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Horses are exposed to various kinds of medication, however, there are limited determinations of plasma clearance (CLp) for the drugs used due to the high cost of equine in vivo studies.Many of the ...

Footrot is a polymicrobial infectious disease in sheep causing severe lameness, leading to one of the industry’s largest welfare problems. The complex etiology of footrot makesin situorin vitroinvestigations difficult. Computational methods offer a solution to understanding the bacteria involved and how they may interact with the host, ultimately providing a way to identify targets for future hypothesis-driven investigative work. Here, we present the first combined global analysis of bacterial community transcripts together with the host immune response in healthy and diseased ovine feet during a natural polymicrobial infection state using metatranscriptomics. The intratissue and surface bacterial populations and the most abundant bacterial transcriptomes were analyzed, demonstrating that footrot-affected skin has reduced diversity and increased abundances of not only the causative bacteriumDichelobacter nodosusbut also other species such asMycoplasma fermentansandPorphyromonas asaccharolytica. Host transcriptomics reveals the suppression of biological processes related to skin barrier function, vascular functions, and immunosurveillance in unhealthy interdigital skin, supported by histological findings that type I collagen (associated with scar tissue formation) is significantly increased in footrot-affected interdigital skin compared to outwardly healthy skin. Finally, we provide some interesting indications of host and pathogen interactions associated with virulence genes and the host spliceosome, which could lead to the identification of future therapeutic targets.

Ovine footrot, is a highly contagious polymicrobial bacterial infection, primarily caused by Dichelobacter nodosus. Preventative bactericidal footbaths are commonly used in the sheep industry to reduce the spread of bacteria. However, their effect on the bacterial community is poorly understood. This is the first study to investigate the impact of 2% Digicur (ProGiene,UK) footbath on the bacterial community of the ovine interdigital skin following a common UK footbathing routine. Swab samples were analysed by qPCR to determine prevalence and load of D. nodosus and numerated on MacConkey agar in the presence or absence of tetracycline and ampicillin to determine phenotypic antimicrobial resistance. Metagenomics were used to determine the impact of a single footbath on the bacterial community and genotypic antimicrobial resistance. The results suggest 2% Digicur is ineffective at reducing the load of D. nodosus when applied as a one off or weekly footbath, however sheep may act as a reservoir for multi-drug resistant bacteria creating opportunities to spread antimicrobial resistance to other sheep and their environment.

Listeria monocytogenes is an important foodborne pathogen in human and veterinary health, causing significant morbidity and mortality including abortion. It has a particular tropism for the gravid uterus, however, the route of infection in reproductive tissues of ruminants (i.e. placentome), is much less clear. In this study, we aimed to investigate a bovine caruncular epithelial cell (BCEC) line as a model for L. monocytogenes infection of the bovine reproductive tract. The BCEC infection model was used to assess the ability of 14 different L. monocytogenes isolates to infect these cells. Lysozyme sensitivity and bacterial survival in 580 μg lysozyme/ml correlated with attenuated ability to proliferate in BCEC (p = 0.004 and p = 0.02, respectively). Four isolates were significantly attenuated compared to the control strain 10403S. One of these strains (AR008) showed evidence of compromised cell wall leading to increased sensitivity to ß-lactam antibiotics, and another (7644) had compromised cell membrane integrity leading to increased sensitivity to cationic peptides. Whole genome sequencing followed by Multi Locus Sequence Type analysis identified that five invasive isolates had the same sequence type, ST59, despite originating from three different clinical conditions. Virulence gene analysis showed that the attenuated isolate LM4 was lacking two virulence genes (uhpT, virR) known to be involved in intracellular growth and virulence. In conclusion, the BCEC model was able to differentiate between the infective potential of different isolates. Moreover, resistance to lysozyme correlated with the ability to invade and replicate within BCEC, suggesting co-selection for surviving challenging environments as the abomasum., Bioinformatics; Genetics; Microbiology; Molecular biology; Listeria monocytogenes; infection model; BCEC; MLST, sequence typing.

Background Footrot and interdigital dermatitis are endemic infectious diseases in all sheep farming regions, impairing welfare and production. The development of efficacious vaccines against the primary causative pathogen has been hampered by the extensive antigenic diversity of Dichelobacter nodosus. Understanding the heterogeneity of the pathogen within and between flocks is essential if the feasibility of bespoke vaccine production is to be assessed for use in the U.K. Results In this study 56 ewe and lamb isolates from 9 flocks were compared by D. nodosus serogroup and Multi Locus Sequence Type which provides significantly enhanced discriminatory power for molecular epidemiology. Serogroup heterogeneity between flocks ranged from two to five unique serogroups per flock. Three flocks contained isolates of two serogroups, two flocks contained isolates of three serogroups and one flock included isolates of five serogroups. Analysis of 25 isolates from one flock with high prevalence of lameness, identified that serogroup and sequence type was significantly correlated with age. Significantly higher proportion of lambs were infected with serogroup B (principally ST85) as opposed to serogroup H (principally ST86), which predominated amongst adult sheep. Conclusions Genomic heterogeneity of the pathogen was significantly lower within flock compared to heterogenicity observed between flocks. Furthermore, this study indicates that within a flock, the host-pathogen dynamics and susceptibility to particular D. nodosus strains may be age dependent.

Background Footrot and interdigital dermatitis are endemic infectious diseases in all sheep farming regions, impairing welfare and production. The development of efficacious vaccines against the primary causative pathogen has been hampered by the extensive antigenic diversity of Dichelobacter nodosus . Understanding the heterogeneity of the pathogen within and between flocks is essential if the feasibility of bespoke vaccine production is to be assessed for use in the U.K. Results In this study 56 ewe and lamb isolates from 9 flocks were compared by D. nodosus serogroup and Multi Locus Sequence Type which provides significantly enhanced discriminatory power for molecular epidemiology. Serogroup heterogeneity between flocks ranged from two to five unique serogroups per flock. Three flocks contained isolates of two serogroups, two flocks contained isolates of three serogroups and one flock included isolates of five serogroups. Analysis of 25 isolates from one flock with high prevalence of lameness, identified that serogroup and sequence type was significantly correlated with age. Significantly higher proportion of lambs were infected with serogroup B (principally ST85) as opposed to serogroup H (principally ST86), which predominated amongst adult sheep. Conclusions Genomic heterogeneity of the pathogen was significantly lower within flock compared to heterogenicity observed between flocks. Furthermore, this study indicates that within a flock, the host-pathogen dynamics and susceptibility to particular D. nodosus strains may be age dependent. Keywords D. nodosus; Footrot; interdigital dermatitis; lameness; sheep; MLST; Serogroup; molecular epidemiology

Listeria monocytogenesis an important foodborne pathogen in human and veterinary health, causing significant morbidity and mortality including abortion. It has a particular tropism for the gravid uterus, however, the route of infection in reproductive tissues of ruminants (i.e. placentome), is much less clear. In this study, we aimed to investigate a bovine caruncular epithelial cell (BCEC) line as a model forL. monocytogenesinfection of the bovine reproductive tract. The BCEC infection model was used to assess the ability of 14 differentL. monocytogenesisolates to infect these cells. Lysozyme sensitivity and bacterial survival in 580 µg lysozyme/ml correlated with attenuated ability to proliferate in BCEC (p=0.004 and p=0.02, respectively). Four isolates were significantly attenuated compared to the control strain 10403S. One of these strains (AR008) showed evidence of compromised cell wall leading to increased sensitivity to ß-lactam antibiotics, and another (7644) had compromised cell membrane integrity leading to increased sensitivity to cationic peptides. Whole genome sequencing followed by Multi Locus Sequence Type analysis identified that five invasive isolates had the same sequence type, ST59, despite originating from three different clinical conditions. Virulence gene analysis showed that the attenuated isolate LM4 was lacking two virulence genes (uhpT,virR) known to be involved in intracellular growth and virulence.In conclusion, the BCEC model was able to differentiate between the infective potential of different isolates. Moreover, resistance to lysozyme correlated with the ability to invade and replicate within BCEC, suggesting co-selection for surviving challenging environments as the abomasum.

1. Scaling factor values for the in vitro-in vivo extrapolation of hepatic metabolic clearance for xenobiotics have not yet been determined in horses. Scaling factors were determined by comparing the total protein and or cytochrome (CYP) P450 content in microsomes and cryopreserved hepatocytes against the content in the liver. 2. Microsomal protein per gram of liver (MPPGL) and hepatocellularity number per gram of liver (HPGL) using CYP P450 content method ranged 41–73 mg/gram of liver (mean= 57 mg/gram of liver, n = 39) and 146–320 × 106 cells/g of liver (mean = 227× 106 cells/g of liver, n = 18), respectively and 156–352 × 106 cells/g of liver (mean = 232× 106 cells/g of liver) using total protein method. 3. A non-monotonic and inverse relationship between age and MPPGL and HPGL, respectively, was observed. Between one and 20 y of age, the liver cell size decreases as age increases. Subsequently, the cell size increases until the hepatocytes of the oldest horses approached the size found in the youngest horses. Hepatocyte density was inversely related to the size of the hepatocytes. 4. This study provides the first extensive and comprehensive data demonstrating the relationship between the size of hepatocytes and HPGL in any species.

The first year of university is critical in shaping persistence decisions (whether students continue with and complete their degrees) and plays a formative role in influencing student attitudes and approaches to learning. Previous educational experiences, especially previous university education, shape the students' ability to adapt to the university environment and the study approaches they require to perform well in highly demanding professional programs such as medicine and veterinary medicine. The aim of this research was to explore the support mechanisms, academic achievements, and perception of students with different educational backgrounds in their first year of veterinary school. Using questionnaire data and examination grades, the effects upon perceptions, needs, and educational attainment in first-year students with and without prior university experience were analyzed to enable an in-depth understanding of their needs. Our findings show that school leavers (successfully completed secondary education, but no prior university experience) were outperformed in early exams by those who had previously graduated from university (even from unrelated degrees). Large variations in student perceptions and support needs were discovered between the two groups: graduate students perceived the difficulty and workload as less challenging and valued financial and IT support. Each student is an individual, but ensuring that universities understand their students and provide both academic and non-academic support is essential. This research explores the needs of veterinary students and offers insights into continued provision of support and improvements that can be made to help students achieve their potential and allow informed “Best Practice.”

Listerial keratoconjunctivitis (‘silage eye’) is a wide spread problem in ruminants causing economic losses to farmers and impacts negatively on animal welfare. It results from direct entry of Listeria monocytogenes into the eye, often following consumption of contaminated silage. An isolation protocol for bovine conjunctival swabbing was developed and used to sample both infected and healthy eyes bovine eyes ( n = 46). L. monocytogenes was only isolated from one healthy eye sample, and suggests that this organism can be present without causing disease. To initiate a study of this disease, an infection model was developed using isolated conjunctiva explants obtained from cattle eyes post slaughter. Conjunctiva were cultured and infected for 20 h with a range of L. monocytogenes isolates ( n = 11), including the healthy bovine eye isolate and also strains isolated from other bovine sources, such as milk or clinical infections. Two L. monocytogenes isolates (one from a healthy eye and one from a cattle abortion) were markedly less able to invade conjunctiva explants, but one of those was able to efficiently infect Caco2 cells indicating that it was fully virulent. These two isolates were also significantly more sensitive to lysozyme compared to most other isolates tested, suggesting that lysozyme resistance is an important factor when infecting bovine conjunctiva. In conclusion, we present the first bovine conjunctiva explant model for infection studies and demonstrate that clinical L. monocytogenes isolates from cases of bovine keratoconjunctivitis are able to infect these tissues.

High throughput sequencing, and quantitative polymerase chain reaction (qPCR), can detect changes in bacterial communities or the genes that they carry, between different environments or treatments. These methods are applied widely to microbiomes in humans, animals, soil and water; an important application is for changes in antimicrobial resistance genes (ARGs). However, at present, there is no statistical method to determine whether observed changes in the overall composition are significant, or result from random variations between samples. Therefore researchers are limited to graphical descriptions. We describe a novel statistical method to determine whether or not observed differences in bacterial populations or their genes are significant. It can be used with data from shotgun metagenomics, 16S characterisation or qPCR. It can also be used for experimental design, to calculate the number of samples needed in future experiments. We show its application to two example data sets. The first is published data on bacterial communities and ARGs in the environment, in which we show that there are significant changes in both ARG and community composition. The second is a new data set on seasonality in bacterial communities and ARGs in hooves from four sheep. While the observed differences are not significant, we show that a minimum group size of eight sheep in a future experiment would provide sufficient power to observe significant changes, should the already observed changes be true. This method has broad uses for statistical testing and experimental design in experiments on changing microbiomes, including for studies on antimicrobial resistance.

Skin infection studies are often limited by financial and ethical constraints, and alternatives, such as monolayer cell culture, do not reflect many cellular processes limiting their application. For a more functional replacement, 3D skin culture models offer many advantages such as the maintenance of the tissue structure and the cell types present in the host environment. A 3D skin culture model can be set up using tissues acquired from surgical procedures or post slaughter, making it a cost effective and attractive alternative to animal experimentation. The majority of 3D culture models have been established for aerobic pathogens, but currently there are no models for anaerobic skin infections. Footrot is an anaerobic bacterial infection which affects the ovine interdigital skin causing a substantial animal welfare and financial impact worldwide. Dichelobacter nodosus is a Gram-negative anaerobic bacterium and the causative agent of footrot. The mechanism of infection and host immune response to D. nodosus is poorly understood. Here we present a novel 3D skin ex vivo model to study anaerobic bacterial infections using ovine skin explants infected with D. nodosus. Our results demonstrate that D. nodosus can invade the skin explant, and that altered expression of key inflammatory markers could be quantified in the culture media. The viability of explants was assessed by tissue integrity (histopathological features) and cell death (DNA fragmentation) over 76 h showing the model was stable for 28 h. D. nodosus was quantified in all infected skin explants by qPCR and the bacterium was visualized invading the epidermis by Fluorescent in situ Hybridization. Measurement of pro-inflammatory cytokines/chemokines in the culture media revealed that the explants released IL1β in response to bacteria. In contrast, levels of CXCL8 production were no different to mock-infected explants. The 3D skin model realistically simulates the interdigital skin and has demonstrated that D. nodosus invades the skin and triggered an early cellular inflammatory response to this bacterium. This novel model is the first of its kind for investigating an anaerobic bacterial infection.

Ovine footrot is a highly prevalent bacterial disease caused by Dichelobacter nodosus and characterised by the separation of the hoof horn from the underlying skin. The role of innate immune molecules and other bacterial communities in the development of footrot lesions remains unclear. This study shows a significant association between the high expression of IL1β and high D. nodosus load in footrot samples. Investigation of the microbial population identified distinct bacterial populations in the different disease stages and also depending on the level of inflammation. Treponema (34%), Mycoplasma (29%) and Porphyromonas (15%) were the most abundant genera associated with high levels of inflammation in footrot. In contrast, Acinetobacter (25%), Corynebacteria (17%) and Flavobacterium (17%) were the most abundant genera associated with high levels of inflammation in healthy feet. This demonstrates for the first time there is a distinct microbial community associated with footrot and high cytokine expression.

Background Toll like receptors are one of the major innate immune system pathogen recognition systems. There is little data on the expression of the TLR10 member of this family in the horse. Results This paper describes the genetic structure of the Equine TLR10 gene and its RNA expression in a range of horse tissues. It describes the phylogenetic analysis of the Equine TLR1,6,10,2 annotations in the horse genome, firmly identifying them in their corresponding gene clades compared to other species and firmly placing the horse gene with other TLR10 genes from odd-toed ungulates. Additional 3’ transcript extensions to that annotated for TLR10 in the horse genome have been identified by analysis of RNAseq data. RNA expression of the equine TLR10 gene was highest in peripheral blood mononucleocytes and lymphoid tissue (lymph nodes and spleen), however some expression was detected in all tissues tested (jejunum, caudal mesenteric lymph nodes, bronchial lymph node, spleen, lung, colon, kidney and liver). Additional data on RNAseq expression of all equine TLR genes (1–4 and 6–10) demonstrate higher expression of TLR4 than other equine TLRs in all tissues. Conclusion The equine TLR10 gene displays significant homology to other mammalian TLR10 genes and could be reasonably assumed to have similar fuctions. Its RNA level expression is higher in resting state PBMCs in horses than in other tissues. Electronic supplementary material The online version of this article (doi:10.1186/s13104-016-2161-9) contains supplementary material, which is available to authorized users.

Ovine footrot is characterised by interdigital dermatitis (ID) and by the separation of the skin and hoof horn (under-running footrot). Dichelobacter nodosus is the essential pathogen causing footrot; the role of other microorganisms in this disease remains unclear. The aims of this study were (i) to investigate the colonisation of D nodosus, Fusobacterium necrophorum and Treponema species in biopsies from the ovine interdigital skin of healthy, ID and footrot-affected feet and (ii) to characterise the virulence of D nodosus strains in those biopsies. Postslaughter biopsy samples (n=241) were collected and analysed by real-time PCR to determine prevalence and load of the different bacterial species. The highest prevalence and load of D nodosus were found on feet with ID. The vast majority of samples contained virulent D nodosus and some samples contained both virulent and benign D nodosus. Notably, the more pathogenic subspecies of F necrophorum was found in samples from UK sheep. Our findings provide further insights into the role bacterial colonisation may play in the early stage of ID and in the progression towards footrot.

Transition into higher education requires students to adjust to a new environment while showing greater independence in managing their own academic and personal life. This is often more difficult for international students who have to adjust to a different country, culture, and potentially another language. A cohort of first-year veterinary medicine students (17% international students) was investigated at a UK university using qualitative and quantitative questionnaires rating first-year experience and support services and statistical analysis of students' assessment performance. While the overall undergraduate perception was that they had learned a lot and progressed well, students in both groups struggled to cope with the workload. The non-UK educated students and students with English as a foreign language also struggled more with teaching delivery in lectures and participation in self-directed group learning and were more likely to feel that the veterinary degree program was too difficult. There was no statistical difference in how British and international students perceived the support system, although it was noticeable that the level of tutorial support was perceived as tutor-dependent. The international students particularly struggled with the assessments in early modules and also with the spot assessment method. However, in the practical assessments, using observed, structured practical exam stations, international and British students performed equally well. Increased support in the initial transition time, especially with regard to communication skills and confidence required for interactive teaching and learning environments such as small-group teaching, as well as increased time for specific assessment types, might benefit the needs of many international students.

Ovine footrot is a degenerative disease of sheep feet leading to the separation of hoof-horn from the underlying skin and lameness. This study quantitatively examined histological features of the ovine interdigital skin as well as their relationship with pro-inflammatory cytokine (IL-1β) and virulent Dichelobacter nodosus in footrot. From 55 healthy and 30 footrot ovine feet, parallel biopsies (one fixed for histology) were collected post-slaughter and analysed for lesions and histopathological analysis using haematoxylin and eosin and Periodic Acid-Schiff. Histological lesions were similar in both conditions while inflammatory scores mirror IL-1β expression levels. Increased inflammatory score corresponded with high virulent D. nodosus load and was significant (p D. nodosus load.

Toll-like receptor-4 (TLR4) is important in protection against lethal Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. Control of the early stages of sublethal S. Typhimurium infection in mice depends on TLR4-dependent activation of macrophages and natural killer (NK) cells to drive an inflammatory response. TLR4 signals through the adapter proteins Mal/MyD88 and TRIF-related adaptor molecule (TRAM)/TIR-domain-containing adaptor-inducing interferon-b (TRIF). In the mouse typhoid model we showed that TLR4 and MyD88, but not Mal or TRIF, are essential for the control of exponential S. Typhimurium growth. TRIF(-/-) mice have a higher bacterial load in comparison with wild-type mice during a sublethal infection because TRIF is important for bacterial killing during the first day of systemic disease. Minimal pro-inflammatory responses were induced by S. Typhimurium infection of macrophages from TLR4(-/-), MyD88(-/-) and TRIF(-/-) mice in vitro. Pro-inflammatory responses from Mal(-/-) macrophages were similar to those from wild-type cells. The pro-inflammatory responses of TRIF(-/-) macrophages were partially restored by the addition of interferon-gamma (IFN-gamma), and TRIF(-/-) mice produced markedly enhanced IFN-gamma levels, in comparison to wild-type mice, probably explaining why bacterial growth can be controlled in these mice. TLR4(-/-), MyD88(-/-), TRIF(-/-) and Mal(-/-) mice all initiated clearance of S. Typhimurium, suggesting that TLR4 signalling is not important in driving bacterial clearance in comparison to its critical role in controlling early bacterial growth in mouse typhoid.

Salmonella enterica serovar Typhimurium (S. Typhimurium) infection causes an inflammatory response through activation of Toll-like receptor 4 by lipopolysaccharide. Dexamethasone, a glucocorticoid analogue, suppresses inflammatory responses by many mechanisms including inhibition of the lipopolysaccharide-induced production of proinflammatory mediators. There is little information on the effect of glucocorticoids on murine salmonellosis. In this study, we treated susceptible BALB/c mice by subcutaneous implantation of slow-release dexamethasone pellets before infection with S. Typhimurium. Dexamethasone promotes bacterial growth early in infection and induces a dose-dependent increase in bacterial growth within mouse livers and spleens. The bacterial load in organs from infected placebo-treated mice was lower than that in dexamethasone-treated mice. Glucocorticoids inhibit lipopolysaccharide-induced inflammation partially through the steroid-inducible protein annexin-A1 (ANXA1). Infection of wild-type and ANXA1 knock-out mice with S. Typhimurium led to similar organ bacterial loads. ANXA1 also did not affect the bacterial load in organs from infected dexamethasone-treated mice. This suggests that glucocorticoids, independently of ANXA1, accelerate S. Typhimurium growth in vivo in susceptible BALB/c mice.

Salmonella enterica serovar typhimurium (S. typhimurium) is an intracellular pathogen that causes macrophage cell death by at least two different mechanisms. Rapid cell death is dependent on the Salmonella pathogenicity island-1 protein SipB whereas delayed cell death is independent of SipB and occurs 18-24 hr post infection. Lipopolysaccharide (LPS) is essential for the delayed cell death. LPS is the main structural component of the outer membrane of Gram-negative bacteria and is recognized by Toll-like receptor 4, signalling via the adapter proteins Mal, MyD88, Tram and Trif. Here we show that S. typhimurium induces SipB-independent cell death through Toll-like receptor 4 signalling via the adapter proteins Tram and Trif. In contrast to wild type bone marrow derived macrophages (BMDM), Tram(-/-) and Trif(-/-) BMDM proliferate in response to Salmonella infection.

Annexin-1 (ANXA1) is a glucocorticoid-regulated protein that modulates the effects of bacterial lipopolysaccharide (LPS) on macrophages. Exogenous administration of peptides derived from the N-terminus of ANXA1 reduces LPS-stimulated inducible nitric oxide synthase (iNOS) expression, but the effects of altering the endogenous expression of this protein are unclear. We transfected RAW264.7 murine macrophage-like cell lines to over-express constitutively ANXA1 and investigated whether this protein modulates the induction of iNOS, cyclooxygenase-2 (COX-2) and tumour necrosis factor-alpha (TNF-alpha) in response to LPS. In contrast to exogenous administration of N-terminal peptides, endogenous over-expression of ANXA1 results in up-regulation of LPS-induced iNOS protein expression and activity. However, levels of iNOS mRNA are unchanged. ANXA1 has no effect on COX-2 or TNF-alpha production in response to LPS. In experiments to investigate the mechanisms underlying these phenomena we observed that activation of signalling proteins classically associated with iNOS transcription was unaffected. Over-expression of ANXA1 constitutively activates extracellular signal regulated kinase (ERK)-1 and ERK-2, components of a signalling pathway not previously recognized as regulating LPS-induced iNOS expression. Inhibition of ERK activity, by the inhibitor U0126, reduced LPS-induced iNOS expression in our cell lines. Over-expression of ANXA1 also modified LPS-induced phosphorylation of the ERK-regulated translational regulation factor eukaryotic initiation factor 4E. Our data suggest that ANXA1 may modify iNOS levels by post-transcriptional mechanisms. Thus differential effects on iNOS expression in macrophages are seen when comparing acute administration of ANXA1 peptides versus the chronic endogenous over-expression of ANXA1.

Campylobacter jejuni can cause an enteritis that is associated with an acute inflammatory response at the gut epithelial surface. The signals inducing inflammation are unknown. C. jejuni can penetrate the intestinal epithelial barrier and may then interact with leucocytes, potentially inducing proinflammatory responses. To investigate this, we studied the interaction of C. jejuni with the human monocytic cell line THP-1 and show that a range of proinflammatory cytokines and chemokines is induced. These include interleukin-1α (IL-1α), IL-1β, IL-6, IL-8, and tumor necrosis factor alpha. Responses can be induced by killed Campylobacter as well as live bacteria and do not depend on the cytolethal distending toxin. C. jejuni infection of THP-1 cells triggers both nuclear translocation of functional NF-κB and stimulation of IL-1α, indicating that NF-κB-dependent and -independent stimulation is occurring. The extent of proinflammatory cytokine stimulation suggests that monocytes might significantly contribute to intestinal inflammation and disease pathology.

Footrot is a common inflammatory bacterial disease affecting the health and welfare of sheep worldwide. The pathogenesis of footrot is complex and multifactorial. The primary causal pathogen is the anaerobic bacterium Dichelobacter nodosus, with Fusobacterium necrophorum also shown to play a key role in disease. Since immune-mediated pathology is implicated, the aim of this research was to investigate the role of the host response in interdigital dermatitis (ID) and footrot. We compared the expression of Toll-like receptors (TLRs) and pro-inflammatory cytokines and the histological appearance of clinically normal in comparison to ID and footrot affected tissues. Severe ID and footrot were characterised by significantly increased transcript levels of pro-inflammatory cytokines TNFα and IL1β and the pattern recognition receptors TLR2 and TLR4 in the interdigital skin. This was reflected in the histopathological appearance, with ID and footrot presenting progressive chronic-active pododermatitis with a mixed lymphocytic and neutrophilic infiltration, gradually increasing from a mild form in clinically normal feet, to moderate in ID and to a focally severe form with frequent areas of purulence in footrot. Stimulation with F. necrophorum and/or D. nodosus extracts demonstrated that dermal fibroblasts, the resident cell type of the dermis, also contribute to the inflammatory response to footrot bacteria by increased expression of TNFα, IL1β and TLR2. Overall, ID and footrot lead to a local inflammatory response given that expression levels of TLRs and IL1β were dependent on the disease state of the foot not the animal.

Weaning is a stressful period for piglets and is associated with gastrointestinal disruption and increased disease susceptibility. This study investigated the influence of probiotics on gut health in young pigs and focused on 1) animal production values, 2) microbiology, 3) gut architecture, mucosal cell distribution and mucus secretion and 4) modulation of stress response. In this study, two litters of piglets were weaned and mixed pair wise with non litter-mates. Two pairs of piglets were fed a successive probiotic supplement of Saccharomyces cerevisiae ssp. boulardii (CNCM I-1079) and Pediococcus acidilactici (CNCM MA 18/5 M), and two pairs a non-supplemented diet. The probiotic regime had positive effects on feed conversion ratio (FCR). Villus length, crypt depth, mucus-producing cell counts and the thickness of the mucus layer remained unaffected in the small intestine; however, Escherichia coli levels were reduced dramatically and transiently compared to non-supplemented diet after 4 weeks supplementation with probiotic.

The Yersinia Ysc apparatus is made of more than 20 proteins, 11 of which have homologues in many type III systems. Here, we characterize YscP from Yersinia enterocolitica. This 515-residue protein has a high proline content, a large tandem repetition and a slow migration in SDS-PAGE. Unlike the products of neighbouring genes, it has a counterpart only in Pseudomonas aeruginosa and it varies even between Yersinia Ysc machineries. An yscPDelta97-465 mutant was unable to secrete any Yop, even under conditions overcoming feedback inhibition of Yop synthesis. Interestingly, a cloned yscPDelta57-324 from Yersinia pestis introduced in the yscPDelta97-465 mutant can sustain a significant Yop secretion and thus partially complemented the mutation. This explains the leaky phenotype observed with the yscP mutant of Y. pestis. In accordance with this secretion deficiency, YscP is required for the delivery of Yop effectors into macrophages. Mechanical shearing, immunolabelling and electron microscopy experiments showed that YscP is exposed at the bacterial surface when bacteria are incubated at 37 degrees C in the presence of Ca2+ and thus do not secrete Yops. At 37 degrees C, when Ca2+ ions are chelated, YscP is released like a Yop protein. We conclude that YscP is a part of the Ysc injectisome which is localized at the bacterial surface and is destabilized by Ca2+ chelation.

Methylglyoxal is a toxic electrophile. In Escherichia coli cells, the principal route of methylglyoxal production is from dihydroxyacetone phosphate by the action of methylglyoxal synthase. The toxicity of methylglyoxal is believed to be due to its ability to interact with the nucleophilic centres of macromolecules such as DNA. Bacteria possess an array of detoxification pathways for methylglyoxal. In E. coli, glutathione-based detoxification is central to survival of exposure to methylglyoxal. The glutathione-dependent glyoxalase I-II pathway is the primary route of methylglyoxal detoxification, and the glutathione conjugates formed can activate the KefB and KefC potassium channels. The activation of these channels leads to a lowering of the intracellular pH of the bacterial cell, which protects against the toxic effects of electrophiles. In addition to the KefB and KefC systems, E. coli cells are equipped with a number of independent protective mechanisms whose purpose appears to be directed at ensuring the integrity of the DNA. A model of how these protective mechanisms function will be presented. The production of methylglyoxal by cells is a paradox that can be resolved by assigning an important role in adaptation to conditions of nutrient imbalance. Analysis of a methylglyoxal synthase-deficient mutant provides evidence that methylglyoxal production is required to allow growth under certain environmental conditions. The production of methylglyoxal may represent a high-risk strategy that facilitates adaptation, but which on failure leads to cell death. New strategies for antibacterial therapy may be based on undermining the detoxification and defence mechanisms coupled with deregulation of methylglyoxal synthesis.

ENTRY to veterinary school as an undergraduate student continues to be highly desirable, with application numbers far outweighing the number of places available in the UK. Applicants have often had ambitions to be vets since early childhood and have undergone extensive work experience placements to gain animal husbandry experience and insight into the profession. However, the veterinary profession, as well as the veterinary degree course, is not only rewarding but also very demanding. Similar to medical students, veterinary students have more pressures compared to students on many other courses (Dent and Rennie 2005). Contributing to this is the course content and high workload; the wide range of skills required; the expectation to behave like a professional and be judged accordingly; having to communicate effectively with a wide range of people; and having to deal with emotions in difficult situations including life/death decisions. Universities, therefore, aim to recruit students who are academic high achievers who can also perform well in problem-based and practical tasks. It is important that students have a good insight into the profession, including its challenging aspects and the high levels of professionalism required. Veterinary undergraduates should also have an awareness of the wide range of career options available in addition to being a veterinary practitioner, and the need to be able to demonstrate effective leadership of …

Veterinary students face an increasing array of pressures and this has been reflected in the findings of several surveys which demonstrate high levels of stress amongst this population (Hafen and others 2008, Mellanby and others 2010, Totemeyer and others 2012). In response to this and the inclusion of ‘student support’ in the National Student Survey, there is an increased need for effective pastoral support in Universities. Improved communication between pastoral support staff and veterinary students is therefore essential to identify the causes of stress and to develop effective support management and solutions. Ultimately, good support will make problems manageable, may improve behaviour and enable successful graduation. It will also provide students with the coping skills to thrive in clinical practice where different stresses apply (Platt and others 2010, 2012, Hawton and others 2011, Bartram and others 2012, ). Furthermore, identifying causes of stress and their effects on student wellbeing will enable veterinary schools and the Royal College of Veterinary Surgeons (RCVS) to promote best practice and inform future changes to the curriculum and student support policies. A workshop was held involving relevant staff from all seven veterinary schools in the UK and representatives from the AVS. The aims were to identify and discuss common issues associated with the …

Exposure to interferon results in the rapid transcriptional induction of genes, many of which function to create an antiviral environment in potential host cells. For the majority of adenoviruses, replication is unaffected by the actions of interferon. It has previously been shown, using non-gastrointestinal cells, that the species F human adenoviruses are sensitive to the action of interferon. Here, we have developed an enterocyte-like cell-culture model to re-evaluate this question, and determined the effects of interferon on species F adenovirus during infection of gastrointestinal cells. We show that species F adenovirus type 40 is sensitive to the effects of interferon in gastrointestinal-like cells, which may help to explain its fastidious growth in culture.

Problem-based or case-based learning is a popular method of instruction in clinical degrees such as veterinary science, nursing, and medicine. It is difficult, however, for students to adapt to this learning method, and this difficulty has been well described. The present study surveyed first-year undergraduate veterinary students at the University of Nottingham about the challenges they faced upon beginning problem-based learning sessions. A surprisingly large percentage of students (36% of females and 38% of males) reported a lack of confidence in speaking in front of the other students as a concern they experienced during their first term. Conversely, only 10% of the female students (and none of the male students) reported overconfidence as a problem. This is in contrast to the perceptions of the staff members who facilitated the sessions who reported that 14% of the students exhibited underconfidence and 14% exhibited overconfidence. The difference between the female and male students' responses as well as the difference between the perceptions of students and those of facilitators is statistically significant (G-test p

Toll-like receptor-4 (TLR4) is important in protection against lethal Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. Control of the early stages of sublethal S. Typhimurium infection in mice depends on TLR4-dependent activation of macrophages and natural killer (NK) cells to drive an inflammatory response. TLR4 signals through the adapter proteins Mal/MyD88 and TRIF-related adaptor molecule (TRAM)/TIR-domain-containing adaptor-inducing interferon-b (TRIF). In the mouse typhoid model we showed that TLR4 and MyD88, but not Mal or TRIF, are essential for the control of exponential S. Typhimurium growth. TRIF(-/-) mice have a higher bacterial load in comparison with wild-type mice during a sublethal infection because TRIF is important for bacterial killing during the first day of systemic disease. Minimal pro-inflammatory responses were induced by S. Typhimurium infection of macrophages from TLR4(-/-), MyD88(-/-) and TRIF(-/-) mice in vitro. Pro-inflammatory responses from Mal(-/-) macrophages were similar to those from wild-type cells. The pro-inflammatory responses of TRIF(-/-) macrophages were partially restored by the addition of interferon-gamma (IFN-gamma), and TRIF(-/-) mice produced markedly enhanced IFN-gamma levels, in comparison to wild-type mice, probably explaining why bacterial growth can be controlled in these mice. TLR4(-/-), MyD88(-/-), TRIF(-/-) and Mal(-/-) mice all initiated clearance of S. Typhimurium, suggesting that TLR4 signalling is not important in driving bacterial clearance in comparison to its critical role in controlling early bacterial growth in mouse typhoid.

Toll-like receptor 4 (TLR4) senses bacterial LPS and is required for the control of systemic Salmonella enterica serovar Typhimurium infection in mice. The mechanisms of TLR4 activation and its downstream signalling cascades are well described, yet the direct effects on the pathogen of signalling via this receptor remain unknown. To investigate this we used microarray-based transcriptome profiling of intracellular S. Typhimurium during infection of primary bone marrow-derived macrophages from wild-type and TLR4-deficient mice. We identified 17 S. Typhimurium genes that were upregulated in the presence of functional TLR4. Nine of these genes have putative functions in oxidative stress resistance. We therefore examined S. Typhimurium gene expression during infection of NADPH oxidase-deficient macrophages, which lack normal oxidative killing mechanisms. We identified significant overlap between the ‘TLR4-responsive’ and ‘NADPH oxidase-responsive’ genes. This is new evidence for a link between TLR4 signalling and NADPH oxidase activity. Interestingly, with the exception of a dps mutant, S. Typhimurium strains lacking individual TLR4- and/or oxidative stress-responsive genes were not attenuated during intravenous murine infections. Our study shows that TLR4 activity, either directly or indirectly, induces the expression of multiple stress resistance genes during the intracellular life of S. Typhimurium.

Pattern recognition receptors are central to the responsiveness of various eukaryotic cell types when they encounter pathogen-associated molecular patterns. IFN-γ is a cytokine that is elevated in humans and other animals with bacterial infection and enhances the LPS-induced production of antibacterial mediators by macrophages. Mice lacking the pattern recognition receptor, TLR4, respond very poorly to stimulation by LPS, but administration of IFN-γ has been described as restoring apparent sensitivity to this stimulatory ligand. In this study, we show that IFN-γ primes murine macrophages stimulated by crude LPS preparations to produce the antibacterial mediator NO, a proportion of which is independent of TLRs 2 and 4. This response is lost in tlr4−/− IFN-γ-primed murine macrophages when the LPS preparation is highly purified. NO is also induced if chemically synthesized muramyl dipeptide, an intermediate in the biosynthesis of peptidoglycan, is used to stimulate macrophages primed with IFN-γ. This is absolutely dependent on the presence of a functional nucleotide oligomerization domain-2 (NOD-2) protein. IFN-γ increases NOD-2 expression and dissociates this protein from the actin cytoskeleton within the cell. IFN-γ priming of macrophages therefore reveals a key proinflammatory role for NOD-2. This study also shows that the effect of IFN-γ in restoring inflammatory responses to Gram-negative bacteria or bacterial products in mice with defective TLR4 signaling is likely to be due to a response to peptidoglycan, not LPS.

Sublethal infection of C57BL/6 mice with Salmonella enterica serovar Typhimurium M525P initiates a strong inflammatory response. We measured organ expression of mRNA for Toll-like receptors and their associated signaling molecules during S. enterica serovar Typhimurium infection. During infection, the Toll-lie receptor 1 (TLR1), TLR2, and TLR9 mRNA levels increased, while TLR6 mRNA expression decreased.

Invasion of macrophages by salmonellae induces cellular responses, with the bacterial inducers likely to include a number of pathogen-associated molecular patterns. LPS is one of the prime candidates, but its precise role in the process, especially when presented as a component of live infecting bacteria, is unclear. We thus investigated this question using the lipid A antagonist E5531, the macrophage-like cell line RAW 264.7, and primary macrophage cultures from C3H/HeJ and Toll-like receptor 4−/− (TLR-4−/−) mice. We show that LPS presented on live salmonellae provides an essential signal, via functional TLR-4, for macrophages to produce NO and TNF-α. Furthermore, the mitogen-activated protein kinase c-Jun N-terminal kinase and p38 are activated, and the transcription factor NF-κB is translocated to the nucleus when RAW 264.7 cells are presented with purified LPS or live salmonellae. Purified LPS stimulates rapid, transitory mitogen-activated protein kinase activation that is inhibited by E5531, whereas bacterial invasion stimulates delayed, prolonged activation, unaffected by E5531. Both purified LPS and bacterial invasion caused translocation of NF-κB, but whereas E5531 always inhibited activation by purified LPS, activation by bacterial invasion was only inhibited at later time points. In conclusion, we show for the first time that production of NO and TNF-α is critically dependent on activation of TLR-4 by LPS during invasion of macrophages by salmonellae, but that different patterns of activation of intracellular signaling pathways are induced by purified LPS vs live salmonellae.

Here, we have investigated the mRNA expression of Toll-like receptor 2 (TLR-2), TLR-4, and MD-2 in spleens and livers of C3H/HeN mice (carrying wild-type TLR-4) and C3H/HeJ mice (carrying mutated TLR-4) in response to Salmonella infection. During Salmonella infections, TLR-4 is activated, leading to increased TLR-2 and decreased TLR-4 expression.

Pathogenic strains of Yersinia spp. inject a set of Yop effector proteins into eukaryotic cells by using a plasmid-encoded type III secretion system. In this study, we analyzed the inflammatory response of human umbilical vein endothelial cells (HUVECs) after infection with different Yersinia enterocolitica strains. We found that both expression of intercellular adhesion molecule 1 and release of the cytokines interleukin-6 (IL-6) and IL-8 by HUVECs are downregulated in a YopP-dependent way, demonstrating that YopP plays a major role in the inflammatory response of these cells. Infection of HUVECs with several low-virulence (biotype 2, 3, and 4) and high-virulence (biotype 1B) Y. enterocolitica strains showed that biotype 1B isolates are more efficient in inhibiting the inflammatory response than low-virulence Y. enterocolitica strains and that this effect depends on the time of contact. We extended the results of Ruckdeschel et al. and found that on the basis of the presence or absence of arginine-143 of YopP (K. Ruckdeschel, K. Richter, O. Mannel, and J. Heesemann, Infect. Immun. 69: 7652-7662, 2001) all the Y. enterocolitica strains used fell into two groups, which correlate with the low- and high-virulence phenotypes. In addition, we found that high-virulence strains inject more YopP into the cytosol of eukaryotic target cells than do low-virulence strains.

Publisher Summary This chapter discusses the methods that are used to elucidate the action of the Yop virulon, an archetype of type III secretion/translocation systems on eukaryotic target cells. This ‘weapon’ is encoded by the pYV plasmid ( Yersinia virulence) and consists of three major parts: (1) type III secretion machinery (Ysc) that directs Yop proteins over the bacterial membranes, (2) translocator Yops that allow delivery of effector Yops in the cytosol of the eukaryotic cell, and (3) Yop effector proteins that interfere with the eukaryotic host cells. Y. enterocolitica strains are routinely grown in tryptic soy broth (TSB) and plated on tryptic soy agar (TSA) containing the required antibiotics. Prior to cell infection, Y. enterocolitica is grown in BHI under selective pressure for maintaining the relevant plasmids. Various methods are used to show translocation of bacterial proteins across the eukaryotic cell membrane. To prove translocation it must be demonstrated that the prokaryotic protein is found inside the eukaryotic cell compartment in the absence of phagocytosed bacteria.

Y. enterocolitica translocates virulence proteins, called Yop effectors, into the cytosol of eukaryotic cells. Here we investigated whether Y. enterocolitica could translocate Yops into a range of eukaryotic cells including neurons and insect cells. Y. enterocolitica translocated the hybrid reporter protein YopE-Cya into each of the eukaryotic cell types tested. In addition, Y. enterocolitica was cytotoxic for each of the adherent cell types. Thus we detected no limit to the range of eukaryotic cells into which Y. enterocolitica can translocate Yops. The Yop effectors YopE, YopH and YopT were each cytotoxic for the adherent cell types tested, showing that not only is Y. enterocolitica not selective in its translocation of particular Yop effectors into each cell type, but also that the action of these Yop effectors is not cell type specific. Invasin and/or YadA, two powerful adhesins were required for translocation of Yop into non-phagocytic cells but not for translocation into macrophages. To use the Yersinia translocation system for broad applications, a Y. enterocolitica translocation strain and vector for the delivery of heterologous proteins into eukaryotic cells was constructed. This strain + vector combination lacks the translocated Yop effectors and allows delivery into eukaryotic cells of heterologous proteins fused to the minimal N-terminal secretion/translocation signal of YopE. Using this strategy translocation of a YopE-Diphtheria toxin subunit A hybrid protein into several cell types has been shown.

Mechanosensitive channels are ubiquitous amongst bacterial cells and have been proposed to have major roles in the adaptation to osmotic stress, in particular in the management of transitions from high to low osmolarity environments. Electrophysiological measurements have identified multiple channels in Escherichia coli cells. One gene, mscL, encoding a large conductance channel has previously been described, but null mutants were without well-defined phenotypes. Here, we report the characterization of a new gene family required for MscS function, YggB and KefA, which has enabled a rigorous test of the role of the channels. The channel determined by KefA does not appear to have a major role in managing the transition from high to low osmolarity. In contrast, analysis of mutants of E.coli lacking YggB and MscL shows that mechanosensitive channels are designed to open at a pressure change just below that which would cause cell disruption leading to death.

The enzyme methylglyoxal synthase (MGS) was partially purified from Escherichia coli extracts, and the amino-terminal sequence of candidate proteins was determined, based on the native protein being a tetramer of about 69 kDa. Database analysis identified an open reading frame in the E. coli genome, YccG, corresponding to a protein of 16.9 kDa. When amplified and expressed from a controlled promoter, it yielded extracts that contained high levels of MGS activity. MGS expressed from the trc promoter accumulated to approximately 20% of total cell protein, representing approximately 900-fold enhanced expression. This caused no detriment during growth on glucose, and the level of methylglyoxal (MG) in the medium rose to only 0.08 mM. High-level expression of MGS severely compromised growth on xylose, arabinose and glycerol. A mutant lacking MGS was constructed, and it grew normally on a range of carbon sources and on low-phosphate medium. However, the mutant failed to produce MG during growth on xylose in the presence of cAMP, and growth was inhibited.

Publisher Summary This chapter reviews the evidence for channels in bacteria and evaluates their roles in cell physiology. Ion channels may play important roles in protection against excessive turgor pressure across the membrane (MscL, MscS, and possibly KefA) and during detoxification of electrophilic compounds (KefB, KefC). Other channels, such as KcsA, may play important roles in potassium acquisition or in balancing the potassium gradient with the membrane potential. The reality of Kch as a channel can only be a subject for speculation. However, if it were to function as an outwardly-rectified channel it might participate in membrane re-polarization during periods of starvation or, given its resemblance to the Ca 2+ -regulated, Drosophila slo channel, there may be a role for this channel in signaling the Ca 2+ status of the cell. Other phenomena in bacteria may have their source in transport systems with channel-like properties. Most prominent among these is the Trk potassium transport system in Escherichia coli. For each of the proteins their open state has different implications for the cell and consequently tight regulation of opening is very important. The porins are open most of the time and are more correctly designated pores.