Additive Bayesian networks for antimicrobial resistance and potential risk factors in non-typhoidal Salmonella isolates from layer hens in Uganda.

BACKGROUND: Multi-drug resistant bacteria are seen increasingly and there are gaps in our understanding of the complexity of antimicrobial resistance, partially due to a lack of appropriate statistical tools. This hampers efficient treatment, precludes determining appropriate intervention points and renders prevention very difficult. METHODS: We re-analysed data from a previous study using additive Bayesian networks. The data contained information on resistances against seven antimicrobials and seven potential risk factors from 86 non-typhoidal Salmonella isolates from laying hens in 46 farms in Uganda. RESULTS: The final graph contained 22 links between risk factors and antimicrobial resistances. Solely ampicillin resistance was linked to the vaccinating person and disposal of dead birds. Systematic associations between ampicillin and sulfamethoxazole/trimethoprim and chloramphenicol, which was also linked to sulfamethoxazole/trimethoprim were detected. Sulfamethoxazole/trimethoprim was also directly linked to ciprofloxacin and trimethoprim. Trimethoprim was linked to sulfonamide and ciprofloxacin, which was also linked to sulfonamide. Tetracycline was solely linked to ciprofloxacin. CONCLUSIONS: Although the results needs to be interpreted with caution due to a small data set, additive Bayesian network analysis allowed a description of a number of associations between the risk factors and antimicrobial resistances investigated.

Effects of plant alkaloids on mitochondrial bioenergetic parameters.

Mitochondria are among the first responders to various stress factors that challenge cell and tissue homeostasis. Various plant alkaloids have been investigated for their capacity to modulate mitochondrial activities. In this study, we used isolated mitochondria from mouse brain and liver tissues to assess nicotine, anatabine and anabasine, three alkaloids found in tobacco plant, for potential modulatory activity on mitochondrial bioenergetics parameters. All alkaloids decreased basal oxygen consumption of mouse brain mitochondria in a dose-dependent manner without any effect on the ADP-stimulated respiration. None of the alkaloids, at 1 nM or 1.25 µM concentrations, influenced the maximal rate of swelling of brain mitochondria. In contrast to brain mitochondria, 1.25 µM anatabine, anabasine and nicotine increased maximal rate of swelling of liver mitochondria suggesting a toxic effect. Only at 1 mM concentration, anatabine slowed down the maximal rate of Ca2+-induced swelling and increased the time needed to reach the maximal rate of swelling. The observed mitochondrial bioenergetic effects are probably mediated through a pathway independent of nicotinic acetylcholine receptors, as quantitative proteomic analysis could not confirm their expression in pure mitochondrial fractions isolated from mouse brain tissue.

Biosecurity risk factors for highly pathogenic avian influenza (H5N8) virus infection in duck farms, France.

Highly pathogenic avian influenza (HPAI) subtype H5N8 outbreaks occurred in poultry farms in France in 2016-2017, resulting in significant economic losses and disruption to the poultry industry. Current evidence on associations between actual on-farm biosecurity risk factors and H5N8 occurrence is limited. Therefore, a retrospective matched case-control study was undertaken to investigate the inter-relationships between on-farm biosecurity practices and H5N8 infection status to provide new insights regarding promising targets for intervention. Data were collected on 133 case and 133 control duck farms (i.e. the most affected species) located in one area of the country that was mostly affected by the disease. Data were analysed using Additive Bayesian Networks which offer a rich modelling framework by graphically illustrating the dependencies between variables. Factors indirectly and directly positively associated with farm infection were inadequate management of vehicle movements (odds ratio [OR] 9.3, 95% credible interval [CI] 4.0-22.8) and inadequate delimitation of farm and units (OR 3.0, 95% CI 1.6-5.8), respectively. Inadequate disposal of dead birds was instead negatively associated with the outcome (OR 0.1, 95% CI 0.0-0.3). The findings highlight that reinforcing farm access control systems and reducing the number of visitors are key biosecurity measures to control farm vulnerability to H5N8 infection and could help setting priorities in biosecurity practices to prevent outbreaks' re-occurrence.

Bayesian Network Modeling Applied to Feline Calicivirus Infection Among Cats in Switzerland.

Bayesian network (BN) modeling is a rich and flexible analytical framework capable of elucidating complex veterinary epidemiological data. It is a graphical modeling technique that enables the visual presentation of multi-dimensional results while retaining statistical rigor in population-level inference. Using previously published case study data about feline calicivirus (FCV) and other respiratory pathogens in cats in Switzerland, a full BN modeling analysis is presented. The analysis shows that reducing the group size and vaccinating animals are the two actionable factors directly associated with FCV status and are primary targets to control FCV infection. The presence of gingivostomatitis and Mycoplasma felis is also associated with FCV status, but signs of upper respiratory tract disease (URTD) are not. FCV data is particularly well-suited to a network modeling approach, as both multiple pathogens and multiple clinical signs per pathogen are involved, along with multiple potentially interrelated risk factors. BN modeling is a holistic approach-all variables of interest may be mutually interdependent-which may help to address issues, such as confounding and collinear factors, as well as to disentangle directly vs. indirectly related variables. We introduce the BN methodology as an alternative to the classical uni- and multivariable regression approaches commonly used for risk factor analyses. We advise and guide researchers about how to use BNs as an exploratory data tool and demonstrate the limitations and practical issues. We present a step-by-step case study using FCV data along with all code necessary to reproduce our analyses in the open-source R environment. We compare and contrast the findings of the current case study using BN modeling with previous results that used classical regression techniques, and we highlight new potential insights. Finally, we discuss advanced methods, such as Bayesian model averaging, a common way of accounting for model uncertainty in a Bayesian network context.

Revealing the structure of the associations between housing system, facilities, management and welfare of commercial laying hens using Additive Bayesian Networks.

After the ban of battery cages in 1988, a welfare control programme for laying hens was developed in Sweden. Its goal was to monitor and ensure that animal welfare was not negatively affected by the new housing systems. The present observational study provides an overview of the current welfare status of commercial layer flocks in Sweden and explores the complexity of welfare aspects by investigating and interpreting the inter-relationships between housing system, production type (i.e. organic or conventional), facilities, management and animal welfare indicators. For this purpose, a machine learning procedure referred to as structure discovery was applied to data collected through the welfare programme during 2010-2014 in 397 flocks housed in 193 different farms. Seventeen variables were fitted to an Additive Bayesian Network model. The optimal model was identified by an exhaustive search of the data iterated across incremental parent limits, accounting for prior knowledge about causality, potential over-dispersion and clustering. The resulting Directed Acyclic Graph shows the inter-relationships among the variables. The animal-based welfare indicators included in this study - flock mortality, feather condition and mite infestation - were indirectly associated with each other. Of these, severe mite infestations were rare (4% of inspected flocks) and mortality was below the acceptable threshold (< 0.6%). Feather condition scored unsatisfactory in 21% of the inspected flocks; however, it seemed to be only associated to the age of the flock, ruling out any direct connection with managerial and housing variables. The environment-based welfare indicators - lighting and air quality - were an issue in 5 and 8% of the flocks, respectively, and showed a complex inter-relationship with several managerial and housing variables leaving room for several options for intervention. Additive Bayesian Network modelling outlined graphically the underlying process that generated the observed data. In contrast to ordinary regression, it aimed at accounting for conditional independency among variables, facilitating causal interpretation.

Does toe clipping for genotyping interfere with later-in-life nociception in mice?

INTRODUCTION: Genetically modified mice are widely used in studies on human and animal physiology and pharmacology, including pain research. The experimental design usually includes comparisons of genetically modified mice with wild-type littermates, requiring biopsy material for genotyping and methods for unequivocal identification of individual mice. Ethical standards and, in some countries, legislation require that both needs are reached with a single procedure. Clipping of the most distal phalanx of up to two toes per paw (toe clipping) is the favored procedure in most research fields, but it may be problematic in sensory physiology and pain research. OBJECTIVES: To systematically investigate whether toe-clipping influences later-in-life nociceptive sensitivity or the susceptibility to neuropathic or inflammatory hyperalgesia. METHODS: We tested in male mice whether the clipping of 2 toes of a hind paw influences nociceptive sensitivities to noxious heat or cold, or to mechanical stimulation under baseline conditions, after peripheral nerve injury (chronic constriction of the sciatic nerve) or during peripheral inflammation induced by subcutaneous zymosan A injection. We tested not only for the presence of significant differences but also specifically addressed bioequivalence using the 2 one-sided t test procedure. We chose a threshold of 25% variation of the control value for nonequivalence, which is usually taken as a threshold for biological relevance in pain tests. RESULTS: Using this value, we found that for all conditions (non-neuropathic and non-inflamed, neuropathic and inflamed), nociceptive sensitivities significantly fell within the equivalence bounds of the non-toe-clipped control mice. CONCLUSIONS: These results suggest that toe clipping does not have long-term effects on nociceptive sensitivities and does not alter the susceptibility of male mice to neuropathic or inflammatory hyperalgesia.

Progression and risk factors of pododermatitis in part-time group housed rabbit does in Switzerland.

In rabbits (Oryctolagus cuniculus L.), pododermatitis is a chronic multifactorial skin disease that appears mainly on the plantar surface of the hind legs. This presumably progressive disease can cause pain leading to poor welfare, yet the progression of this disease has not been thoroughly assessed on the level of individual animals. The aim of this longitudinal study thus was to investigate the possible risk factors and the progression of pododermatitis in group housed breeding does in Switzerland on litter and plastic slats. Three commercial rabbit farms with part-time group housing on litter and plastic slats were visited every four weeks throughout one year. During every visit, the same 201 adult female breeding rabbits (67 does per farm) were evaluated for the presence and severity of pododermatitis. Additionally, the does' age, parity, body weight, reproductive state, hybrid, claw length, cleanliness and moisture of the paws and the temperature and humidity inside the barns were recorded as potential risk factors. The risk factors were analysed through general linear models and additive Bayesian network (ABN) modelling using a directed acyclic graph (DAG) for visualising associations between potential risk factors. The progression of pododermatitis was analysed with a transition matrix. Relative humidity inside the barns, body weight, number of kindlings, age, and claw length were the most important risk factors, all being positively associated with pododermatitis. In contrast to expectations, the cleanliness of the left hind paw was negatively associated with the occurrence of pododermatitis, but the effect was small. In young does, the severity of pododermatitis quickly increased and in some rabbits proceeded to ulcerated spots. It was shown that 60.00%, 14.17% and 3.33% of ulcerated lesions recovered to a state without ulceration within 4, 8 or >12 weeks, respectively.

Pododermatitis in group housed rabbit does in Switzerland-Prevalence, severity and risk factors.

In rabbits (Oryctolagus cuniculus L.), pododermatitis is a chronic multifactorial skin disease that appears mainly on the plantar surface of the hind legs. In later stages, it causes pain leading to poor welfare of affected animals. Pododermatitis is commonly observed in commercial rabbit production in breeding does housed with wire mesh flooring. However, the prevalence in breeding does that are housed in groups on litter and plastic slats is not known. The aim of this cross-sectional study was to investigate the frequency, the severity and possible risk factors of pododermatitis in group housed breeding does in Switzerland on litter and plastic slats. Between June and September 2016 about 30% of all adult female breeding rabbits (1090 animals in total) were evaluated for the presence and the severity of pododermatitis on 17 commercial rabbit farms with group housing. The latter was done with a tagged visual-analogue-scale. Additionally, various animal-related (e.g. hybrid, age or body weight) and environmental risk factors (e.g. temperature, relative humidity or wet area per pen) known from the literature were recorded. The risk factors were analysed with generalized linear models, additive Bayesian network (ABN) models resulting in directed acyclic graphs (DAGs) and random forests with variable importance plots. On average, 25% of the rabbits displayed ulcerative pododermatitis likely to be painful on at least one hind leg, while the prevalence varied between farms from 4 to 49%. The age, body weight and claw-length of the animals were positively associated with pododermatitis as the most important risk factors. The best model explained 37.4% of the observed variance in the primary outcome measure for pododermatitis. These findings demonstrate that pododermatitis is prevalent in female breeding does even in group housing systems with litter and plastic slats. However, the results of this cross-sectional study also indicate that important risk factors may have been missed or were not recorded precisely enough. Thus, more in-depth research is needed to assess risk factors of pododermatitis in view of effectively preventing the occurrence of this painful disease.

Characterization of the Vitrocell® 24/48 aerosol exposure system for its use in exposures to liquid aerosols.

BACKGROUND: The Vitrocell® 24/48 is an advanced aerosol exposure system that has been widely used and characterized for exposure studies of cigarette smoke, but not for exposure to liquid aerosols with a low gas-vapor phase content such as the ones generated by electronic cigarettes. An experimental system characterization for this specific application was therefore performed. METHODS: Glycerol model aerosols of different particle size distributions, produced by a condensation monodisperse aerosol generator, were used for exposing small volumes of phosphate-buffered saline in the Vitrocell® 24/48. Disodium fluorescein, added as a tracer in the aerosol, allowed the exact aerosol mass deposition to be quantified fluorometrically. RESULTS: The aerosol mass delivery efficiency within the system showed variations in the range of ±25%. Aerosol dilution was not fully reflected in aerosol delivery, the achieved aerosol delivery should therefore be determined experimentally. Quartz crystal microbalances underestimated the deposition of liquid aerosols. Unequal delivery of particles of different sizes was detectable, although this effect is unlikely to be relevant under applied experimental conditions. CONCLUSIONS: The Vitrocell® 24/48 aerosol exposure system can be used for exposures to liquid aerosols, such as those generated by electronic cigarettes. However, our results indicate that, compared with aerosol studies of cigarettes, a higher variability is to be expected.

A new fluorescence-based method for characterizing in vitro aerosol exposure systems.

Knowledge of how an in vitro aerosol exposure system delivers a test aerosols to the biological test system is among the most crucial prerequisites for the interpretation of exposure experiments and relies on detailed exposure system characterization. Although various methods for this purpose exist, many of them are time consuming, require extensive instrumentation, or offer only limited ability to assess the performance of the system under experimental settings. We present the development and evaluation of a new, highly robust and sensitive fluorometry-based method for assessing the particle size specific delivery of liquid aerosols. Glycerol aerosols of different mean particle sizes and narrow size distributions, carrying the fluorophore disodium fluorescein, were generated in a condensation monodisperse aerosol generator. Their detailed characterization confirmed their stability and the robustness and reproducibility of their generation. Test exposures under relevant experimental settings in the Vitrocell® 24/48 aerosol exposure system further confirmed their feasibility for simulating exposures and the high sensitivity of the method. Potential applications of the presented method range from the experimental confirmation of computationally simulated particle dynamics, over the characterization of in vitro aerosol exposure systems, to the detailed description of aerosol delivery in test systems of high complexity.

A systems toxicology approach for comparative assessment: Biological impact of an aerosol from a candidate modified-risk tobacco product and cigarette smoke on human organotypic bronchial epithelial cultures.

This study reports a comparative assessment of the biological impact of a heated tobacco aerosol from the tobacco heating system (THS) 2.2 and smoke from a combustible 3R4F cigarette. Human organotypic bronchial epithelial cultures were exposed to an aerosol from THS2.2 (a candidate modified-risk tobacco product) or 3R4F smoke at similar nicotine concentrations. A systems toxicology approach was applied to enable a comprehensive exposure impact assessment. Culture histology, cytotoxicity, secreted pro-inflammatory mediators, ciliary beating, and genome-wide mRNA/miRNA profiles were assessed at various time points post-exposure. Series of experimental repetitions were conducted to increase the robustness of the assessment. At similar nicotine concentrations, THS2.2 aerosol elicited lower cytotoxicity compared with 3R4F smoke. No morphological change was observed following exposure to THS2.2 aerosol, even at nicotine concentration three times that of 3R4F smoke. Lower levels of secreted mediators and fewer miRNA alterations were observed following exposure to THS2.2 aerosol than following 3R4F smoke. Based on the computational analysis of the gene expression changes, 3R4F (0.13 mg nicotine/L) elicited the highest biological impact (100%) in the context of Cell Fate, Cell Proliferation, Cell Stress, and Inflammatory Network Models at 4 h post-exposure. Whereas, the corresponding impact of THS2.2 (0.14 mg nicotine/L) was 7.6%.