Antimicrobial resistance- and pathogen patterns in the fecal microbiota of sows and their offspring in German commercial pig farms

Reducing antibiotic use is one of the biggest challenges in pig farming, as antibiotics have been used for years to control typical problems such as newborn or post-weaning diarrhea. The pressure a one health approach has created on animal production regarding antimicrobial resistance is an opportunity to find other strategies against enterobacterial pathogens in suckling and weaned piglets. A farm-specific approach could have a good success due to the individual farm structures in Germany and other countries. In this study, non-metric multidimensional scaling, hierarchical clustering, and latent class analysis were used to determine the impact of antibiotic use on antibiotic resistance patterns and pathogen prevalence in 20 German pig farms. This may help to develop individualized health strategies. 802 fresh fecal samples were collected from sows and piglets from 20 piglet production and rearing farms at different production times (sows antepartum and postpartum, suckling piglets, weaned piglets). In addition, the use of antibiotics was recorded. DNA extracts were subjected to quantitative real-time qPCR with primers specific for antibiotic resistance genes (int1, sul1-3, dfrA1, mcr-1, blaCTX-M), and virulence factors of relevant bacteria (C. difficile, C. perfringens, Salmonella, Escherichia/Shigella/Hafnia, E. coli). Linear and logistic regression models were used to analyze the relationship between different antibiotics and the major genes contributing to the clustering of observations for the different animal groups. Clustering revealed different farm clusters for sows, suckling piglets, and weaned piglets, with the most remarkable diversity in antibiotic use among weaned piglets. Amoxicillin, lincomycin, and enrofloxacin were identified as the most probable cause of increased odds of the presence of relevant antibiotic resistance genes (mcr1, dfrA1, blaCTX-M). Still, direct effects of a specific antibiotic on its associated resistance gene were rare. Enrofloxacin and florfenicol favored the occurrence of C. difficile in sows. The E. coli fimbriae genes were less affected by antibiotic use in sows and piglets, but the F4 fimbriae gene could be associated with the integrase 1 gene in piglets. The results confirm that multidrug-resistant enterobacteria are widespread in German pig farms and give awareness of the impact of current antibiotic use while searching for alternative health strategies

GPR35 Activation Reduces Ca2+ Transients and Contributes to the Kynurenic Acid-Dependent Reduction of Synaptic Activity at CA3-CA1 Synapses

Limited information is available on the brain expression and role of GPR35, a Gi/o coupled receptor activated by kynurenic acid (KYNA). In mouse cultured astrocytes, we detected GPR35 transcript using RT-PCR and we found that KYNA (0.1 to 100 µM) decreased forskolin (FRSK)-induced cAMP production (p<0.05). Both CID2745687 (3 µM, CID), a recently described GPR35 antagonist, and GPR35 gene silencing significantly prevented the action of KYNA on FRSK-induced cAMP production. In these cultures, we then evaluated whether GPR35 activation was able to modulate intracellular Ca(2+) concentration ([Ca(2+)]i ) and [Ca(2+)]i fluxes. We found that both KYNA and zaprinast, a phosphodiesterase (PDE) inhibitor and GPR35 agonist, did not modify either basal or peaks of [Ca(2+)]i induced by challenging the cells with ATP (30 µM). However, the [Ca(2+)]i plateau phase following peak was significantly attenuated by these compounds in a store-operated Ca(2+) channel (SOC)-independent manner. The activation of GPR35 by KYNA and zaprinast was also studied at the CA3-CA1 synapse in the rat hippocampus. Evoked excitatory post synaptic currents (eEPSCs) were recorded from CA1 pyramidal neurons in acute brain slices. The action of KYNA on GPR35 was pharmacologically isolated by using NMDA and α7 nicotinic receptor blockers and resulted in a significant reduction of eEPSC amplitude. This effect was prevented in the presence of CID. Moreover, zaprinast reduced eEPSC amplitude in a PDE5- and cGMP-independent mechanism, thus suggesting that glutamatergic transmission in this area is modulated by GPR35. In conclusion, GPR35 is expressed in cultured astrocytes and its activation modulates cAMP production and [Ca(2+)]i. GPR35 activation may contribute to KYNA effects on the previously reported decrease of brain extracellular glutamate levels and reduction of excitatory transmission

Leveraging Accelerometer Data for Lameness Detection in Dairy Cows: A Longitudinal Study of Six Farms in Germany

Lameness in dairy cows poses a significant challenge to improving animal well-being and optimizing economic efficiency in the dairy industry. To address this, employing automated animal surveillance for early lameness detection and prevention through activity sensors proves to be a promising strategy. In this study, we analyzed activity (accelerometer) data and additional cow-individual and farm-related data from a longitudinal study involving 4860 Holstein dairy cows on six farms in Germany during 2015–2016. We designed and investigated various statistical models and chose a logistic regression model with mixed effects capable of detecting lameness with a sensitivity of 77%. Our results demonstrate the potential of automated animal surveillance and hold the promise of significantly improving lameness detection approaches in dairy livestock

Avaliação das propriedades eletrofisiológica de neurônios hipocampais humanos no modelo de 0-Mg2+

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Antimicrobial resistance- and pathogen patterns in the fecal microbiota of sows and their offspring in German commercial pig farms

Reducing antibiotic use is one of the biggest challenges in pig farming, as antibiotics have been used for years to control typical problems such as newborn or post-weaning diarrhea. The pressure a one health approach has created on animal production regarding antimicrobial resistance is an opportunity to find other strategies against enterobacterial pathogens in suckling and weaned piglets. A farm-specific approach could have a good success due to the individual farm structures in Germany and other countries. In this study, non-metric multidimensional scaling, hierarchical clustering, and latent class analysis were used to determine the impact of antibiotic use on antibiotic resistance patterns and pathogen prevalence in 20 German pig farms. This may help to develop individualized health strategies. 802 fresh fecal samples were collected from sows and piglets from 20 piglet production and rearing farms at different production times (sows antepartum and postpartum, suckling piglets, weaned piglets). In addition, the use of antibiotics was recorded. DNA extracts were subjected to quantitative real-time qPCR with primers specific for antibiotic resistance genes (int1, sul1-3, dfrA1, mcr-1, blaCTX-M), and virulence factors of relevant bacteria (C. difficile, C. perfringens, Salmonella, Escherichia/Shigella/Hafnia, E. coli). Linear and logistic regression models were used to analyze the relationship between different antibiotics and the major genes contributing to the clustering of observations for the different animal groups. Clustering revealed different farm clusters for sows, suckling piglets, and weaned piglets, with the most remarkable diversity in antibiotic use among weaned piglets. Amoxicillin, lincomycin, and enrofloxacin were identified as the most probable cause of increased odds of the presence of relevant antibiotic resistance genes (mcr1, dfrA1, blaCTX-M). Still, direct effects of a specific antibiotic on its associated resistance gene were rare. Enrofloxacin and florfenicol favored the occurrence of C. difficile in sows. The E. coli fimbriae genes were less affected by antibiotic use in sows and piglets, but the F4 fimbriae gene could be associated with the integrase 1 gene in piglets. The results confirm that multidrug-resistant enterobacteria are widespread in German pig farms and give awareness of the impact of current antibiotic use while searching for alternative health strategies

Estudo comparativo dos padrões de acomodação dos neurônios hipocampais de ratos e tecido humano

03

ESTABLISHMENT AND CHARACTERIZATION OF A CANCER STEM CELL LINE FROM A RARE BONE SARCOMA

Objective: To establish of a bone cancer stem cell (CSC) line from a rare human bone disease, the high grade osteoblastic osteosarcoma (OS). Material and Methods: Central high grade osteoblastic OS samples were collected at the “Unit Ortopedia Oncologica e Ricostruttiva”, AOUC Careggi, Florence, with informed consent approved by the local Ethical Committee. First, primary human cancer cell cultures of osteoblastic OS have been established. After that, the subpopulations of CSCs have been isolated from these, with the sphere-formation assay. Hence, the cancer stem cell phenotype has been evaluated by several cellular assays/stainings, by flow cytometry analysis and by analyzing the pro-files expressions of a few genes related to CSCs. Results: We have established primary cell cultures of high grade osteoblastic OS from each samples collected. Consequently, from these we have started to isolate CSCs. Nowadays we have established a OS-CSC line, called as OSA5-CSC. The cancer stem cell phenotype from OSA5-CSC line was confirmed by observing the capacity of the OSA5-CSCs to differentiate into osteoblasts and into adipocytes, by showing the positive presence of the MSCs and ofthe ESCs markers into the cell line, and by evaluating a good rate (14 %) as clonogenic capacity. The presence of the ESC markers was confirmed also by their gene expression together with the expression of CD133. On the other hand, the malignant phenotype was verify by demonstrating the presence of the expression of genes involved in invasion/migration process (i.e., AXL and EZR) and in the pluripotency of CSC (i.e., MYC), by the agar soft and bythe ALDH1A1 assays. Conclusions: In conclusion, we have collected a few biopsies of this rare bone disease. We have established primary cell cultures from these. We have completely characterized and studied a OS-CSC line at cellular and molecular level. Nowadays we are concluding the characterization of the OS-CSCs isolated from the relative cell cultures and we are starting the study of micro-RNAs related to their phenotype