovibase from 2019.The available data on bTB included in this study cover the period 2015 to 2020 and so includes periods during and after implementation of the new SOPs from 2014 and the enhancements to The cumulative budget for BTEC program operations addressing bTB and brucellosis from 2015 to 2020 was 5.06 M USD  with an From 2015 to 2020, a total of 3995 farm visits were conducted by the BTEC program across the 4 Divisions of Fiji. This corresponded to 1,280 unique identity farms, the vast majority of which were tested more than once in this period. On average 172 new farms  were enrolled each year from 2016–2020. On average, 21,560 cattle  from 455 farms  were tested per year during the 6 years of this study .A total of 83,602 individual animals were tested during the 6 years of the study with some of them tested more than once according to the BTEC unique identity animal numbers from 2015 to 2020. This number might underrepresent or overrepresent the total number of individual animals tested before 2018, because during this period a hard copy list of animal ID numbers to be tested was not taken on each farm test visit and no record of lost metal ear tags or replacement ear tag ID numbers was kept. It is also likely that there were missing hard copies of field sheets for 2018.Central and Western Division were visited each year from 2015, while testing commenced in Northern division from 2017 and Eastern division in 2020, so that 2020 was the only year in which all 4 divisions were visited . All proThe majority of animals tested were in Central Division with an average of 17,373 cattle tested per year between 2015 - 2020 . Then, in decreasing order of total number of animals tested were: Western Division  tested in 6 years, Northern Division  tested in 4 years, and the Eastern Division with only 104 animals tested in 2020 .From 2019 to 2020, based on the 2020 FAC, BTEC had a greater average coverage of dairy cattle (30.3%) than beef cattle (19.1%) across Fiji, and a greater average coverage of cattle in the Central Division (59.8%) compared to Western (8.0%), Northern (13.5%) and Eastern Divisions (2.5%) . In CentBefore 2015, holding facilities were not visited by BTEC, but during the study period and with the increased restrictions on movement of untested cattle, farmers reclaiming their cattle from holding facilities requested testing of their animals before return to their farm. The number of bTB tests conducted in holding facilities increased gradually from 2015 to 2019, but in 2020 the number of tests decreased because animals in these facilities were sent directly to the abattoir for slaughter .A total of 5,219 bTB positive tests were identified nationally from 2015 to 2020, corresponding to 5,198 unique identity animals (21 of which had multiple tests) from a total population of 83,602 (6.2%) cattle that were tested . This reOn average, 870 cattle  from 131 farms  tested positive per year during the study. bTB positive cattle were identified in 3 of 4 geographic divisions, and in these 3 divisions testing was performed consistently over 4 or more consecutive years .Between 2015 and 2020, a total of 4,724 unique identity cattle out of a total population of 64,663 (7.3%) that were tested were detected as bTB reactors in Central Division. They came from 336 unique identity farms, which represented 38.3% of the total of 877 unique identity farms that were tested.For Central Division, testing was conducted each year in the 5 provinces  . On averbTB positive cattle were detected consistently in all provinces, but particularly in Tailevu and Naitasiri, which had the most reactors during the study. In Tailevu, on average 554 cattle  from 51 farms  tested positive each year. In Naitasiri, on average 163 cattle  from 45 farms  tested positive to bTB each year. In Serua, on average 45 cattle  from 8 farms  tested positive to bTB each year. In Rewa, on average 27 cattle  from 5 farms  tested positive to bTB each year. In Namosi, on average 4 cattle  from one farm  were bTB positive each year.Between 2015 and 2020, a total of 250 unique identity cattle out of a total population of 14,385 (1.7%) that were tested were detected as bTB reactors in Western Division. They came from 85 unique identity farms, which represented 24.6% of the total of 346 unique identity farms that were tested.For Western Division, testing was conducted from 2015 to 2020 in the 4 provinces  .Between 2017 and 2020, a total of 225 unique identity cattle out of a total population of 5,557 (4.1%) that were tested were detected as bTB reactors in Northern Division. They came from 21 unique identity farms, which represented 42.0% of the total of 50 unique identity farms that were tested.For Northern Division, testing was conducted from 2017 to 2020 across its 3 provinces  .Cattle in the Eastern Division (Lomaiviti province) were tested only in 2020, with no bTB positives detected from among the 140 cattle tested.P < 0.0001). In beef farms, on average 206 cattle  from 49 farms  tested positive to bTB each year ] that were tested were detected as bTB reactors on dairy farms. They came from 234 unique identity dairy farms, which represented 53.8% of the total 435 unique identity farms that were tested.Dairy farms were covered by the BTEC program only in Central Division and Western Division . In CentIn Central Division, between 2015 and 2020, a total of 3,912 unique identity dairy cattle out of a total of 50,237 (7.79%) that were tested were detected as bTB reactors on dairy farms. They came from 209 unique identity dairy farms, which represented 61.65% of the total 339 unique identity farms that were tested. On average, a total of 76 farms  tested positive each year .In Western Division, between 2015 and 2020, a total of 44 unique identity dairy cattle out of a total of 1,985 (2.2%) that were tested were detected as bTB reactors on dairy farms. They came from 25 unique identity dairy farms, which represented 26.0% of the total 96 unique identity farms that were tested. On average, a total of 7 farms  tested positive each year .P < 0.0001) than the other sizes. For Central Division, from 2015 to 2020, on average, 559 cattle  from 41 commercial farms , 63 cattle  from 25 semi-commercial farms , and 33 cattle  from 9 subsistence farms  were detected bTB positive each year during the study  of the tested dairy farms maintained their clear status, another 8.4% (20/239) reverted to infected after one year or more of being bTB clear, and most farms remained infected during these 2 years. Five of the farms that reverted to infected status had untested cattle during the time that they were considered to be clear from bTB.Untested animals were recorded only during 2019 and 2020. Before this, the farmer may have informed the BTEC team of missing animals but there was no way to capture the information. In 2019, the proportion of farms with untested animals in Central Division was higher in Naitasiri and Tailevu provinces with similar distribution across beef and dairy cattle .Missing animals were recorded only during 2019 and 2020. An increase in the number of reported missing animals occurred in 2020 across all farm sizes of operation.In 2019 and 2020, on average, the commercial farm category had the highest number of missing cattle, being 1,729 (21.2%) cattle from 67 (80.5%) farms compared to 895 (30.2%) from 79 (68.1%) semi-commercial farms and 167 (24.7%) from 34 (64.6%) subsistence farms .The highest numbers of reactors with visible lesions were found in 2015 and 2018 when there were 784 and 852 animals, respectively, while the lowest numbers of reactors with visible lesions were found in 2017 and 2020 when there were 250 and 186, respectively. The number of animals with NVL fluctuated considerably between years, ranging from 423 to 760. From 2018 to 2020, there was a decrease in the proportion of reactors with visible lesions compared to NVL .M. bovis. In 2018, 66.2% of the cultures from the NVL reactors were positive, i.e., a lower percentage compared to 2019 and 2020 results. In 2019, 14 bacterial cultures in Löwenstein-Jensen pyruvate were submitted for Mycobacterium species identification by PCR and 92.8% were confirmed to be positive for M. bovis.In 2018, the proportion of bovine tissue samples (visible lesions and NVL) sent to the laboratory that were presumptively positive for M. bovis by culture was 66.7%. In 2019 and 2020, only samples from NVL reactors were sent to Fiji Veterinary Laboratory for culture. The percentage of samples positive for bacterial isolation in Löwenstein-Jensen pyruvate agar was 90% (463/512) in 2019, and 68% (354/520) in 2020 . During In 2019, most of the animals that were moved from non-clear farms to any other farm between different provinces were sent to Central Division. Ninety-five farms in 9 provinces and 3 divisions sent a total of 153 animals to 83 farms in Central Division. Eventually, three of these moved animals were identified to be bTB positive and slaughtered at FMIB Nasinu abattoir in Central Division .In 2020, there was an apparent reduction in such movements with only thirteen farms in 4 provinces and 2 divisions moving a total of 34 animals to 13 farms in Central Division . EventuaIn 2019, of the animals tested in each division, 0.8% of animals in Central Division, 0.8% in Western Division and 0.3% in Northern Division had been moved from non-clear farms in different provinces in the same or a different division. However, in 2020, such movements decreased to 0.2% from Central Division and 0.04% from Western Division .In 2019, most of the animals that were moved from non-clear farms to any other farm in the same province occurred in Central Division. In total, 39 localities moved 263 animals across 102 farms in the Central division. Eventually, fifteen of these moved animals—two that tested positive to bTB before being moved and thirteen that subsequently tested positive to bTB—were sent slaughter at FMIB Nasinu abattoir in Central Division . Of the In Western Division, in total, 6 localities across Ba, Nadroga and Ra provinces moved 7 animals to 6 farms. In Northern Division, in total, 2 localities across Bua and Cakaudrove provinces moved 4 animals to 3 farms .In 2020, animal movements from non-clear farms within a province were registered only in Central Division. In total, 21 localities across Naitasiri and Tailevu provinces, moved 164 animals to 47 farms in Central Division. Of the animal population tested, 1.6% from Naitasiri and 0.4% from Tailevu were moved between non-clear farms. Eventually, four of these moved animals- two that tested positive to bTB before being moved, and two that subsequently tested positive to bTB—were sent slaughter at FMIB Nasinu abattoir in Central Division .In 2019, most of the animals moved from clear farms to any other farm between different provinces were sent to Central Division. Eleven farms in 5 provinces and 2 divisions sent a total of 17 animals to 14 farms in Central Division. None of these animals subsequently tested positive to bTB or were sent to slaughter at FMIB Nasinu abattoir . In 2020In 2019, all the animal movements from clear farms to any other farm in the same province occurred in Central Division. In total, 18 localities moved 59 animals across 33 farms in Central Division. Eventually, 1 animal moved across Naitasiri province, subsequently tested positive to bTB, and was sent for slaughter at FMIB Nasinu abattoir in Central Division . In 2020According to Mussman , in lessM. bovis, but the details will vary from country to country. In Fiji, untested cattle and uncontrolled cattle movements were important contributors, whereas a wildlife reservoir has not yet been identified . The important role of the MOA BTEC and BAF officers must be emphasized; in relation to provision of accurate information on bTB transmission and how regular whole herd testing and reactor removal along with cattle movement restrictions reduces transmission. Alongside the bTB control, the government extension officers need to provide services sought by farmers such as production advice and access to bTB-free replacement stock, and practical assistance to ensure testing of all cattle can occur and reactors be promptly sent to slaughter. For example, lack of on-farm animal handling facilities contributes to incomplete testing and farmers can be encouraged to setup simple holding yards to hold animals for further testing and hold reactors until sent to slaughter. This will assist in lowering the numbers of untested and missing animals. Additionally for compliant farmers with clear farms, it would be beneficial to provide a partial subsidy for fencing to minimize the risk of bTB recurrence posed by straying cattle .To foster trust, respect, and farmer belief in the accuracy of information provided on bTB, MOA and BAF officers need to adhere to the SOPs and at the same time seek to develop a framework for communication with the farming community. It is important to examine the psychosocial factors that might influence the decision of farmers about compliance with the BTEC program. A study in Spain conducted interviews to identify the major themes related to how veterinarians and farmers were affected by their BTEC program. Participants mentioned that some of the weak points of the program were communication flow issues. This combined with the complexity of the bTB epidemiology and gaps in stakeholder knowledge contributed to disbelief and distrust in the bTB control program . A studyBovibase to encourage the usage of its other components by the MOA Regulatory section, BAF, and FDL will allow for more effective monitoring of cattle movement, BTEC testing and herd production improvements in Fiji.The restriction of animal movement from non-clear farms needs to be ensured across Fiji because many such movements were discovered in this study. Continuous cooperation is needed from BAF to decrease animal movements from infected farms and clear farms that still have a high risk for bTB. Consistent and prompt implementation of penalties by BAF for non-compliance is necessary for bTB control in Fiji. Continuous enhancement of Bovibase. However, the implementation of Bovibase has enabled the recording of data on BTEC activities with better identification and follow-up of participating farms and animals, recording of untested and missing animals, and stringent farm bTB status classification. The consistent use of Bovibase will play a critical role in harmonizing data between all cattle industry stakeholders and promote confidence in the information received about farms, geographic areas, and cattle movements  to characterize patterns of cattle movement and quantify the role of high-risk farms. Research objectives should be a topic for a 3rd BTEC Forum discussion with all stakeholders.Developing a dedicated research program funded by the government and the cattle industry in collaboration with international agencies will have the benefit of capacity building while providing important evidence to inform decision making on diagnostic and control protocols, and evaluation of the cost-benefit of implementation of bTB control at industry and farm levels. Immediate activities to enhance diagnostic capability and to address the presence of residual infected cattle, could include collaborations in repeating testing over time using SID, enhancing microbiological culture methods, introducing procedures for cell-mediated immune assays (interferon gamma release assay) to be evaluated alongside the SID test and establishing proficiency for PCR testing in-country at the Fiji Veterinary Laboratory. Moving beyond presumptive  for bTB . MaintenBovibase will play a critical role in harmonizing data. bTB remains a serious concern for the Fiji dairy industry and a public health risk for those that consume unpasteurized milk; there is value in One Health research on zoonotic bTB in high-risk communities, to support the human TB control program in Fiji. Implementation of a control strategy targeting the main contributors to bTB persistence that were identified in this study, along with maintenance of a comprehensive reporting and traceability system, industry awareness, and government support, will enable more farms to achieve and maintain a bTB clear status. Capacity building through scientific research that provides data to underpin policy and procedures in BTEC will be required. As in all countries, control of bTB in Fiji is a long-term objective that must have multiple stakeholder engagement and regular review to measure success.Relative to the stages of control reached in the decades-long bTB control programs in high-income countries, Fiji is in the early part of its control efforts. The program has been progressively improved and protocols have been enhanced based on technical analysis and stakeholder review. Reflecting this early phase, and consistent with the available financial and human resources, testing for bTB did not reach the majority of cattle in Fiji during the study period. Testing revealed that bTB was highly prevalent, with about 8% of dairy cattle and 62% of dairy farms infected. Against a high baseline prevalence of bTB, there was no visible downward trend in bTB apparent prevalence over 6 years. The factors contributing to this situation were objectively determined to be persistent infections, which were partly due to the significant number of untested animals and uncontrolled animal movements, and the particular importance of large farms. Data management was critical in the control program and is still evolving. There was complexity in the datasets that were compiled in this study, the lack of a consistent unique farm identification number and individual animal identification problems being responsible for time lost in correlating field-testing data, abattoir and laboratory data. However, the implementation, enhancement, and consistent use of jenny-ann.toribio@sydney.edu.au.The data analyzed in this study is subject to the following licenses/restrictions. The original contributions presented in the study are included in the article/The proposal for this collaborative research was developed by J-AT, AG, EB, and RW. EB, SS, and VS facilitated collation, encoding and validation of field data and provided local documents for referencing. AG constructed and cleaned the dataset for analysis then performed all analysis and interpretation of results. EB and AR provided information about Bovibase contributing to validation of cleaned data and interpretation of the results. J-AT and RW supervised the analysis, presentation of the results and documentation of the outcomes and findings of the study. All authors contributed to the content and the preparation of this manuscript.The Sydney School of Veterinary Science at the University of Sydney funded costs associated with data collation and validation for this study. J-AT was supported by scholarships from the Secretaría Nacional de Ciencia Tecnología e Innovación (SENACYT) and the Instituto para la Formación y Aprovechamiento de los Recursos Humanos (IFARHU) of the Republic of Panama, and the James Ramage Wright Supplementary Scholarship of the University of Sydney.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.


Tumors of the craniocervical junction (CCJ) are complicated pathologies with high patient mortality or low quality of life. In the pediatric population, these tumors are less prevalent, with various symptomatic presentations that include motor and neurological manifestations. Three of the most common neoplasms at the CCJ in children are meningiomas, schwannomas, and chordomas. In this review, we will characterize the tissue biomarkers, clinical presentation, treatment methods, and surgical outcomes for these pediatric tumors at the CCJ. A comprehensive literature review was used using the PubMed Database. Keywords used were “craniocervical junction”, “pediatric”, “meningiomas”, schwannomas”, and “meningiomas”. Articles that were not related to the CCJ, included only adult cases, and non-English studies were filtered. Our search yielded a total of 11 studies, with a total of 239 pediatric patients with tumors at the CCJ. These studies were broken down as five for meningiomas, one for schwannomas, and eight for chordomas. In conclusion, resection of pediatric neoplasms at the CCJ is challenging due to anatomical limitations and the size of the patient. Within the CCJ, chordomas were the most prevalent tumor type, with schwannomas being the least prevalent. Literature findings indicate that genetic mutations of the NF2 gene associated with neurofibromatosis type II, as well as incomplete tumor resection, are predictors of poor outcomes. Further developments of monoclonal antibody chemotherapy and endoscopic approaches could expand treatment options for aggressive pediatric neoplasms at the skull base. The craniocervical junction (CCJ) is a complex anatomical region composed of bony, neural, vascular, lymphatic, and supportive structures that change over the course of pediatric development. Due to the anatomic complexity and low incidence, treatment of tumors localized to the skull base within the CCJ in pediatric populations pose unique treatment challenges to neurosurgeons and the rest of the care team. Advances in the understanding of pediatric neoplasm growth, imaging detection modalities, and treatment options in this region have led to reductions in postoperative morbidity and mortality. However, treatment of these tumors is still associated with a significant risk of recurrence, postoperative complications, and mortality . The useThe bony structures of the CCJ include the occipital skull base, atlas, and axis. The articulation between the CCJ and the subaxial cervical spine gives rise to complex movement of the head while simultaneously housing and protecting vital neurovascular structures. Postoperative instability of the CCJ increases the risk of catastrophic consequences, such as nerve or arterial compression and noncommunicating hydrocephalus . TherefoNeoplasms that arise within the CCJ originate from osseous structures, soft tissue, and neural tissue . While tPathophysiology and biomarker analysisMeningiomas normally originate from meningothelial cells of the arachnoid mater with corresponding dural attachment and are present anywhere along the exterior brain and ventricular system. Most meningioma mutations are sporadic with associated chromosomal deletions with some associated with genetic predispositions . NeurofiSchwannomas arise from Schwann cells in peripheral nerves and have the highest genetic association with NF2, with vestibular schwannomas being a hallmark of the disease. In NF2 and many sporadic schwannomas, loss-of-function mutations in the NF2 gene on chromosome 22 lead to decreased expression of the protein merlin. In highly proliferative cellular environments, Schwann cell-mediated dephosphorylation of merlin induces cessation of tumor proliferation . A lack Chordomas are benign primary tumors of soft tissue and bone that arise from embryological remnants of the primitive notochord . The mosPediatric meningiomasClinical PresentationMeningiomas account for 37.6% of benign central nervous system tumors and 50% of benign brain tumors . MeningiTreatmentMeningioma surgical treatment should be carefully planned with preoperative pediatric anatomic considerations to avoid serious surgical complications. For example, due to forward displacement of pterion in children, a standard burr hole can penetrate the periorbita rather than the anterior fossa . When apLiterature OutcomesMenezes et al. conducted an analysis of pediatric CCJ tumors. Out of 38 children, 5 had meningiomas, making them the second most prevalent tumor type . In a fuPediatric schwannomasClinical PresentationSchwannomas are the most common benign nerve sheath tumor with an incidence of about one for a population of 100,000 and represent about 5-10% of all intracranial tumors ,34. SchwTreatmentDuring planning of the surgical approach to the region of the CCJ, it is imperative to consider the medulla and spinal cord, the lower cranial and upper spinal nerves, the vertebral arteries with their branches, the veins along with the dural sinuses, and the ligaments and muscles uniting the atlas, axis, and occipital bone ,38. SurgLiterature OutcomesOutcomes describing pediatric CCJ schwannomas are very limited. In the study on CCJ pediatric tumors by Menezes et al., two of the tumors were schwannomas, one localized to the foramen magnum and the other to the C2 spinal nerve . The patPediatric chordomasClinical PresentationChordomas are slow-growing infiltrative tumors that arise from remnants of notochordal origin . These aChordomas have the highest rate of presentation in adults between the ages of 40 and 70, with males being affected twice as much as females, with Caucasians being the most impacted . About 5TreatmentThere are multiple surgical approaches to chordomas found in the CCJ. These include but are not limited to midline transoral, transmaxillary, transmandibular, high anterolateral retropharyngeal, upper cervical lateral, lateral petrous, and subtemporal approaches. More recently, less invasive approaches, such as conventional and expanded endoscopic endonasal approaches have been explored ,47. SkulLiterature OutcomesIn the study by Menezes et al., chordomas were the most prevalent neoplasm at the CCJ with eight cases. Isolated nerve palsy was the most common finding for these chordoma patients. Literature findings demonstrate that chordomas near CCJ anatomical structures such as the clivus or foramen magnum have a varying prevalence. For example, Borba et al. conducted a literature review and found that out of 79 pediatric chordomas, 21 had chordomas affecting the clivus or spheno-occipital region, with 65% having an atypical histological pattern. All these cases had fatal outcomes by the time of their last follow-up . A studyNeoplasms within the craniocervical junction of the skull base are a complex pathology that requires careful prognostic evaluation and management. Surgical resection has proven to be challenging due to anatomical constraints and the size of the patient. Future implementation of robotic or hybrid techniques may be warranted for improving outcomes. Among the meningiomas, schwannomas, and chordomas, the latter have shown the most versatile management methods with incorporation of molecular targets, warranting the investigation of future therapies to include pathophysiological inhibitors. Biomarker analysis should be further established for predictors of poor outcomes and early interventions. Nevertheless, the surgical management of pediatric neoplasms at the CCJ has indicated positive outcomes, prolonging pediatric mortality and preserving the quality of life.