10 research outputs found
Endotoxin induced muscle wasting in avian and murine skeletal muscle
No full textThis project was aimed to elucidate the sub-cellular and molecular regulation of Lipopolysaccharide (LPS) induced muscle protein turnover (protein synthesis (PS) and protein degradation) in two in vitro models, C2C12 murine myotubes and avian primary skeletal muscle cell line. In addition, the effect of natural challenge of chicken with Salmonella serotypes gallinarium or Enteritidis on mRNA expression levels in skeletal muscle was assessed. LPS (1 μgml-1) transiently decreased PS rate by 50% compared with control cells. This effect was mediated via decreased phosphorylation of translation initiation mediators (p70S6K, 4E-BP1 and eIF-4E). This effect was preceded by decreased Akt and mTOR phosphorylation. Although, LPS significantly increased p38, Erk1/2 and their down stream target Mnk1, however, this effect was not sufficient to abolish LPS-induced decreased PS. The role of Akt and MAPKs (p38 or Erk1/2) was verified using specific pathway inhibitors. Inhibition of Akt by LY0294002 (PI3-K/Akt inhibitor) dramatically decreased PS by 80% compared with control cells. Incubation of C2C12 myotubes with SB203580 (p38 inhibitor) or with PD098059 (MEK/Erk inhibitor) alone significantly decreased the PS rate at the 3 h time point by -63 ± 12.48% and -64 ± 5.05% respectively compared with control cells (P < 0.01). In contrast, LPS (1 μgml-1) significantly increased the chymotrypsin-like enzyme at all the time points. This effect was preceded by a significant increase in the IkB-α phosphorylation and nuclear translocation of NF-kB, and significant increase in TNF-α, atrogin-1, MuRF1 and TLR4 mRNA expression. Of note, increased atrogin-1 mRNA is the prominent feature of our septic model. The data presented in chapter 4 and 5 showed that, there is no absolute correlation between the expression levels of atrogens (atrogin-1 and MuRF1) and the overall proteolytic activity in LPS-stimulated C2C12 myotubes. The beneficial roles of the curcumin were evaluated LPS-stimulated C2C12 myotubes for 3 h. Incubation of C2C12 myotubes with LPS (1 μgml-1) and curcumin (25 μM) significantly decreased the LPS-induced chymotrypsin-like enzyme activity. This effect was mediated via decreased p38 and IkB-α phosphorylation. Although, curcumin blocked LPS-induced decreased Akt and p70S6K phosphorylation and significantly increased Erk1/2 phosphorylation, however, curcumin still had no effect on LPS-induced decreased protein synthesis. The effect of the LPS on the muscle protein turnover in the avian primary skeletal muscle was summarised in chapter (7). Incubation of avian primary skeletal cells with LPS (1 μgml-1) for 3 h, significantly decreased the proteasomal activity and increased PS rate. The difference in response to LPS between C2C12 myotubes and avian primary skeletal muscle cells could be attributed to the different incubation parameters mainly the presence of insulin in case of avian primary cells. Finally, the effect of natural challenge of chicken with S. Gallinarum or S. Enteritidis on skeletal muscle mRNA expression was summarised in chapter 9. Natural challenge of chicken with S. Gallinarum or S. Enteritidis had no effect on the expression of many atrophic genes in chicken skeletal muscle (gastrocnemius and pectoral muscle). The data collected from this project showed that, LPS is a strong catabolic stimulus significantly decreased PS along with increased protein breakdown rates in skeletal muscle. This effect was mediated via two main pathways PI3-K/Akt and MAPKs (p38 or Erk1/2) and the cross talk between them is exists. The better understanding of these signalling cascades and their cross talk will be the starting point for developing the appropriate and safe therapeutic intervention in order to decrease the sepsis-induced muscle proteolysis.EThOS - Electronic Theses Online ServiceGBUnited Kingdo
Endotoxin induced muscle wasting in avian and murine skeletal muscle
No full textThis project was aimed to elucidate the sub-cellular and molecular regulation of Lipopolysaccharide (LPS) induced muscle protein turnover (protein synthesis (PS) and protein degradation) in two in vitro models, C2C12 murine myotubes and avian primary skeletal muscle cell line. In addition, the effect of natural challenge of chicken with Salmonella serotypes gallinarium or Enteritidis on mRNA expression levels in skeletal muscle was assessed.
LPS (1 μgml-1) transiently decreased PS rate by 50% compared with control cells. This effect was mediated via decreased phosphorylation of translation initiation mediators (p70S6K, 4E-BP1 and eIF-4E). This effect was preceded by decreased Akt and mTOR phosphorylation. Although, LPS significantly increased p38, Erk1/2 and their down stream target Mnk1, however, this effect was not sufficient to abolish LPS-induced decreased PS.
The role of Akt and MAPKs (p38 or Erk1/2) was verified using specific pathway inhibitors. Inhibition of Akt by LY0294002 (PI3-K/Akt inhibitor) dramatically decreased PS by 80% compared with control cells. Incubation of C2C12 myotubes with SB203580 (p38 inhibitor) or with PD098059 (MEK/Erk inhibitor) alone significantly decreased the PS rate at the 3 h time point by -63 12.48% and -64 5.05% respectively compared with control cells (P < 0.01).
In contrast, LPS (1 μgml-1) significantly increased the chymotrypsin-like enzyme at all the time points. This effect was preceded by a significant increase in the IkB-α phosphorylation and nuclear translocation of NF-kB, and significant increase in TNF-α, atrogin-1, MuRF1 and TLR4 mRNA expression. Of note, increased atrogin-1 mRNA is the prominent feature of our septic model. The data presented in chapter 4 and 5 showed that, there is no absolute correlation between the expression levels of atrogens (atrogin-1 and MuRF1) and the overall proteolytic activity in LPS-stimulated C2C12 myotubes.
The beneficial roles of the curcumin were evaluated LPS-stimulated C2C12 myotubes for 3 h. Incubation of C2C12 myotubes with LPS (1 μgml-1) and curcumin (25 μM) significantly decreased the LPS-induced chymotrypsin-like enzyme activity. This effect was mediated via decreased p38 and IkB-α phosphorylation. Although, curcumin blocked LPS-induced decreased Akt and p70S6K phosphorylation and significantly increased Erk1/2 phosphorylation, however, curcumin still had no effect on LPS-induced decreased protein synthesis.
The effect of the LPS on the muscle protein turnover in the avian primary skeletal muscle was summarised in chapter (7). Incubation of avian primary skeletal cells with LPS (1 μgml-1) for 3 h, significantly decreased the proteasomal activity and increased PS rate. The difference in response to LPS between C2C12 myotubes and avian primary skeletal muscle cells could be attributed to the different incubation parameters mainly the presence of insulin in case of avian primary cells.
Finally, the effect of natural challenge of chicken with S. Gallinarum or S. Enteritidis on skeletal muscle mRNA expression was summarised in chapter 9. Natural challenge of chicken with S. Gallinarum or S. Enteritidis had no effect on the expression of many atrophic genes in chicken skeletal muscle (gastrocnemius and pectoral muscle).
The data collected from this project showed that, LPS is a strong catabolic stimulus significantly decreased PS along with increased protein breakdown rates in skeletal muscle. This effect was mediated via two main pathways PI3-K/Akt and MAPKs (p38 or Erk1/2) and the cross talk between them is exists. The better understanding of these signalling cascades and their cross talk will be the starting point for developing the appropriate and safe therapeutic intervention in order to decrease the sepsis-induced muscle proteolysis
Prevalence and antimicrobial resistance of Bacillus cereus isolated from beef products in Egypt
No full textFoodborne pathogens have the main concern in public health and food safety. Bacillus cereus food poisoning is one of the most important foodborne pathogens worldwide. In the present study, a total of 200 random beef product samples were collected from different supermarkets located at Menofia and Cairo governorates were examined for the presence of B. cereus. In addition, the presence of some virulence encoding genes was evaluated using Multiplex PCR. Finally, the antibiogram testing was conveyed to illustrate the resistance pattern of the confirmed B. cereus. The data showed that B. cereus was recovered from 22.5%, 30%, 25%, 37.5% and 15% of the minced meat, burger, sausage, kofta, and luncheon respectively. Among the 20 examined isolates 18/20 (90%) were harbor hblC enterotoxin encoding gene compared with 20/20 (100) were have cytK enterotoxin encoding gene. The isolated strains of B. cereus were resistant to penicillin G and sensitive to oxacillin, clindamycin, vancomycin, erythromycin, gentamicin, ciprofloxacin, and ceftriaxone. In all, the obtained data showed the importance of emerging B. cereus in disease control and prevention programs, and in regular clinical and food quality control laboratories in Egypt
Isolation and characterization of Salmonella Enteritidis and Salmonella Typhimurium from chicken meat in Egypt
No full textIntroduction: Salmonella enterica serovars Enteritidis and Typhimurium represent the major serovars associated with human salmonellosis. Contamination of meat products with these serovars is considered the main source of infection. Methodology: In this study, 100 raw chicken meat samples were investigated for the presence of Salmonella spp., which were subsequently identified based on biochemical and serological tests as well as matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) profile. Furthermore, the isolated serovars were examined using multiplex polymerase chain reaction (PCR) for the presence of virulence genes suspected to have a role in infection. Results: S. Enteritidis was isolated from two samples (2%), while S. Typhimurium was isolated from three samples (3%) of chicken meat. Of the 17 examined virulence genes using multiplex PCR, the sitC, sopB, sifA, lpfC, spaN, sipB, invA, spiA, and msgA genes were detected in S. Enteritidis. However, the sitC, iroN, sopB, sifA, lpfC, spaN, sipB, invA, and tolC genes were successfully amplified in S. Typhimurium. Conclusions: The detection of S. Enteritidis and S. Typhimurium in meat, even at low incidence, has important implications. In addition, the data presented here is the first attempt to identify a wide range of virulence genes in Egyptian Salmonella isolates recovered from meat products. A strict public health and food safety regime is urgently needed in order to decrease the human health hazard risk associated with salmonellosis.</jats:p
Effect of Dietary Selenium Nanoparticles Supplementation on Hematological, Serum Biochemical, Oxidant-Antioxidant Biomarkers, and Proinflammatory Cytokines in Broilers Challenged with Salmonella Typhimurium
No full textThe current study evaluated the efficacy of Selenium Nanoparticles (Se-NPs) on hemato-biochemical, antioxidant biomarkers, and immunological responses induced by S. Typhimurium in broiler chickens. Chicks (N=120) were divided into six groups. Group 1: received no treatment and set as a control group. Group 2: fed Se-NPs enriched diet (0.5 mg/kg diet). Group 3: subjected to oral challenge with 3.5x108 CFU/mL/1 ml/bird of S. Typhimurium. Group 4: administrated Se-NPs (0.5 mg/kg diet) then on day 21 was subjected to 3.5x108 CFU/mL/1 ml/bird of S. Typhimurium. Group 5: vaccinated by a SERVAC Tri Sal. 0.1ml subcutaneous (s/c) injection on day 3 then subjected to 3.5x108 CFU/mL/1 ml/bird of S. Typhimurium on day 21. Group 6: treated from day 1 with Se-NPs (0.5 mg/kg diet) till the end of the experiment and vaccinated by a SERVAC Tri Sal. 0.1ml (s/c) on day 3 and then subjected to 3.5x108 CFU/mL/1 ml/bird of S. Typhimurium on day 21. The results showed that S. Typhimurium significantly decreased erythrogram, lymphocytes count, total protein, albumin, A/G ratio, glucose, cholesterol, triglyceride, serum iron, and TIBC, GPX, SOD, TAC, and IL-10 expression compared to the control. Meanwhile, S. Typhimurium significantly increased TLC, heterophils, monocytes, serum ferritin, liver enzymes (ALT, AST), renal products (creatinine, uric acid), MDA, IL6 expression. Conversely, the dietary Se-NPs supplementation and/or Salmonella vaccine to the infected broiler induced, to various degrees, improvement in hemato-biochemical, antioxidant biomarkers, and proinflammatory responses compared to challenged group. In conclusion, dietary Se-NPs supplementation offered a direct protection against S. Typhimurium infection for sustaining poultry production and correspondingly protecting human health
Phenotypical and Genotypical Assessment Techniques for Identification of Some Contagious Mastitis Pathogens
No full textMastitis is one of the most economic disease affecting dairy cows worldwide. Its classic diagnosis using bacterial culture and biochemical findings is a difficult and prolonged method. In this research, using of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) permitted identification of different microorganisms with high accuracy and rapidity (only 24 hours for microbial growth and analysis). During the application of MALDI-TOF MS, one hundred twenty strains of Staphylococcus and Streptococcus species isolated from milk of cows affected by clinical and subclinical mastitis were identified, and the results were compared with those obtained by traditional methods as API and VITEK 2 Systems. 37 of totality 39 strains (~95%) of Staphylococcus aureus (S. aureus) were exactly detected by MALDI TOF MS and then confirmed by a nuc-based PCR technique, whereas accurate identification was observed in 100% (50 isolates) of the coagulase negative staphylococci (CNS) and Streptococcus agalactiae (31 isolates). In brief, our results demonstrated that MALDI-TOF MS is a fast and truthful technique which has the capability to replace conventional identification of several bacterial strains usually isolated in clinical laboratories of microbiology
Phenotypic and Genotypic Characterization of Bacteriocinogenic Enterococci Against Clostridium botulinum
No full textDevelopment of Salmonella Enteritidis vaccine candidate based on streptomycin independent suppressor and metabolic drift rifampicin resistance-attenuating markers
No full textSalmonella is one of the most frequent food-borne pathogens and remains public health threat globally. The control of Salmonella in poultry, the main reservoir of non-typhoidal salmonellae, is a fundamental approach to ensure the safety of poultry products for human consumption. In the present study, a new live attenuated Salmonella enterica serovar Enteritidis vaccine candidate containing three attenuating markers based on streptomycin-independent (Sm-id) suppressor, and metabolic drift antibiotic resistance (MD- “res”) was developed. The streptomycin dependent (Smd) mutants were derived from Salmonella Enteritidis wild-type strain using streptomycin. Then the Sm-id mutants were derived from the isolated Smd mutants and designated “Smd→Sm-id”. A third MD- “res” marker was generated from Smd→Sm-id using rifampicin (Rif) and designated “Smd→Sm-id→Rif”. The colony sizes of these mutants were stable after more than 50 serial passages on blood agar; reversion to virulence can be almost excluded. The safety and efficacy of Smd→Sm-id and Smd→Sm-id→Rif were evaluated in one-day-old commercial layer chicks. Both mutants proved to be safe in terms of clinical signs, mortalities, lesion scores of visceral organs and rapid clearance when administered orally at a dose of 108 colony forming unit (CFU), whereas birds inoculated with 108 CFU Salmonella Enteritidis wild-type strain showed diarrhea, mortalities (3/40) and necrosis in liver and spleen. Chickens vaccinated with the developed mutants showed no seroconversion; however, wild-type strain induced a significant seroconversion at 3-week-postvaccination (wpv). The developed mutants protected chickens against challenge with 108 CFU of Salmonella Enteritidis wild-type strain at 3-wpv. Vaccinated birds showed neither clinical signs nor mortalities during two-week post-challenge. In addition, the challenge strain could not be detected in pooled liver and spleen samples (0/5) at 7th day post-inoculation (dpi). However, non-vaccinated challenged birds showed diarrhea and the challenge strain was re-isolated from pooled liver and spleen samples (3/5) at 7th dpi. In conclusion, the developed mutants are safe and fully protected immunized chickens following heterologous challenge. It is obvious that the genetic characterization of these mutants and evaluation of different vaccination regimes are still in demand
Characterization of Sunflower Oil Extracts from the Lichen Usnea barbata
No full textThe increasing global emergence of multidrug resistant (MDR) pathogens is categorized as one of the most important health problems. Therefore, the discovery of novel antimicrobials is of the utmost importance. Lichens provide a rich source of natural products including unique polyketides and polyphenols. Many of them display pharmaceutical benefits. The aim of this study was directed towards the characterization of sunflower oil extracts from the fruticose lichen, Usnea barbata. The concentration of the major polyketide, usnic acid, was 1.6 mg/mL extract as determined by NMR analysis of the crude mixture corresponding to 80 mg per g of the dried lichen. The total phenolics and flavonoids were determined by photometric assays as 4.4 mg/mL (gallic acid equivalent) and 0.27 mg/mL (rutin equivalent) corresponding to 220 mg/g and 13.7 mg/g lichen, respectively. Gram-positive (e.g., Enterococcus faecalis) and Gram-negative bacteria, as well as clinical isolates of infected chickens were sensitive against these extracts as determined by agar diffusion tests. Most of these activities increased in the presence of zinc salts. The data suggest the potential usage of U. barbata extracts as natural additives and mild antibiotics in animal husbandry, especially against enterococcosis in poultry
Molecular Detection, Serotyping, and Antibiotic Resistance of Shiga Toxigenic <i>Escherichia coli</i> Isolated from She-Camels and In-Contact Humans in Egypt
No full textThis study aims to determine the prevalence of STEC in she-camels suffering from mastitis in semi-arid regions by using traditional culture methods and then confirming it with Serological and molecular techniques in milk samples, camel feces, as well as human stool samples for human contacts. In addition, an antibiotic susceptibility profile for these isolates was investigation. Mastitic milk samples were taken after California Mastitis Test (CMT) procedure, and fecal samples were taken from she-camels and human stool samples, then cultured using traditional methods to isolate Escherichiacoli. These isolates were initially classified serologically, then an mPCR (Multiplex PCR) was used to determine virulence genes. Finally, both camel and human isolates were tested for antibiotic susceptibility. Out of a total of 180 she-camels, 34 (18.9%) were mastitic (8.3% clinical and 10.6% sub-clinical mastitis), where it was higher in camels bred with other animals. The total presence of E. coli was 21.9, 13.9, and 33.7% in milk, camel feces, and human stool, respectively, whereas the occurrence of STEC from the total E. coli isolates were 36, 16, and 31.4% for milk, camel feces, and stool, respectively. Among the camel isolates, stx1 was the most frequently detected virulence gene, while hlyA was not detected. The most detected virulence gene in human isolates was stx2 (45.5%), followed by stx1. Camel STEC showed resistance to Oxytetracycline only, while human STEC showed multiple drug resistance to Amoxicillin, Gentamycin, and Clindamycin with 81.8, 72.7, and 63.6%, respectively. Breeding camels in semi-arid areas separately from other animals may reduce the risk of infection with some bacteria, including E. coli; in contrast, mixed breeding with other animals contributes a significant risk factor for STEC emergence in camels
