The prevalence of non-Alcoholic Fatty Liver Disease (NAFLD) has now reached epidemic proportions, but the role of gene-lifestyle interactions in its pathogenesis remains poorly understood. While evidence for an inverse association between odd-chain length fatty acids (OCFA) and cardiometabolic diseases, suggests a possible link between OCFAs and NAFLD, little is known about the impact of diet, gut microbiota and peroxisomal biogenesis on the metabolism of OCFAs. We hypothesized that suboptimal diet, altered gut microbiota and peroxisomal biogenesis could promote the development of NAFLD by impairing the metabolism of OCFAs. This thesis aimed to understand the effect of dietary fat/protein on the genetic and metabolic regulation of lipids and OCFAs in relation to NAFLD, using a high fat diet (HFD) model, well established in the literature for inducing obesity and insulin resistance in mice within 4 weeks, and a low protein diet (LPD) model, known to promote NAFLD. Under specific pathogen free or normal husbandry conditions, a HFD reduced serum OCFA in mice after 4 and 12 weeks of feeding, and down-regulated the activity of several key enzymes in fatty acid metabolism (desaturases, lyase, elongase). Liver histology also showed deposition of lipid droplets and higher expression of peroxin 14 protein in HFD fed mice (Chapter 3). The characterisation of gut microbiota revealed an alteration in propionate-producing bacteria, Lachnospiraceae and Clostridiales, in HFD fed mice (Chapter 4). Mice fed with carbohydrate rich-LPD for 7 weeks resulted in lower levels of serum OCFA, increased CD36 mRNA and peroxin 14 expressions. However, OCFA did not change in the reduced and quality carbohydrate-LPD after 8 weeks (Chapter 5). In conclusion, these findings provide evidence that HFD and carbohydrate rich-LPD reduced OCFA via changes in gut microbiota and peroxisomal biogenesis in the liver and increases our understanding of how suboptimal diets contributes to NAFLD.
Probiotics represent a non-invasive, environmentally-friendly alternative to reduce infectious diseases in wildlife species. Our aim was to evaluate the potential of typical gut commensals, such as lactic acid bacteria (LAB), as wildlife probiotics. The selected LAB were isolated from European badgers (Meles meles); a wildlife reservoir of bovine tuberculosis, and comprised four different genera: Enterococcus; Weissella; Pediococcus; and Lactobacillus. The enterococci displayed a phenotype and genotype that correlate with the production of antibacterial peptides and stimulation of antiviral responses. However, these isolates carry virulence and antibiotic resistance genes. Weissella showed some anti-mycobacterial activity due to their ability to produce lactate and ethanol. Interestingly, lactobacilli and pediococci modulated pro-inflammatory phagocytic responses that associate with protection against pathogens; and these responses agreed with the presence of immunomodulatory markers in their genomes. Although both lactobacilli and pediococci showed tolerance to antibiotics, this resistance was naturally acquired and almost all isolates possessed a strong phylogenetic relationship with isolates from food and healthy animals. Our results show that LAB display probiotic benefits that depend on the genera. Lactobacilli and pediococci are probably the most interesting candidates as probiotics against infectious diseases in wildlife because of their food-grade status and ability to modulate protective innate immune responses.
The objective of this study was to evaluate the efficacy of plant essential oils (EOs) in combination and to investigate the effect of food ingredients on their efficacy. The EOs assessed in combination included basil, lemon balm, marjoram, oregano, rosemary, sage and thyme. Combinations of EOs were initially screened against Bacillus cereus, Escherichia coli, Listeria monocytogenes and Pseudomonas aeruginosa using the spot-on-agar test. The influence of varying concentrations of EO combinations on efficacy was also monitored using E. coli. These preliminary studies showed promising results for oregano in combination with basil, thyme or marjoram. The checkerboard method was then used to quantify the efficacy of oregano, marjoram or thyme in combination with the remainder of selected EOs. Fractional inhibitory concentrations (FIC) were calculated and interpreted as synergy, addition, indifference or antagonism. All the oregano combinations showed additive efficacy against B. cereus, and oregano combined with marjoram, thyme or basil also had an additive effect against E. coli and P. aeruginosa. The mixtures of marjoram or thyme also displayed additive effects in combination with basil, rosemary or sage against L. monocytogenes. The effect of food ingredients and pH on the antimicrobial efficacy of oregano and thyme was assessed by monitoring the lag phase and the maximum specific growth rate of L. monocytogenes grown in model media. The model media included potato starch (0, 1, 5 or 10%), beef extract (1.5, 3, 6 or 12%), sunflower oil (0, 1, 5 or 10%) and TSB at pH levels of 4, 5, 6 or 7. The antimicrobial efficacy of EOs was found to be a function of ingredient manipulation. Starch and oils concentrations of 5% and 10% had a negative impact on the EO efficacy. On the contrary, the EOs were more effective at high concentrations of protein, and at pH 5, by comparison with pH 6 or 7. This study suggests that combinations of EOs could minimize application concentrations and consequently reduce any adverse sensory impact in food. However, their application for microbial control might be affected by food composition, therefore, careful selection of EOs appropriate to the sensory and compositional status of the food system is required. This work shows that EOs might be more effective against food-borne pathogens and spoilage bacteria when applied to ready to use foods containing a high protein level at acidic pH, as well as lower levels of fats or carbohydrates.
The objectives of this study were to evaluate the antimicrobial activity of plant essential oils (EOs) against foodborne pathogens and key spoilage bacteria pertinent to ready-to-eat vegetables and to screen the selected EOs for sensory acceptability. The EOs basil, caraway, fennel, lemon balm, marjoram, nutmeg, oregano, parsley, rosemary, sage, and thyme were evaluated. The bacteria evaluated were Listeria spp., Staphylococcus aureus, Lactobacillus spp., Bacillus cereus, Salmonella, Enterobacter spp., Escherichia coli, and Pseudomonas spp. Quantitative antimicrobial analyses were performed using an absorbance-based microplate assay. Efficacy was compared using MIC, the half maximum inhibitory concentration, and the increase in lag phase. Generally, gram-positive bacteria were more sensitive to EOs than were gram-negative bacteria, and Listeria monocytogenes strains were among the most sensitive. Of the spoilage organisms, Pseudomonas spp. were the most resistant. Oregano and thyme EOs had the highest activity against all the tested bacteria. Marjoram and basil EOs had selectively high activity against B. cereus, Enterobacter aerogenes, E. coli, and Salmonella, and lemon balm and sage EOs had adequate activity against L. monocytogenes and S. aureus. Within bacterial species, EO efficacy was dependent on strain and in some cases the origin of the strain. On a carrot model product, basil, lemon balm, marjoram, oregano, and thyme EOs were deemed organoleptically acceptable, but only oregano and marjoram EOs were deemed acceptable for lettuce. Selected EOs may be useful as natural and safe additives for promoting the safety and quality of ready-to-eat vegetables.
The aim of this study was to optimise the antimicrobial efficacy of plant essential oils (EOs) for control of Listeria spp. and spoilage bacteria using food model media based on lettuce, meat and milk. The EOs evaluated were lemon balm, marjoram, oregano and thyme and their minimum inhibitory concentrations (MIC) were determined against Enterobacter spp., Listeria spp., Lactobacillus spp., and Pseudomonas spp. using the agar dilution method and/or the absorbance based microplate assay. MICs were significantly lower in lettuce and beef media than in TSB. Listeria strains were more sensitive than spoilage bacteria, and oregano and thyme were the most active EOs. EO combinations were investigated using the checkerboard method and Oregano combined with thyme had additive effects against spoilage organisms. Combining lemon balm with thyme yielded additive activity against Listeria strains. The effect of simple sugars and pH on antimicrobial efficacy of oregano and thyme was assessed in a beef extract and tomato serum model media. EOs retained greater efficacy at pH 5 and 2.32% sugar, but sugar concentrations above 5% did not negatively impact EO efficacy. In addition to proven antimicrobial efficacy, careful selection and investigation of EOs appropriate to the sensory profile of foods and composition of the food system is required. This work shows that EOs might be more effective against food-borne pathogens and spoilage bacteria when applied to foods containing a high protein level at acidic pH, as well as moderate levels of simple sugars.
J Gutierrez, J O'Donovan, A Proctor, C Brady, PX Marques, S Worrall, JE Nally, M McElroy, H Bassett, J Fagan, S Maley, D Buxton, D Sammin, BK Markey (2012)Application of quantitative real-time polymerase chain reaction for the diagnosis of toxoplasmosis and enzootic abortion of ewes, In: JOURNAL OF VETERINARY DIAGNOSTIC INVESTIGATION24(5)pp. 846-854
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PX Marques, J O' Donovan, EJ Williams, J Gutierrez, S Worrall, M McElroy, A Proctor, C Brady, D Sammin, H Bassett, D Buxton, S Maley, BK Markey, JE Nally (2012)Detection of Toxoplasma gondii antigens reactive with antibodies from serum, amniotic, and allantoic fluids from experimentally infected pregnant ewes, In: VETERINARY PARASITOLOGY185(2-4)pp. 91-100
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Dietary protein insufficiency has been linked to excessive triglyceride storage (TG) and non-alcoholic fatty liver disease (NAFLD) in developing countries. Hepatic TG accumulation following a low-protein diet may be due to altered peroxisomal, mitochondrial and gut microbiota function. Hepatic peroxisomes and mitochondria normally mediate metabolism of nutrients to provide energy and substrates for lipogenesis. Peroxisome biogenesis and activities can be modulated by odd (OCFA) and short-chain (SCFA) fatty acids that are derived from gut bacteria e.g. propionate and butyrate. Also produced during amino acid metabolism by peroxisomes and mitochondria, propionate and butyrate correlate with reduced risk of obesity, insulin resistance and NAFLD. In this horizon-scanning review, we have compiled available evidence on the effects of protein malnutrition on OCFA production, arising from loss in mitochondrial, peroxisomal and gut microbiota function, and its association with lipid accumulation in the liver. The methyl donor amino acid composition of dietary protein is an important contributor to liver function and lipid storage; the presence and abundance of dietary branched chain amino acids can modulate the composition and metabolic activity of the gut microbiome and on the other hand, can affect protective OCFA and SCFA production in the liver. In preclinical animal models fed with low protein diets, specific amino acid supplementation can ameliorate fatty liver disease. The association between low dietary protein intake and fatty liver disease is underexplored and merits further investigation, particularly in vulnerable groups with dietary protein restriction in developing countries.
J O'Donovan, A Proctor, J Gutierrez, S Worrell, J Nally, P Marques, C Brady, M McElroy, D Sammin, D Buxton, S Maley, H Bassett, B Markey (2012)Distribution of lesions in fetal brains following experimental infection of pregnant sheep with Toxoplasma gondii., In: Vet Pathol49(3)pp. 462-469
Six ovine fetal brains were harvested 33 to 35 days postchallenge from 5 ewes, each of which was given 3000 Toxoplasma gondii oocysts on day 90 of pregnancy. Histopathologic examination of transverse sections taken at 13 levels in the fetal brains revealed the presence of toxoplasmosis-related lesions in all 6 brains. However, lesions were not randomly distributed (P = .007); they were most numerous at the level of the optic tract, the rostral margin of the pons, and 4 mm caudal to the ansate sulcus and were absent in all sections at the level of the caudal cerebellum. Lesion distribution may be due to hemodynamic factors, differences in the expression of endothelial surface receptor molecules at the level of the blood-brain barrier, or the presence of localized permissive/inhibitory factors within the brain. The results have implications for the selection of areas of brain from aborted ovine fetuses to be examined histopathologically for laboratory diagnosis.
Type-I interferon (IFN-I) cytokines are produced by immune cells in response to microbial infections, cancer and autoimmune diseases, and subsequently, trigger cytoprotective and antiviral responses through the activation of IFN-I stimulated genes (ISGs). The ability of intestinal microbiota to modulate innate immune responses is well known, but the mechanisms underlying such responses remain elusive. Here we report that the intracellular sensors stimulator of IFN genes (STING) and mitochondrial antiviral signaling (MAVS) are essential for the production of IFN-I in response to lactic acid bacteria (LAB), common gut commensal bacteria with beneficial properties. Using human macrophage cells we show that LAB strains that potently activate the inflammatory transcription factor NF-κB are poor inducers of IFN-I and conversely, those triggering significant amounts of IFN-I fail to activate NF-κB. This IFN-I response is also observed in human primary macrophages, which modulate CD64 and CD40 upon challenge with IFN-I-inducing LAB. Mechanistically, IFN-I inducers interact more intimately with phagocytes as compared to NF-κB-inducers, and fail to activate IFN-I in the presence of phagocytosis inhibitors. These bacteria are then sensed intracellularly by the cytoplasmic sensors STING and, to a lesser extent, MAVS. Accordingly, macrophages deficient for STING showed dramatically reduced phosphorylation of TANK-binding kinase (TBK)-1 and IFN-I activation, which resulted in lower expression of ISGs. Our findings demonstrate a major role for intracellular sensing and STING in the production of IFN-I by beneficial bacteria and the existence of bacteria-specific immune signatures, which can be exploited to promote cytoprotective responses and prevent overreactive NF-κB-dependent inflammation in the gut.
S Worrall, DJ Sammin, HF Bassett, CR Reid, J Gutierrez, PX Marques, JE Nally, J O'Donovan, EJ Williams, A Proctor, BK Markey (2011)Interferon-γ expression in trophoblast cells in pregnant ewes challenged with Chlamydophila abortus., In: J Reprod Immunol90(2)pp. 214-219
Pregnant ewes were challenged with Chlamydia abortus at 91-98 days of gestation and euthanised at 14, 21 and 28 days post-challenge. IFNγ mRNA labelling appeared to be co-localised with Chlamydial lipopolysaccharide within trophoblast cells in discrete areas lining the primary villi in the limbus and hilar zone of the placentomes from challenged sheep on days 21 and 28 post-infection. The presence of IFNγ was also demonstrated by immunohistochemistry. No labelling was seen in tissues from the non-infected ewes. The presence of IFNγ in trophoblast cells from infected ewes may indicate an attempt to restrict the replication of the organism and be an important trigger for the inflammatory responses that develop on the fetal side of the placenta in enzootic abortion.
PX Marques, P Souda, J O'Donovan, J Gutierrez, EJ Gutierrez, S Worrall, M McElroy, A Proctor, C Brady, D Sammin, HF Basset, JP Whitelegge, BE Markey, JE Nally (2010)Identification of immunologically relevant proteins of Chlamydophila abortus using sera from experimentally infected pregnant ewes., In: Clin Vaccine Immunol17(8)pp. 1274-1281
Chlamydophila abortus is an intracellular pathogen and the etiological agent of enzootic abortion of ewes (EAE). C. abortus has a biphasic development cycle; extracellular infectious elementary bodies (EB) attach and penetrate host cells, where they give rise to intracellular, metabolically active reticulate bodies (RB). RB divide by binary fission and subsequently mature to EB, which, on rupture of infected cells, are released to infect new host cells. Pregnant ewes were challenged with 2 x 10(6) inclusion forming units (IFU) of C. abortus cultured in yolk sac (comprising both EB and RB). Serum samples were collected at 0, 7, 14, 21, 27, 30, 35, 40, and 43 days postinfection (dpi) and used to identify antigens of C. abortus expressed during disease. Additionally, sera from fetal lambs were collected at 30, 35, 40, and 43 dpi. All serum samples collected from experimentally infected pregnant ewes reacted specifically with several antigens of EB as determined by one-dimensional (1-D) and 2-D gel electrophoresis; reactive antigens identified by mass spectrometry included the major outer membrane protein (MOMP), polymorphic outer membrane protein (POMP), and macrophage infectivity potentiator (MIP) lipoprotein.
J Gutierrez, J O'Donovan, E Williams, A Proctor, C Brady, PX Marques, S Worrall, JE Nally, M McElroy, H Bassett, D Sammin, D Buxton, S Maley, BK Markey (2010)Detection and quantification of Toxoplasma gondii in ovine maternal and foetal tissues from experimentally infected pregnant ewes using real-time PCR., In: Vet Parasitol172(1-2)pp. 8-15
A real-time PCR (rt-PCR) targeting the 529-bp repeat element (RE) of Toxoplasma gondii was used to detect and quantify the parasite burden in maternal and foetal tissues in 18 seronegative ewes infected with 3000 toxoplasma oocysts on day 90 of pregnancy. The infected ewes were sacrificed in groups of 4-6 at 21, 25, 33 and 35 days post-challenge. Ten sham inoculated pregnant ewes were used as controls. T. gondii was not detected in the control ewes or their foeti. The parasite was only detected in the maternal tissues in a few of the challenged ewes on a small number of occasions where it was identified in spleen and uterine lymph nodes. T. gondii was detected in the foetal spleen and liver at the early sacrifice times but only sporadically thereafter. In the case of amniotic, allantoic and foetal aqueous humor samples T. gondii was only detected on a small number of occasions. However, it was found in the majority of the foetal lung and placentome samples throughout the study period, while placentomes and foetal brains contained high levels of the parasite during the later stages. Histopathological examination of placentome and brain tissue from the foeti in the present study revealed a strong correlation between histopathological lesions and quantities of the parasite DNA detected. These results indicate that the cotyledonary component of the foetal membranes is the sample of choice for the diagnosis of T. gondii by rt-PCR, followed by foetal lung and brain.
J Gutierrez, EJ Williams, J O'Donovan, C Brady, AF Proctor, PX Marques, S Worrall, JE Nally, M McElroy, HF Bassett, DJ Sammin, BK Markey (2011)Monitoring clinical outcomes, pathological changes and shedding of Chlamydophila abortus following experimental challenge of periparturient ewes utilizing the natural route of infection, In: VETERINARY MICROBIOLOGY147(1-2)pp. 119-126
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PX Marques, J O'Donovan, P Souda, J Gutierrez, EJ Williams, S Worrall, M McElroy, A Proctor, C Brady, D Sammin, H Basset, JP Whitelegge, BK Markey, JE Nally (2011)Amniotic and allantoic fluids from experimentally infected sheep contain immunoglobulin specific for Chlamydophila abortus, In: VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY140(1-2)pp. 1-9
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Enterocin P (EntP), a sec-dependent bacteriocin from Enterococcus faecium P13, was produced by Lactococcus lactis. The EntP structural gene (entP) with or without the EntP immunity gene (entiP) was cloned in (1), plasmid pMG36c under control of the lactococcal constitutive promoter P32, (2) in plasmid pNG8048e under control of the inducible PnisA promoter, and (3) in the integration vector pINT29. Introduction of the recombinant vectors in L. lactis resulted in production of biologically active EntP in the supernatants of L. lactis subsp. lactis IL1403 and L. lactis subsp. cremoris NZ9000, and the coproduction of nisin A and EntP in L. lactis subsp. lactis DPC5598. The level of production of EntP, detected and quantified by specific anti-EntP antibodies and a noncompetitive indirect enzyme-linked immunosorbent assay, by the recombinant L. lactis strains depended on the host strain, the expression vector, and the presence of the entiP gene in the constructs of the recombinant L. lactis strains. The highest amount of EntP was produced with derivatives containing entP and entiP, for both L. lactis IL1403 and L. lactis NZ9000. These derivatives produced up to five- to six-fold more EntP than E. faecium P13. Mass spectrometry analysis revealed that EntP purified from L. lactis IL1403 (pJP214) has a molecular mass identical to that purified from E. faecium P13, suggesting that the synthesis, processing, and secretion of EntP progresses efficiently in recombinant L. lactis hosts.
Samples of the intestinal content and carcasses of wood pigeons (Columba palumbus) were evaluated for enterococci with antimicrobial activity. Enterococcus faecium comprised the largest enterococcal species with antagonistic activity, followed by Enterococcusfaecalis and Enterococcus columbae. PCR amplification of genes coding bacteriocins and determination of their nucleotide sequence, and the use of specific antipeptide bacteriocin antibodies and a noncompetitive indirect enzyme-linked immunosorbent assay, permitted characterization of enterococci coding that described bacteriocins and their expression. The efaAfm determinant was the only virulence gene detected in E. faecium, whereas E. faecalis showed a larger number of virulence determinants, and E. columbae did not carry any of the virulence genes examined. Although all E. faecalis isolates manifested a potent direct antimicrobial activity, no activity was detected in supernatants of producer cells. Purification of the antagonistic activity of E. columbae PLCH2 showed multiple chromatographic fragments after matrix-assisted laser desorption-ionization time-of-flight mass spectrometry analysis, suggesting the active peptide(s) had not yet purified to homogeneity. Bacteriocinogenic E. faecium and E. columbae isolates may be considered hygienic for production of enterocins and potentially safe due to their low incidence of potential virulence genes and susceptibility of most relevant clinical antibiotics. However, the presence among the enterococci of E. faecalis strains with a potent antagonistic activity and multiple virulence factors is an issue that must be considered further.
PX Marques, P Souda, J O'Donovan, J Gutierrez, EJ Williams, S Worrall, M McElroy, A Proctor, C Brady, D Sammin, HF Basset, JP Whitelegge, BE Markey, JE Nally (2010)Identification of immunologically relevant proteins of Chlamydophila abortus using sera from experimentally infected pregnant ewes (Clinical and Vaccine Immunology (2010) 17, 8, (1274-1281)), In: Clinical and Vaccine Immunology17(9)pp. 1491-?
The objectives of this study were to evaluate the efficacy of plant essential oils (EOs) for control of the natural spoilage microflora on ready-to-eat (RTE) lettuce and carrots whilst also considering their impact on organoleptic properties. Initial decontamination effects achieved using EOs were comparable to that observed with chlorine and solution containing oregano recorded a significantly lower initial TVC level than the water treatment on carrots (p < 0.05). No significant differences were found between the EO treatments and chlorine considering gas composition, color, texture and water activity of samples. The sensory panel found EO treatments acceptable for carrots throughout storage, while lettuce washed with the EO solutions were rejected for overall appreciation by Day 7. Correlating microbial and sensory changes with volatile emissions identified 12 volatile quality markers. Oregano might be a suitable decontamination alternative to chlorine for RTE carrots, while the identification of volatile quality markers is a useful complement to sensory and microbiological assessments in the monitoring of organoleptic property changes and shelf-life of fresh vegetables. Industrial relevance: There is industrial demand for natural alternatives to chlorine, which is commonly used for decontamination of fresh produce but which has limitations with respect to antimicrobial efficacy and possible formation of carcinogenic compounds in water. Plant essential oils have proven antimicrobial and other bioactive properties, however their usefulness in foods can be mitigated by their high sensory impact. This study examined the application of EOs for fresh produce decontamination addressing control of spoilage microflora and improving shelf-life characteristics whilst also considering the impact on organoleptic properties. The effectiveness of oregano as a decontamination treatment was comparable with that of chlorine. Carrot discs treated with the EO regimes were acceptable in terms of sensory quality and appreciation, therefore oregano could offer a natural alternative for the washing and preservation of fresh produce. Combining EOs with other natural preservatives might minimize doses and reduce the impact on organoleptic properties of fresh vegetables. © 2008 Elsevier Ltd. All rights reserved.