Νευροτοξικότητα και ανοσοτοξικότητα εξωπαρασιτοκτόνων

The aims of the present in vitro study were to evaluate the capacity of two widely used pesticides, diazinon-oxon (DZO) and fipronil (FIP), to cause neurotoxicity in mouse N2a neuroblastoma and rat C6 glioma cells and immunotoxicity in human lymphocytic Jurkat and human promyelotic THP-1 cells. Sub-lethal concentrations of DZO and FIP, established by cell viability assays, were used in all experiments to study the mechanisms underlying their possible toxicity. Western blotting analysis revealed that the inhibition of axon-like processes in N2a neuronal cells and inhibition of outgrowth extensions in C6 glioma cells was present only in cells treated with DZO. This was associated with modifications of a variety of cytoskeletal proteins, which play a major role in cell development, growth and maintenance. Densitometric scanning of C6 cell extracts demonstrated that exposure to DZO decreased glial fibrillary acidic protein (GFAP) and tubulin and microtubule associated protein (MAP1B). Densitometric scanning of Western blots of lysates of N2a neurobla-stoma cells revealed that exposure to DZO increased the expression of phosphorylated neurofilament heavy chain (pNFH) and heat shock protein-70 (HSP70), and reduced growth-associated protein-4 (GAP-43). On the other hand, FIP caused severe disruption of the developmentally important ERK 1/2-MAP kinase pathway, characterized by major reductions in MAPK kinase, mainly ERK 1/2, without significant changes in total or phosphorylated NFH. The present study also examined the possible immunotoxic effects of DZO and FIP by interfering with the production of some important cytokines. The study investigated the effects of exposure to DZO and FIP on cytokine levels, gene transcription, and expression of some cell signalling pathways in human lymphocytic Jurkat and human promyelotic THP-1 cells. Jurkat cells exposed to 10 μM DZO significantly enhanced IFN-γ mRNA expression and increased NF-kB protein levels. The MAPK signalling pathway also appears to be affected by DZO in Jurkat cells, as pERK1, tERK1 and tERK2 protein levels were decreased. Significant up-regulation of TNF-α mRNA was observed in THP-1 cells exposed to 1 μM or 5 μM DZO for 6 h and exposure of THP-1 cells to DZO (5 μM) also up-regulated IL-1β mRNA and caused a slight increase in cytokine production. Exposure to DZO caused significant reductions of pERK2 (10 μM) and tERK2 (5 μM) indicating that the MAPK pathway was involved. The present study demonstrated for the first time that FIP has inhibitory effects on the production of IL-2 by Jurkat cells, which was also characterized by downregulation of IL-2 mRNA and by interference with the MAPK pathway. In addition, exposure to FIP caused reduced IFN-γ secretion in Jurkat cells and reduced IL-1β cytokine release and IL-1β mRNA expression in THP-1 cellsSidiropoulou E, Διδακτορική διατριβή (Doctor in Philosophy): Neurotoxicity and immunotoxicity of pesticides, Faculty of Health and Life Sciences, School of Veterinary Science, University of Liverpool (2014). Σκοπός της διατριβής ήταν η διερεύνηση των βιοχημικών φαινομένων και μηχανισμών τοξικότητας δύο ευρείας χρήσης εξωπαρασιτοκτόνων παγκοσμίως, του fipronil, και του μεταβολίτη του diazinon, diazinon-oxon, στο νευρικό και ανοσοποιητικό σύστημα. Τα παρασιτοκτόνα αυτά έχουν την δυνατότητα να προκαλέσουν τοξικώσεις σε ζώα αλλά και στον άνθρωπο.Όσον αφορά το νευρικό σύστημα, χρησιμοποιήθηκαν κυτταρικές σειρές από επίμυ (C6 γλοιωματικά κύτταρα), ποντικό (N2α-νευροβλαστωματικά). Αρχικά, εφαρμόστηκε η φασματοφωτομετρική μέθοδος αναγωγής της ουσίας MTT (cell viability assays) για να προσδιορίσουμε συγκεντρώσεις των παρασιτοκτόνων που δεν προκαλούν κυτταρικό θάνατο (1, 5 και 10μΜ). Στη συνέχεια, εφαρμόστηκε η ανοσοχημική μέθοδος κατά Western (Western blotting analysis) όπου με τη χρήση αντισωμάτων προσδιορίστηκαν τα επίπεδα πρωτεινών που σχετίζονται με την δυναμική του κυτταρικού σκελετού. Περαιτέρω, μελετήθηκε η επίδραση των δύο αυτών ουσιών στον σχηματισμό νευραξονικών προεκβολών (rat C6 glioma cells) ή νευραξόνων (mouse N2a-neuroblastoma cells)(inhibition of axon-like processes), που προσδιορίστηκε ποσοτικά με το μικροσκόπιο. Τέλος, προσδιορίστηκαν τα επίπεδα της ακετυλοχολινεστεράσης -ενός σημαντικού ενζύμου για την λειτουργία του νευρικού συστήματος-, σε όλες τις υπό διερεύνηση συγκεντρώσεις των ουσιών αυτών.Πιο συγκεκριμένα, διερευνήθηκε η επίδραση του βασικού μεταβολίτη του εξωπαρασιτοκτόνου diazinon, diazinon-oxon (DZO), σε νευροβλαστωματικά κύτταρα N2a ποντικού, υπό διαφοροποίηση. Σε συγκεντρώσεις που δεν επηρεάζουν την βιωσιμότητά τους βρέθηκε ότι προκαλείται αναστολή του νευροαξονικών προεκβολών τους μετά από 24 ώρες έκθεση. Εφαρμόζοντας την μέθοδο Western Blotting analysis, προσδιορίστηκε η πυκνότητα πρωτεινών όπως της βαριάς αλύσου (NF-H) των νευρικών νηματίων και βρέθηκε αύξηση της φωσφορυλίωσης τους μόνο στις δύο υψηλότερες δόσεις, συγκριτικά με κύτταρα μάρτυρες. Σε αντίθεση, η πρωτείνη ανάπτυξης του νευράξονα-43 (GAP-43) παρουσίασε σημαντική μείωση σε όλες τις συγκεντρώσεις. Η νευροτοξική δράση αυτή του DZO βρέθηκε οτι δεν σχετίζεται με παράλληλη αναστολή της ακετυλοχολινεστεράσης (AChE).Περαιτέρω, ερευνήθηκε η επίδραση του μεταβολίτη DZO σε κυτταροσκελετικές πρωτείνες οπως η σωληνίνη, η GFAP (glial fibrillary acidic protein) και η κυτταροσκελετική πρωτείνη MAP1B που σχετίζεται με τους μικροσωληνίσκους σε υπό διαφοροποίηση γλοιωματικά κύτταρα C6 επίμυος. Η κατά Western ανάλυση έδειξε μείωση των πρωτεινών αυτών στη μεγαλύτερη συγκέντρωση του, ενώ η πρωτείνη GFAP βρέθηκε μειωμένη σε όλες τις συγκεντρώσεις. Αξιοσημείωτο είναι ότι το DZO προκαλεί αναστολή των προεκβολών των C6 κυττάρων.Παρόμοια, το FIP προκάλεσε σημαντική αναστολή των νευροαξονικών προεκβολών στα N2a κύτταρα χωρίς πάλι να επηρεάζει την βιωσιμότητά τους. Επίσης, επηρέασε σημαντικά την οδό κυτταρικής σηματοδότησης της ERK1/2-MAP κινάσης που παίζει ρόλο στην διαφοροποίηση αυτών των κυττάρων αλλά και μείωσε τα επίπεδα των φωσφορυλιωμένων μορφών των ERK1/2 και MEK1/2. Τα αποτελέσματα αυτά δείχνουν ότι και το FIP ασκεί τοξική δράση στο νευρικό σύστημα

Efficacy of a Dietary Polyherbal Formula on the Performance and Gut Health in Broiler Chicks after Experimental Infection with Eimeria spp.

One-hundred and fifty, one-day-old Ross-308 female chicks were randomly allocated to five equal treatments: NCONTR negative control—not challenged; PCONTR positive control—challenged; PHERB1 and PHERB2 diets were supplemented with phytogenic formula (1 and 2 g/kg feed, respectively)—challenged; PSALIN diet was supplemented with salinomycin (60 mg/kg feed)—challenged. Challenge was made by oral inoculation with 3.5 × 104 E. acervulina, 7.0 × 103 E. maxima and 5.0 × 103 E. tenella oocysts, at 14 days of age. One week post inoculation, bloody diarrhea, oocysts numbers, and intestinal lesions were evaluated, along with intestinal microbiota, viscosity, and pH of digesta, and histopathology. PHERB2 had a comparable (p ≤ 0.001) growth performance and feed conversion ratio to PSALIN. PHERB1 and PHERB2 had similar (p ≤ 0.001) oocyst counts to PSALIN and lower than PCONTROL. PHERB2 and PSALIN had lower (p ≤ 0.001) jejunal, ileal, and cecal lesion scores compared to PCONTR. PHERB1 and PHERB2 had higher (p ≤ 0.001) jejunal and cecal lactobacilli and lower (p ≤ 0.001) coliform counts compared to other treatments. PCONTR had lower (p ≤ 0.001) jejunum villus height, height to crypt ratio, and villus goblet cells. Breast and thigh meat resistance to oxidation was improved (p ≤ 0.001) in PHERB1 and PHERB2 compared to the PCONTR. The polyherbal formula exerted a substantial improvement on growth performance and intestinal health of the Eimeria-challenged birds

Effects of Cornus and Its Mixture with Oregano and Thyme Essential Oils on Dairy Sheep Performance and Milk, Yoghurt and Cheese Quality under Heat Stress

The effect of a diet supplemented with a novel cornus extract, enriched with essential oils of oregano and thyme, on the performance of Chios cross-bred dairy sheep was investigated during the summer period. The plant extracts were prepared using a “green” method based on aqueous extraction. A total of 45 lactating ewes were allocated into three equal groups in a randomized block design. The three groups were fed the same feed allowance, roughage based on Lucerne hay and wheat straw and a concentrate based on cereals and oil cakes (the control diet). The diet of two groups was fortified with cornus extract, with or without oregano and thyme essential oils, at a level 0.515 g of plant extract/essential oils per kg of concentrate. Individual milk yield was recorded weekly and feed refusals were recorded on a pen basis daily, during a six-week period of lactation. Milk samples were analyzed for the chemical composition of protein, fat, lactose and solids-not-fat constituents, somatic cell counts and total viable bacteria counts. Moreover, the milk of each group was used for yoghurt and Feta cheese production. The lipid oxidative stability, protein carbonyl content and fatty acid composition of milk, yoghurt and cheese samples were also evaluated. The results showed that the incorporation of novel plant extracts and essential oils increased the milk production per ewe. Dietary supplementation with cornus extracts and essential oils lowered lipid and protein oxidation in milk, yoghurt and cheese samples, compared to the control. However, diet supplementation with herbal extracts did not affect the fatty acid profile in milk, cheese and yoghurt or the serum biochemical parameters. In conclusion, dietary supplementation with cornus in combination with oregano and thyme has the potential to improve feed utilization and the performance of high-yield dairy Chios cross-bred ewes reared under heat stress

Untargeted UHPLC-MS metabolic profiling as a valuable tool for the evaluation of eggs quality parameters after dietary supplementation with oregano, thyme, sideritis tea and chamomile on brown laying hens

Introduction Bioactive constituents of medicinal-aromatic plants used as feed additives may affect the metabolic profile and oxidative stability of hen eggs. Objectives To determine the effects of dietary supplementation with a mixture of dried oregano, thyme, sideritis tea and chamomile on laying hen performance, egg quality parameters, and oxidative stability in the egg yolk were monitored. Methods In this trial 432 hens were allocated in two treatments (unsupplemented vs. supplemented with the mixture) and fed for 42 days. Eggs were collected at the end of the trial period, egg yolk was separated, extracted, and the total phenolic content (TPC) and oxidative stability was measured. Furthermore, LC-MS metabolic profile of eggs was studied and pathway analysis was elaborated in MetaboAnalyst to facilitate annotation of features. Results Overall, egg production and feed conversion ratio were not affected by the supplementation. However, eggs from the supplemented treatment showed improved shell thickness and strength, and yolk resistance to oxidation. Moreover, LC-MS metabolomic analysis of egg yolk of supplemented and unsupplemented layers showed significant variations and tight clustering in unsupervised principal component analysis due to different chemical profiling of egg yolk. LC-MS study showed that secondary metabolites of aromatic plants did not transfer into yolk, nevertheless the feed supplementation impacted the pathway metabolism of tyrosine, phenylalanine, propanate, and the biosynthesis of aminoacyl-tRNA, phenylalanine, tyrosine and tryptophan. Conclusions The dietary supplementation of layers with a mixture of dried medicinal aromatic plants affected shell thickness and strength, the lipid and protein oxidative stability and increased tyrosine and phenylalanine content in eggs

Effect of an Herbal Mixture of Oregano, Garlic, Sage and Rock Samphire Extracts in Combination with Tributyrin on Growth Performance, Intestinal Microbiota and Morphology, and Meat Quality in Broilers

The present study investigated the effects of two feed additives, the first containing an herbal mixture of oregano, garlic, sage, and rock samphire extracts and the second containing tributyrin (glyceryl tributyrate) when fed to broiler chickens. A total of 360 one-day-old chicks were randomly allocated to four treatments (6 replicate pens of 15 chicks). One treatment served as the unsupplemented control, whereas the feeds of the other three treatments were supplemented either with the herbal additive (3 g/kg), the tributyrin additive (1 g/kg), or both additives. The duration of the trial was 37 days. Data were collected on growth performance, intestinal microbiota and morphology, and some meat quality parameters. The combined supplementation improved (p p Escherichia coli, total Enterobacteriaceae, and Clostridium spp. compared to the other treatments. Fecal coccidial oocyst counts were also reduced (p p < 0.05) breast and thigh meat resistance to oxidation. In conclusion, the combined dietary supplementation with the examined feed additives could be utilized to improve the performance and intestinal health of broiler chickens

In Vitro Antioxidant, Antimicrobial, Anticoccidial, and Anti-Inflammatory Study of Essential Oils of Oregano, Thyme, and Sage from Epirus, Greece

Origanum vulgare subsp. hirtum, Thymus vulgaris, and Salvia fructicosa are aromatic plants commonly found in Mediterranean countries and are traditionally used in Greece as a remedy for humans, since they are well known as potent antibacterial, antioxidant, and anti-inflammatory agents. Essential oils (EOs) derived from plants cultivated in the mountainous region of Epirus, Greece, were investigated for their inhibitory activity against key microorganisms with relevance to avian health, while also assessing their antioxidant and anti-inflammatory activity. The total phenolic content (TPC) of the EOs was estimated according to the Folin–Ciocalteu method, while the antioxidant capacity was tested through the EOs’ ability to scavenge free radicals by means of the DPPH, ABTS, and FRAP assays. Antibacterial and anti-inflammatory effects were examined by the agar disc diffusion method and the lipoxygenase (LOX) inhibition test, respectively. Furthermore, the EOs’ ability to inhibit the invasion of sporozoites of Eimeria tenella (Wisconsin strain) along with any toxic effects were assayed in Madin–Darby bovine kidney (MDBK) cells. The antioxidant activity of the EOs was observed in descending order: oregano > thyme > sage. The antimicrobial effects of thyme and oregano were equivalent and higher than that of sage, while the anti-inflammatory effect of thyme was higher compared to both sage and oregano. The intracellular invasion of sporozoites was evaluated by the detection of E. tenella DNA by qPCR from cell monolayers harvested at 2 and 24 h post-infection. Parasite invasion was inhibited by the addition of oregano essential oil at the concentration of 100 μg/mL by 83% or 93% after 2 or 24 h, respectively, and was higher compared to the addition of thyme and sage, which had similar effects, but at a less intensive level. The cytotoxic assessment of all three essential oils revealed that they had no effect on MDBK cells compared to dimethyl sulfoxide (DMSO), used as the control substance. The supplementation of oregano, thyme, and sage essential oils had a potent antioxidant, anti-inflammatory, antimicrobial, and anticoccidial in vitro effect that is comparable to synthetic substances or approved drugs, justifying the need for further evaluation by in vivo studies in broilers reared in the absence of antimicrobial and anticoccidial drugs or synthetic antioxidant and/or anti-inflammatory compounds

Effect of an Herbal Mixture of Oregano, Garlic, Sage and Rock Samphire Extracts in Combination with Tributyrin on Growth Performance, Intestinal Microbiota and Morphology, and Meat Quality in Broilers

The present study investigated the effects of two feed additives, the first containing an herbal mixture of oregano, garlic, sage, and rock samphire extracts and the second containing tributyrin (glyceryl tributyrate) when fed to broiler chickens. A total of 360 one-day-old chicks were randomly allocated to four treatments (6 replicate pens of 15 chicks). One treatment served as the unsupplemented control, whereas the feeds of the other three treatments were supplemented either with the herbal additive (3 g/kg), the tributyrin additive (1 g/kg), or both additives. The duration of the trial was 37 days. Data were collected on growth performance, intestinal microbiota and morphology, and some meat quality parameters. The combined supplementation improved (p &lt; 0.05) weigh gain, feed conversion ratio, and the European Efficiency Factor. In the cecum, the combined supplementation lowered (p &lt; 0.05) the microbial populations of aerobes, anaerobes, Escherichia coli, total Enterobacteriaceae, and Clostridium spp. compared to the other treatments. Fecal coccidial oocyst counts were also reduced (p &lt; 0.01) by the combined supplementation. The herbal mixture supplementation improved (p &lt; 0.05) breast and thigh meat resistance to oxidation. In conclusion, the combined dietary supplementation with the examined feed additives could be utilized to improve the performance and intestinal health of broiler chickens