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Birch (Betula sp.) bark is a well-known natural source of betulin (Bet) and betulinic acid (BAc), both of which have several bioactive properties. The evaluation of the extraction performance, relative to these lupane-type triterpenoids, provided by different biosolvents requires the development of a high-resolution and high-sensitivity liquid chromatography-mass spectrometry (LC-MS) approach that is also compatible with challenging extractants such as natural deep eutectic solvents (NADESs). In this work, an LC-MS method was developed and analytically characterized prior to its application for the quantitation of Bet and BAc in birch bark extracts obtained using conventional solvents (methanol and acetone) and biosolvents (limonene and NADESs). High precision (RSD < 3.3%), sensitivity (LOD: 23 ng mL−1 and 29 ng mL−1 for Bet and BAc, respectively), and accuracy (95–102% recovery) were found for this optimized method, using an acidulated water–methanol mixture as the mobile phase and sodium acetate as an additive. Extraction experiments conducted at 55 °C revealed that the NADESs, particularly thymol:1-octanol (1:1 molar ratio), outperformed the other solvents and were highly effective for the recovery of both triterpenoids (17.50 mg g−1 and 0.92 mg g−1 of Bet and BAc, respectively). This method can also be applied to similar extracts obtained from other biomasses.

Please confirm that an ethics committee approval has been applied for or granted: Not relevant (see information at the bottom of this page)Background and AimsAortic-bifemoral bypass is a surgery chosen for patients with Leriche syndrome or severe peripheral arteriopathy. This procedure implies a laparotomy supra and infraumbilical. That translates into a severe pain during postoperative period. Therefore, pain management becomes a key pilar for early recovery. Cardiovascular anesthesiologists usually choose low thoracic epidural to control pain. However, the circumstances of some patients make it a non-feasible technique. In those cases, abdominal wall blocks are a valid alternative reducing pain, morbidity and the length of stay in hospital.MethodsWe expose a case in which a bilateral transverse abdominis block with a single shot technique was performed on a patient who was elected for aortic-bifemoral bypass.ResultsA woman 61 years old is elected for aortic-bifemoral bypass due to Leriche syndrome. In our hospital our gold-standard technique is thoracic epidural at a t10-t11 level. However, in this case she had systemic sclerosis, so we decided to perform a bilateral transverse abdominis block with a posterior approach at the level of Petit´s triangle. We administered levobupivacaine 0,25% with a volume of 40 ml in total. During the first 48 hours in the ICU, she received an elastomeric pump consisting of dexketoprofen, metamizole and ondansetron. She didn’t have irruptive pain either she got any opioid rescue analgesia.Abstract #36493 Figure 1Transverse abdominis plane block with a posterior approach towards the Petit´s triangleConclusionsBilateral transverse abdominis plane block is a valid alternative to thoracic epidural in aortic-bifemoral bypass. Transverse abdominis plane block with a posterior approach can give a sensory block from T7 until L1.

Please confirm that an ethics committee approval has been applied for or granted: Not relevant (see information at the bottom of this page) Application for ESRA Abstract Prizes: I apply as an Anesthesiologist (Aged 35 years old or less)Background and AimsClavicle fractures are a pathology with a relatively low incidence (2-3% of all fractures). Only a percentage of cases require surgical treatment. Among the different anaesthetic approaches, general anaesthesia associated with locoregional techniques is generally the gold standard. Classically, the regional block of choice has been the interscalene block. However, the development of ultrasound-guided peripheral blocks allows more interesting analgesic options, such as the clavipectoral fascia block described by anaesthesiologist Dr Luis Valdés in 2017.MethodsAbout 10 cases of clavicle fractures. Patients aged between 28 and 42 years, ASA I except for one ASA II patient due to type I obesity. All cases were scheduled surgeries for open osteosynthesis for acromioclavicular fracture-dislocation. Balanced general anaesthesia combined with CPB block at the mid-clavicular level along with ultrasound-guided superficial cervical plexus block was performed under standard monitoring and standard premedication.ResultsNo adverse effects or anaesthetic complications were reported. The dose administered was 15 ml bupivacaine 0.5% for CPB and 5 ml bupivacaine 0.5% for the superficial cervical plexus block.There was no evidence of motor block of the operated limb. Immediate postoperative VAS was 0 in all cases and no rescue analgesia was required in the first 24 hours, only the usual multimodal analgesia.Abstract #36477 Figure 1IMAGEN 1ConclusionsCPB associated with superficial cervical plexus block is an effective analgesic alternative for clavicular surgery. It is a safe ultrasound-guided block, which makes it a valid alternative to multimodal intravenous analgesia. Further studies are needed to demonstrate the efficacy, advantages and complications associated with this locoregional technique.

Bovine tuberculosis (bTB) caused by complex (MTC) remains a significant concern for public health. Direct real-time PCR and droplet digital PCR (ddPCR) are proposed as alternative tools to enhance diagnostic precision and efficiency. This study aims to assess the diagnostic performance of a ddPCR assay targeting IS for the detection of MTC DNA in both microbiological culture and fresh lymph node (LN) tissue samples obtained from cattle, in comparison with the established reference standard, the microbiological culture followed by real-time PCR. The fresh LNs (N=100) were collected each from a different cattle carcass at the slaughterhouse. The limit of detection of ddPCR-IS was set to 101 copies per 20 μl reaction. DdPCR-IS detected 44 out of 49 reference-standard positive samples and yielded negative results in 47 out of 51 reference-standard negative samples, resulting in adjusted sensitivity (Se) and specificity (Sp) of 90.76% [95% confidence interval (CI): 82.58 - 98.96%)], and 100% (95% CI: 100%) respectively. The estimated adjusted false negative rate (FNR) was 9.23% (95% CI: 1.04 - 17.42%) and the false positive rate (FPR) was 0% (95% CI: 0%). When directly applied from fresh bovine LN tissues, ddPCR-IS6110 identified 47 out of 49 reference-standard positive samples as ddPCR-IS6110-positive and 42 out of 51 reference-standard negative samples as ddPCR-IS -negative, resulting in adjusted Se and Sp values of 94.80% [95% (CI): 88.52 - 100%] and 100% (95% CI: 100%), respectively. The adjusted FNR was 5.20% (95% CI: 0 - 11.50%) and the FPR was 0% (95% CI: 0%). Noteworthy, ddPCR-IS disclosed as positive 9 samples negative to reference-standard. DdPCR-IS proved to be a rapid, highly sensitive, and specific diagnostic tool as an alternative to reference-standard method.

Carvacrol is a compound present in essential oils with proven antimicrobial activity against numerous pathogens. We firstly determine the post-antibiotic effect (PAE) of carvacrol (1×, 2×, 4× MIC) and post-antibiotic sub-minimum inhibitory concentration (MIC) effect (1× + 0.25× MIC and 2× + 0.25× MIC) for two concentrations of Salmonella Typhimurium ATCC14028 (106 and 108 CFU/mL). Prior to testing, the minimum concentration and exposure time to achieve the bacterial inhibition (MIC 0.6 mg/mL and 10 min) were determined by broth microdilution and time–kill curve methods, respectively. At the MIC, carvacrol did not generate any PAE. At twice the MIC, the PAE was 2 h with the standard inoculum (106 CFU/mL) and 1 h with the high-density inoculum (108 CFU/mL). At 4× MIC concentrations, the PAE was higher in both cases > 43.5 h. Continuous exposure of post-antibiotic phase bacteria (1× and 2× MIC) to carvacrol at 0.25× MIC (0.15 mg/mL) resulted in an increase in PAE (PA-SME) above 43.5 h with both inocula. These results suggest that the PA-SME of carvacrol for S. Typhimurium can be significantly prolonged by increasing the sub-MICs, which would allow dose spacing, reduce adverse effects and improve its efficacy in the treatment of infected animals and as a disinfectant in agri-food facilities.

Tuberculosis-like lesions (TBL) in pigs have been associated with microorganisms other than mycobacteria. In this work a histopathological and microbiological evaluation of TBL in pigs is shown. A total of 352 samples belonging to 171 pigs totally condemned at slaughterhouse due to generalized TBL were sampled and selected for analysis. Pyogranulomatous (56.2%) and granulomatous lesions (20.2%) were observed in all analysed organs. Most of the granulomas observed in both lymph nodes and lungs belonged to more advanced stages of development (stages III and IV) whereas in the liver and the spleen most of lesions belonged to intermediate stages (stages II and III). Different microorganisms were simultaneously detected from TBL in the 42.7% of the animals. Mycobacterium tuberculosis complex (MTC) (38%), coryneform bacteria (40.3%) and streptococci (28.1%) were the main groups of microorganisms detected after bacteriological analysis, with Trueperella pyogenes and Streptococcus suis as the most frequently isolated species. Mycobacteria belonging to MTC were the most frequently detected pathogens in granulomatous and pyogranulomatous lesions in submandibular lymph nodes (32.7%) and coryneform bacteria were the microorganisms more frequently isolated from lungs (25.9%) and spleen samples (37.2%). These results may provide new insights into the pathogenesis and diagnosis of this pathology. The importance of coryneform bacteria and streptococci in such processes must be evaluated in future studies.

Rapid and accurate diagnostic tools, such as Real-Time PCR (qPCR), need to be implemented as a confirmatory test in the framework of bovine tuberculosis (bTB) surveillance and control programs, shortening the turnaround time to confirm bTB infection. The present study aimed to evaluate a direct qPCR from fresh tissue samples targeting the insertion sequence IS 6110 using individually homogenized bovine lymph nodes compared with microbiological culture. Retropharyngeal, tracheobronchial, and mesenteric lymph nodes fresh tissue samples ( n = 687) were collected from 230 different cattle carcasses at the slaughterhouse. Only 23 of the 230 examined animals showed tuberculosis-like lesions, with 62 of 230 considered as positive. Among these 62 animals, 61 resulted as culture-positive, whereas 48 were qPCR-positive. Thus, this qPCR targeting IS 6110 showed an apparent diagnostic sensitivity and specificity values of 77.1% [95% confidence interval (CI): 66.5–87.6%] and 99.4% (95% CI: 98.3–100.6%), respectively, and a positive predictive value of 97.9% (95% CI: 93.9–102.0%) and negative predictive value of 92.3% (95% CI: 88.4–96.2%). Positive and negative likelihood ratios were 130.2 and 0.2, respectively, and the agreement between microbiological culture and this qPCR was almost perfect (κ = 0.82). These results highlight this qPCR targeting IS 6110 as a suitable complementary method to confirm bTB in animals with either tuberculosis-like lesions or non-tuberculosis-like lesions, decreasing the number of samples subjected to microbiological culture and, hence, its overall associated costs and the turnaround time (under 48 h) to confirm bTB infection. Besides, sampling mesenteric lymph node, which is uncommonly sampled, together with tracheobronchial and retropharyngeal ones, is advisable during postmortem inspection in bTB surveillance programs at the slaughterhouse, especially in areas with a low bTB prevalence scenario.

The diagnosis of bovine tuberculosis (bTB) is based on the single intradermal tuberculin test (SIT), interferon gamma, and compulsory slaughter of reactor animals. Culture and PCR from fresh tissue are regarded as gold standard techniques for post-mortem confirmation, with the former being time-consuming and presenting moderate to low sensitivity and the latter presenting promising results. Histopathology has the advantage to identify and categorize lesions in both reactor and non-reactor animals. Therefore, this study aims to highlight the role of histopathology in the systematic diagnosis of bTB to shorten the time to disclose positive animals. Blood (212) and lymph node (681) samples were collected for serological, bacteriological, and histopathological analyses from a total of 230 cattle subjected to the Spanish bTB eradication program. Seventy-one lymph nodes and 59 cattle yielded a positive result to bacteriology, with 59 lymph nodes and 48 cattle presenting a positive result in real-time PCR from fresh tissue. Roughly 19% (40/212) of sera samples gave a positive result to ELISA. Tuberculosis-like lesions (TBLs) were observed in 11.9% (81/681) of the lymph nodes and 30.9% (71/230) of cattle. Noteworthy, TBLs were evidenced in 18 out of 83 SIT − and real-time PCR and bacteriology negative animals, with 11/18 disclosing a positive result to Ziehl-Neelsen technique and two of them to ddPCR from paraffin blocks targeting IS 6110 . Six out of these 11 ZN + corresponded with mesenteric LN and were confirmed positive to paratuberculosis. Histopathology yielded a sensitivity of 91.3% (CI 95 83.2–99.4%) and a specificity of 84.4% (CI 95 78.6–89.3%) with good agreement (κ = 0.626) when compared with real-time PCR. Our results confirm that histopathology allows a rapid confirmation of real-time PCR and bacteriology, emphasizing its contribution to bTB control and monitoring.

Trueperella pyogenes is an opportunistic pathogen, responsible for important infections in pigs and significant economic losses in swine production. To date, there are no available commercial vaccines to control diseases caused by this bacterium. In this work, we performed a comparative proteomic analysis of 15 T. pyogenes clinical isolates, by “shaving” live cells, followed by LC-MS/MS, aiming at the identification of the whole set of surface proteins (i.e., the “pan-surfome”) as a source of antigens to be tested in further studies as putative vaccine candidates, or used in diagnostic tools. A total of 140 surface proteins were detected, comprising 25 cell wall proteins, 10 secreted proteins, 23 lipoproteins and 82 membrane proteins. After describing the “pan-surfome”, the identified proteins were ranked in three different groups based on the following criteria: to be (i) surface-exposed, (ii) highly conserved and (iii) widely distributed among different isolates. Two cell wall proteins, three lipoproteins, four secreted and seven membrane proteins were identified in more than 70% of the studied strains, were highly expressed and highly conserved. These proteins are potential candidates, alone or in combination, to obtain effective vaccines against T. pyogenes or to be used in the diagnosis of this pathogen.

Q Fever is a zoonosis caused by Coxiella burnetii that affects domestic and wild ruminants, leading to reproductive disorders. In humans, the disease can manifest with acute and chronic clinical manifestations. Veterinarians, as healthcare professionals in close contact with animals, serve both as the first line of defence in preventing infection at the animal–human interface and as an important sentinel group for the rapid detection of outbreaks. The aim of this study was to assess the knowledge, perception, and attitude of veterinarians in Southern Spain regarding Q Fever. To this end, an online survey was designed, validated, and conducted among veterinarians in the province of Malaga, with a final participation of 97 individuals, predominantly from the private sector (clinic, livestock, agri-food, etc.). The data obtained reflected a general lack of knowledge about the disease, particularly concerning its epidemiology and infection prevention. Regarding perception and attitude, a significant percentage of respondents stated they did not use protective equipment when handling susceptible animals and only sought information about the disease in response to outbreak declarations. The study emphasised the significance of promoting training in zoonotic diseases during and after graduation, the relevance of official channels in occupational risk prevention, and the utility of epidemiological surveys as a tool to identify and address potential gaps in knowledge related to this disease.