Påvisande av Helicobacter spp hos hund

The purpose of this study was to develop a reliable molecular-genetic method to determine the different species of helicobacter in dogs. The study is part of a larger project to map the prevalence of Helicobacter spp in healthy and sick dogs in Sweden, and to determine the possible connection of Helicobacter spp infection with gastrointestinal diseases in dogs. Several published studies have reported on the prevalence of Helicobacter spp in dogs. The problem is that three of the most common species are so alike that a 16SrRNA-PCR with sequencing is not able to differentiate between them. In this study, DNA has been purified from samples and then a Multiplex PCR has been performed. Multiplex PCRs use multiple primers in one single PCR. The primers have been attached with fluorescent markers, and capillary electrophoresis is used to illustrate the results. In this way, very similar species can be differentiated from each other. This paper also includes a short review of previous studies made in this field and the different methods used for diagnosis of infection with Helicobacter spp. Samples from 17 dogs were collected and analyzed. Multiplex PCR was used on 12 healthy dogs. For the Multiplex PCR non-invasivly collected samples were used, namely faeces and mouth swabs. The remaining 5 dogs were patients with different gastrointestinal problems. From these dogs, in addition to faeces and mouth swab samples, biopsies were taken from the stomach and duodenum. The study shows that the Multiplex PCR works well, but that the capillary electrophoresis is not able to illustrate the results. Also, all samples from all dogs were analyzed in a helicobacteria-specific 16SrRNA PCR and this showed Helicobacter spp in nearly all samples, the frequency of mixed helicobacter-infection being as common in mouth swabs as it was in faeces samples. This unexpected result further emphasises the need to develop the Multiplex PCR-technology, as normal sequencing cannot be used in severe mixed infections. Thus, the Multiplex PCR should prove to be a diagnostically reliable method, but further development and testing are needed.Syftet med denna studie var att utveckla en molekylärgenetiskt pålitlig metod för att artbestämma helicobacterbakterier hos hund. Studien är en del av ett större projekt där man avser kartlägga helicobacterförekomsten hos friska och sjuka hundar i Sverige samt undersöka bakteriens betydelse för uppkomsten av gastrointestinala sjukdomar hos hund. Ett flertal studier har rapporterat om förekomst av Helicobacter spp hos hundar, men ett stort problem är att de allra vanligaste arterna är så pass lika varandra att en vanlig 16S rRNA-PCR (Polymerase Chain Reaktion) med sekvensering ej kan skilja dem åt. Detta arbete beskriver hur DNA har renats fram från provmaterial varpå en Multiplex-PCR metod med multipla primrar tillämpats i en enda PCR-reaktion. Dessa primrar innehåller fluorescerande ämnen som kan åskådliggöras med hjälp av kapillärelektrofores. Härigenom kan arter som liknar varann skiljas åt. Arbetet ger även en kort inblick i tidigare studier som gjorts inom området, samt de olika metoder som finns för diagnos av infektion med Helicobacter spp. Prov från 17 hundar analyserades och Multiplex-PCR utfördes på 12 friska hundar. Till Multiplex-PCR-reaktionen användes icke-invasivt tagna prover, faecesprov samt munskrapprov. De övriga fem hundarna var patienthundar som inkom till kliniken för olika gastrointestinala störningar. Från dessa hundar analyserades, förutom faeces- och munskrapprov, även biopsier från magsäck och duodenum. Resultaten visar att Multiplex-PCR-reaktionen fungerar bra men att kapillärelektroforesen ej kan åskådliggöra resultatet. Sammanfattningsvis kommer Multiplex-PCR vara en diagnostiskt pålitlig metod men vidare utveckling och studier krävs. Samtliga prover från samtliga hundar analyserades i en helicobacterspecifik 16S rRNA PCR och det visade sig finnas Helicobacter spp hos nästan alla de hundar som provtogs. Dessutom var frekvensen blandinfektion i munprov liknande den som ses i faecesprov, vilket ej var förväntat. Detta accentuerar ytterligare behovet av att utveckla Multiplex-PCR metoden då vanlig sekvensering inte kan användas vid kraftiga blandinfektioner

Dynamic telomerase gene suppression via network effects of GSK3 inhibition

<b>Background</b>: Telomerase controls telomere homeostasis and cell immortality and is a promising anti-cancer target, but few small molecule telomerase inhibitors have been developed. Reactivated transcription of the catalytic subunit hTERT in cancer cells controls telomerase expression. Better understanding of upstream pathways is critical for effective anti-telomerase therapeutics and may reveal new targets to inhibit hTERT expression. <b>Methodology/Principal Findings</b>: In a focused promoter screen, several GSK3 inhibitors suppressed hTERT reporter activity. GSK3 inhibition using 6-bromoindirubin-3′-oxime suppressed hTERT expression, telomerase activity and telomere length in several cancer cell lines and growth and hTERT expression in ovarian cancer xenografts. Microarray analysis, network modelling and oligonucleotide binding assays suggested that multiple transcription factors were affected. Extensive remodelling involving Sp1, STAT3, c-Myc, NFκB, and p53 occurred at the endogenous hTERT promoter. RNAi screening of the hTERT promoter revealed multiple kinase genes which affect the hTERT promoter, potentially acting through these factors. Prolonged inhibitor treatments caused dynamic expression both of hTERT and of c-Jun, p53, STAT3, AR and c-Myc. <b>Conclusions/Significance</b>: Our results indicate that GSK3 activates hTERT expression in cancer cells and contributes to telomere length homeostasis. GSK3 inhibition is a clinical strategy for several chronic diseases. These results imply that it may also be useful in cancer therapy. However, the complex network effects we show here have implications for either setting

Identification of a selective G1-phase benzimidazolone inhibitor by a senescence-targeted virtual screen using artificial neural networks

Cellular senescence is a barrier to tumorigenesis in normal cells and tumour cells undergo senescence responses to genotoxic stimuli, which is a potential target phenotype for cancer therapy. However, in this setting, mixed-mode responses are common with apoptosis the dominant effect. Hence, more selective senescence inducers are required. Here we report a machine learning-based in silico screen to identify potential senescence agonists. We built profiles of differentially affected biological process networks from expression data obtained under induced telomere dysfunction conditions in colorectal cancer cells and matched these to a panel of 17 protein targets with confirmatory screening data in PubChem. We trained a neural network using 3517 compounds identified as active or inactive against these targets. The resulting classification model was used to screen a virtual library of ~2M lead-like compounds. 147 virtual hits were acquired for validation in growth inhibition and senescence-associated β-galactosidase (SA-β-gal) assays. Among the found hits a benzimidazolone compound, CB-20903630, had low micromolar IC50 for growth inhibition of HCT116 cells and selectively induced SA-β-gal activity in the entire treated cell population without cytotoxicity or apoptosis induction. Growth suppression was mediated by G1 blockade involving increased p21 expression and suppressed cyclin B1, CDK1 and CDC25C. Additionally, the compound inhibited growth of multicellular spheroids and caused severe retardation of population kinetics in long term treatments. Preliminary structure-activity and structure clustering analyses are reported and expression analysis of CB-20903630 against other cell cycle suppressor compounds suggested a PI3K/AKT-inhibitor-like profile in normal cells, with different pathways affected in cancer cells

Extended Spectrum Beta-Lactamase/AmpC-Producing E Coli in Dogs Treated with Antimicrobials in Surgical Wards

The aim of this study is to investigate the prevalence and carriage of Extended Spec - trum Beta-Lactamase (ESBL) and AmpC- producing strains of E. coli and Klebsi - ella spp in hospitalized dogs treated with antimicrobials. Tissue and fecal samples from 66 dogs were analyzed for presence of AmpC or ESBL producing bacteria. The dogs had to have been admitted to the surgical ward for at least 24 hours and have received antimicrobial treatment. Samples were plated onto bovine blood agar and after incubation for 24 + 24 h, five colonies morphologically consistent with E.coli and Klebsiella spp , were selected and recultured onto media containing antimicrobials. Dogs carrying ESBL/AmpC- producing bacteria were retested for rectal colonisation at 3-6 months intervals for up to 16 months. Five (7.6%) dogs carried bacterial strains posi - tive for ESBL/AmpC- producing- genes in feces. All tissue samples were negative. One dog, previously positive for bla CMY-2 , carried ESBL genotype bla TEM-52 , in the second sample. Four dogs remained posi - tive throughout the testing. None of the dogs had signs of infection or symptoms associ - ated with the carriage of ESBL or plasmid mediated-AmpC- producing bacteria. Seven unique MLVA-types were identified. The results from this study show fecal car - riage for as long as 16 months of ESBL/ AmpC- producing E.coli in dogs treated with antimicrobials. Although clonal spread could not be verified in this study, the risk of dissemination of multiresistant bacteria in animal hospitals and in the community must be considered

Consumer attitudes to injurious pecking in free range egg production

Free range egg producers face continuing problems from injurious pecking (IP) which has financial consequences for farmers and poor welfare implications for birds. Beak trimming has been practised for many years to limit the damage caused by IP, but with the UK Government giving notification that they intend to ban beak trimming in 2016, considerable efforts have been made to devise feasible housing, range and management strategies to reduce IP. A recent research project investigated the efficacy of a range of IP reducing management strategies, the mean costs of which came to around 5 pence per bird. Here, the results of the above project’s consumer survey are presented: consumers’ attitudes to free range egg production are detailed showing that, whilst consumers had a very positive attitude towards free range eggs, they were especially uninformed about some aspects of free range egg production. The contingent valuation technique was used to estimate the price premium consumers would be prepared to pay to ensure that hens do not suffer from IP: this was calculated as just over 3% on top of the prevailing retail price of free range eggs. These findings reinforce other studies that have found that whilst consumers are not generally well-informed about certain specific welfare problems faced by animals under free range conditions, they are prepared to pay to improve animal welfare. Indeed, the study findings suggest that producers could obtain an additional price premium if they demonstrate the welfare provenance of their eggs, perhaps through marketing the eggs as coming from birds with intact beaks. This welfare provenance issue could usefully be assured to consumers by the introduction of a mandatory, single, accredited EU-wide welfare-standards labelling scheme

The ability of laying pullets to negotiate two ramp designs as measured by bird preference and behaviour

Background Laying hens are often kept in barn or free-range systems where they must negotiate level changes in the house to access resources. However, collisions and resultant keel fractures are commonplace. Producers sometimes add ramps to make raised areas more accessible but designs vary and very little research has investigated bird preference or behaviour when using different ramp designs, or the effect of ramp design on falls and collisions. Methods Two ramp designs were studied in an experimental setting—a ramp made of plastic poultry slats (grid ramp, GR) and a ramp made of wooden rungs (ladder ramp, LR). Sixty-four young female hens were trained to move to a food reward and this was used to test their behavioural responses when first negotiating the two different ramps during individual tests. Both upward and downward transitions were studied. Ramp preference was also tested using a room that replicated a commercial single-tier system with both types of ramp available. Birds were placed in this room in groups of 16 for three days and their use of the ramps studied. Results A greater percentage of birds successfully completed (reached the reward bowl) on the GR than the LR during both upward (58% vs 37%) and downward (83% vs 73%) transitions, and a smaller percentage of birds made zero attempts to use the GR than the LR (upwards: 13% vs 56%, downwards: 8% vs 26%). When making a downward transition, more hesitation behaviours were seen (head orientations, stepping on the spot, moving away) for the LR. However, more head orientations were seen for the GR during the upward transition. Birds were more likely to abort attempts (an attempt began when a bird placed both feet on the ramp) to move up the GR than the LR. Birds took longer to negotiate the LR than the GR in both directions, and more pauses were seen during a successful upward transition on the LR. Birds were more likely to move down the GR by walking/running whereas birds tended to jump over the entire LR. More collisions with the food reward bowl were seen for the LR. In the group tests, birds preferred to use the GR, with more transitions seen at all timepoints. However, in these tests, birds preferred to rest on the LR with greater numbers of birds counted on this type of ramp during scan sampling at all timepoints. Discussion Behavioural results suggest that the GR was easier for the birds to use than the LR, particularly on the downward transition. The GR was also less likely to result in collisions. However, the upward transition may be more difficult on the GR for some birds, potentially because of the inability to pause on a level surface during the transition. The results suggest that the GR was preferred by pullets for moving between a raised area and the ground but the LR was preferred for resting

Consumer perceptions of free-range laying hen welfare

Purpose – The purpose of this paper is to understand which factors and resources free-range egg consumers believe are important for hen welfare. Design/methodology/approach – An online survey was distributed via the mailing list of a UK free-range egg brand receiving 6,378 responses. The survey was mostly five-point Likert-scale based. The same survey was also distributed to a group of animal welfare specialists receiving 34 responses. Findings – Respondents bought free-range eggs because hens are “happier” (74.2 per cent) and “healthier” (69.0 per cent) and because they believed such eggs to taste better (57.9 per cent). They rated all the suggested factors that might contribute to hen welfare as “important” or “very important” (on average) but believed outside access and fresh air to be most important. Respondents rated the suitability of resources relating to behavioural needs high (“suitable” or “very suitable”) indoors and shelter as the most suitable outdoors. Consumers differed from welfare specialists in their views on factors contributing to hen welfare, but their views on resource suitability were similar. Research limitations/implications – The sample was biased towards free-range egg consumers who had expressed an interest in a brand marketed as high welfare. Originality/value – This is the first study to ask consumers what they consider to be important for hen welfare and how they think hen welfare can be improved. Because consumers can affect on-farm welfare through their purchasing habits assessing the degree of agreement between consumers and animal welfare specialists is important.</p