RESUMEN
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 makes in situ or in vitro investigations 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 bacterium Dichelobacter nodosus but also other species such as Mycoplasma fermentans and Porphyromonas 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.
Asunto(s)
Bacterias/inmunología , Panadizo Interdigital/inmunología , Interacciones Huésped-Patógeno/inmunología , Inmunidad/inmunología , Ovinos/inmunología , Animales , Colágeno Tipo I/inmunología , Panadizo Interdigital/microbiología , Ovinos/microbiología , Enfermedades de las Ovejas/inmunología , Enfermedades de las Ovejas/microbiología , Piel/inmunología , Piel/microbiología , Transcriptoma/inmunología , Virulencia/inmunologíaRESUMEN
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 CLp values generated come from the equine sports industry for determining drug plasma screening limits in the control of medications at the time of competition.The kinetics of omeprazole metabolism were investigated in freshly isolated and cryopreserved equine hepatocytes and hepatic microsomes (n = 3 horses).The Vmax, Km and intrinsic clearance (CLint) of omeprazole were determined via the substrate depletion method as well as Km values for the formation of three metabolites.The CLint values were extrapolated to in vivo hepatic plasma clearance (CLH) using the well stirred and parallel tube models.Clp for omeprazole was successfully predicted using freshly isolated or cryopreserved equine hepatocytes, while microsomes under-predicted.Equine microsomes were used to perform a drug-drug interaction (DDI) study between omeprazole and chloramphenicol. The average inhibitor constant Ki, assuming competitive inhibition, was 15.4 ± 5 µM.To the authors' knowledge, this is the first report showing the successful extrapolation of drug CLp in the horse using equine hepatocytes and the prediction of a DDI using microsomes.
Asunto(s)
Caballos/metabolismo , Omeprazol/metabolismo , Animales , Criopreservación , Interacciones Farmacológicas , Hepatocitos , Hígado/metabolismo , Microsomas Hepáticos/metabolismo , Omeprazol/farmacologíaRESUMEN
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.
Asunto(s)
Envejecimiento/fisiología , Sistema Enzimático del Citocromo P-450/metabolismo , Hepatocitos , Hígado , Microsomas Hepáticos/enzimología , Animales , Hepatocitos/citología , Hepatocitos/enzimología , Caballos , Hígado/citología , Hígado/enzimologíaRESUMEN
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."
Asunto(s)
Educación en Veterinaria , Aprendizaje , Percepción , Estudiantes/psicología , Logro , Educación en Veterinaria/estadística & datos numéricos , Inglaterra , Facultades de Medicina Veterinaria , Estudiantes/estadística & datos numéricosRESUMEN
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.
Asunto(s)
Adenoviridae/efectos de los fármacos , Adenoviridae/patogenicidad , Infecciones por Adenovirus Humanos/prevención & control , Antivirales/farmacología , Tracto Gastrointestinal/virología , Interferones/farmacología , Adenoviridae/crecimiento & desarrollo , Técnicas de Cultivo de Célula , Quimioprevención/métodos , Humanos , Modelos TeóricosRESUMEN
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.
Asunto(s)
Diversidad Cultural , Educación en Veterinaria , Aprendizaje , Estudiantes/psicología , Estudios de Cohortes , Inglaterra , Humanos , Percepción , Facultades de Medicina Veterinaria , Encuestas y CuestionariosRESUMEN
Ovine footrot is a complex multifactorial infectious disease, causing lameness in sheep with major welfare and economic consequences. Dichelobacter nodosus is the main causative bacterium; however, footrot is a polymicrobial disease with Fusobacterium necrophorum, Mycoplasma fermentans and Porphyromonas asaccharolytica also associated. There is limited understanding of the host response involved. The proinflammatory mediators, interleukin (IL)-1ß and C-X-C Motif Chemokine Ligand 8 (CXCL8), have been shown to play a role in the early response to D. nodosus in dermal fibroblasts and interdigital skin explant models. To further understand the response of ovine skin to bacterial stimulation, and to build an understanding of the role of the cytokines and chemokines identified, primary ovine interdigital fibroblasts and keratinocytes were isolated, cultured and stimulated. The expression of mRNA and protein release of CXCL8 and IL-1ß were measured after stimulation with LPS, D. nodosus or F. necrophorum, which resulted in increased transcript levels of IL-1ß and CXCL8 in the M. fermentans-free cells. However, only an increase in the CXCL8 protein release was observed. No IL-1ß protein release was detected, despite increases in IL-1ß mRNA, suggesting the signal for intracellular pre-IL-1ß processing may be lacking when culturing primary cells in isolation. The keratinocytes and fibroblasts naturally infected with M. fermentans showed little response to the LPS, a range of D. nodosus preparations or heat-inactivated F. necrophorum. Primary single cell culture models complement ex vivo organ culture models to study different aspects of the host response to D. nodosus. The ovine keratinocytes and fibroblasts infected with M. fermentans had a reduced response to the experimental bacterial stimulation. However, in the case of footrot where Mycoplasma spp. are associated with diseased feet, this natural infection gives important insights into the impact of multiple pathogens on the host response.
RESUMEN
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.
Asunto(s)
Antiinfecciosos , Dichelobacter nodosus , Panadizo Interdigital , Infecciones por Bacterias Gramnegativas , Enfermedades de las Ovejas , Animales , Antiinfecciosos/farmacología , Dichelobacter nodosus/genética , Panadizo Interdigital/epidemiología , Panadizo Interdigital/microbiología , Glutaral/farmacología , Infecciones por Bacterias Gramnegativas/epidemiología , Infecciones por Bacterias Gramnegativas/microbiología , Infecciones por Bacterias Gramnegativas/veterinaria , Prevalencia , Ovinos , Enfermedades de las Ovejas/tratamiento farmacológico , Enfermedades de las Ovejas/epidemiología , Enfermedades de las Ovejas/microbiologíaRESUMEN
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<.05).
Asunto(s)
Competencia Clínica , Educación en Veterinaria , Aprendizaje Basado en Problemas , Estudiantes de Medicina , Estudios de Cohortes , Barreras de Comunicación , Femenino , Humanos , Masculino , Encuestas y Cuestionarios , Reino UnidoRESUMEN
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.
RESUMEN
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.
Asunto(s)
Factor 88 de Diferenciación Mieloide/inmunología , Infecciones por Salmonella/inmunología , Salmonella typhimurium/crecimiento & desarrollo , Receptor Toll-Like 4/inmunología , Animales , Células Cultivadas , Interferón gamma/biosíntesis , Hígado/microbiología , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/biosíntesis , Infecciones por Salmonella/microbiología , Salmonella typhimurium/aislamiento & purificación , Transducción de Señal/inmunología , Bazo/microbiología , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
High throughput genomics technologies are applied widely to microbiomes in humans, animals, soil and water, to detect changes in bacterial communities or the genes they carry, between different environments or treatments. We describe a method to test the statistical significance of differences in bacterial population or gene composition, applicable to metagenomic or quantitative polymerase chain reaction data. Our method goes beyond previous published work in being universally most powerful, thus better able to detect statistically significant differences, and through being more reliable for smaller sample sizes. It can also be used for experimental design, to estimate how many samples to use in future experiments, again with the advantage of being universally most powerful. We present three example analyses in the area of antimicrobial resistance. The first is to published data on bacterial communities and antimicrobial resistance genes (ARGs) in the environment; we show that there are significant changes in both ARG and community composition. The second is to new data 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 would provide sufficient power to observe significance of similar changes in further experiments. The third is to published data on bacterial communities surrounding rice crops. This is a much larger data set and is used to verify the new method. Our method has broad uses for statistical testing and experimental design in research on changing microbiomes, including studies on antimicrobial resistance.
Asunto(s)
Bacterias , Bioestadística/métodos , Farmacorresistencia Bacteriana/genética , Pezuñas y Garras/microbiología , Microbiota/genética , Oryza/microbiología , Aguas del Alcantarillado/microbiología , Animales , Bacterias/clasificación , Bacterias/genética , China , ADN Bacteriano/genética , Genes Bacterianos/genética , Metagenoma/genética , Rizosfera , Ovinos/microbiología , Microbiología del Suelo , Reino Unido , Estados UnidosRESUMEN
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.
Asunto(s)
Anexina A1/metabolismo , Dexametasona/farmacología , Glucocorticoides/farmacología , Salmonelosis Animal/inmunología , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/patogenicidad , Animales , Anexina A1/genética , Hígado/efectos de los fármacos , Hígado/microbiología , Hígado/patología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Salmonelosis Animal/microbiología , Salmonelosis Animal/patología , Salmonella typhimurium/crecimiento & desarrollo , Bazo/efectos de los fármacos , Bazo/microbiología , Bazo/patologíaRESUMEN
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-1ß) 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-1ß expression levels. Increased inflammatory score corresponded with high virulent D. nodosus load and was significant (p < 0.0001) in footrot feet with an inflammatory score of 3 compared to scores 1 and 2. In addition, in contrast to healthy tissues, localisation of eubacterial load extended beyond follicular depths in footrot samples. The novel inflammatory cell infiltration scoring system in this study may be used to grade inflammatory response in the ovine feet and demonstrated an association between severity of inflammatory response and increased virulent D. nodosus load.
RESUMEN
Dichelobacter nodosus (D. nodosus) is the causative pathogen of ovine footrot, a disease that has a significant welfare and financial impact on the global sheep industry. Previous studies into the phylogenetics of D. nodosus have focused on Australia and Scandinavia, meaning the current diversity in the United Kingdom (U.K.) population and its relationship globally, is poorly understood. Numerous epidemiological methods are available for bacterial typing; however, few account for whole genome diversity or provide the opportunity for future application of new computational techniques. Multilocus sequence typing (MLST) measures nucleotide variations within several loci with slow accumulation of variation to enable the designation of allele numbers to determine a sequence type. The usage of whole genome sequence data enables the application of MLST, but also core and whole genome MLST for higher levels of strain discrimination with a negligible increase in experimental cost. An MLST database was developed alongside a seven loci scheme using publically available whole genome data from the sequence read archive. Sequence type designation and strain discrimination was compared to previously published data to ensure reproducibility. Multiple D. nodosus isolates from U.K. farms were directly compared to populations from other countries. The U.K. isolates define new clades within the global population of D. nodosus and predominantly consist of serogroups A, B and H, however serogroups C, D, E, and I were also found. The scheme is publically available at https://pubmlst.org/dnodosus/.
Asunto(s)
Educación en Veterinaria , Salud Mental , Animales , Curriculum , Facultades de Medicina VeterinariaRESUMEN
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 IL1ß 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.
Asunto(s)
Panadizo Interdigital/microbiología , Microbiota , Enfermedades de las Ovejas/microbiología , Acinetobacter/aislamiento & purificación , Acinetobacter/patogenicidad , Animales , Estudios de Casos y Controles , Coinfección/metabolismo , Coinfección/microbiología , Dichelobacter nodosus/patogenicidad , Panadizo Interdigital/metabolismo , Interleucina-1beta/metabolismo , Mycoplasma/aislamiento & purificación , Mycoplasma/patogenicidad , Porphyromonas/aislamiento & purificación , Porphyromonas/patogenicidad , Ovinos , Enfermedades de las Ovejas/metabolismo , Treponema/aislamiento & purificación , Treponema/patogenicidadRESUMEN
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 IL1ß 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.
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Dichelobacter nodosus/crecimiento & desarrollo , Panadizo Interdigital/microbiología , Infecciones por Bacterias Gramnegativas/veterinaria , Cultivo Primario de Células/métodos , Enfermedades de las Ovejas/microbiología , Enfermedades Cutáneas Bacterianas/veterinaria , Animales , Biopsia , Infecciones por Bacterias Gramnegativas/microbiología , Interleucina-1beta/análisis , Interleucina-8/análisis , Queratinocitos/metabolismo , Modelos Biológicos , Ovinos , Enfermedades Cutáneas Bacterianas/microbiología , Factores de Tiempo , Técnicas de Cultivo de TejidosRESUMEN
Despite reports of the successful isolation of primary equine hepatocytes, there are no published data regarding the successful cryopreservation of these isolated cells. In this study, a detailed description of the procedures for isolation, cryopreservation, and recovery of equine hepatocytes are presented. Furthermore, the intrinsic clearance (Clint) and production of metabolites for three drugs were compared between freshly isolated and recovered cryopreserved hepatocytes. Primary equine hepatocytes were isolated using a two-step collagenase perfusion method, with an average cell yield of 2.47 ± 2.62 × 106 cells/g of perfused liver tissue and viability of 84.1 ± 2.62%. These cells were cryopreserved with William's medium E containing 10% fetal bovine serum with 10% DMSO. The viability of recovered cells, after a 30% Percoll gradient, was 77 ± 11% and estimated recovery rate was approximately 27%. These purified cells were used to determine the in vitro Clint of three drugs used in equine medicine; omeprazole, flunixin, and phenylbutazone, via the substrate depletion method. Cryopreserved suspensions gave a comparable estimation of Clint compared to fresh cells for these three drugs as well as producing the same metabolites. This work paves the way for establishing a bank of cryopreserved equine hepatocytes that can be used for estimating pharmacokinetic parameters such as the hepatic metabolic in vivo clearance of a drug as well as producing horse-specific drug metabolites.
RESUMEN
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.