Reduction In Number Of Animals Research Articles

A novel rodent model of severe renal ischemia reperfusion injury

Renal ischemia reperfusion injury (IRI) is a major problem, currently without treatments in clinical use. This reflects the failure of animal models to mimic the severity of IRI observed in clinical practice. Most described models lack both the ability to inflict a permanent reduction in renal function and the sensitivity to demonstrate the protective efficacy of different therapies in vivo. To test novel cell-based therapies, we have developed a model of renal IRI in Fisher 344 rats. Animals were subjected to 120 min of unilateral warm ischemia, during which they underwent an intra-renal artery infusion of therapeutic agents or vehicle. At either 2 or 6 weeks post-surgery, animals underwent terminal glomerular filtration rate (GFR) studies by inulin clearance to most accurately quantify renal function. Harvested kidneys underwent histological analysis. Compared to sham operations, saline treated animals suffered a long-term reduction in GFR of ≈50%. Histology revealed short- and long-term disruption of renal architecture. Despite the injury severity, post-operative animal losses are <5%. This model produces a severe, consistent renal injury that closely replicates the pathological processes encountered in clinical medicine. Renal artery infusion mimics the route likely employed in clinical transplantation, where the renal artery is accessible. Inulin clearance characterizes GFR, allowing full assessment of therapeutic intervention. This model is useful for screening therapeutic agents prior to testing in a transplant model. This reduces animal numbers needed to test drugs for clinical transplantation and allows for refinement of dosing schedules.

Biosensor for Hepatocellular Injury Corresponds to Experimental Scoring of Hepatosplenic Schistosomiasis in Mice.

Severe hepatosplenic injury of mansonian schistosomiasis is caused by Th2 mediated granulomatous response against parasite eggs entrapped within the periportal tissue. Subsequent fibrotic scarring and deformation/sclerosing of intrahepatic portal veins lead to portal hypertension, ascites, and oesophageal varices. The murine model of Schistosoma mansoni (S. mansoni) infection is suitable to establish the severe hepatosplenic injury of disease within a reasonable time scale for the development of novel antifibrotic or anti-infective strategies against S. mansoni infection. The drawback of the murine model is that the material prepared for complex analysis of egg burden, granuloma size, hepatic inflammation, and fibrosis is limited due to small amounts of liver tissue and blood samples. The objective of our study was the implementation of a macroscopic scoring system for mice livers to determine infection-related organ alterations of S. mansoni infection. In addition, an in vitro biosensor system based on the detection of hepatocellular injury in HepG2/C3A cells following incubation with serum of moderately (50 S. mansoni cercariae) and heavily (100 S. mansoni cercariae) infected mice affirmed the value of our scoring system. Therefore, our score represents a valuable tool in experimental schistosomiasis to assess severity of hepatosplenic schistosomiasis and reduce animal numbers by saving precious tissue samples.

Animals and the 3Rs in toxicology research and testing: The way forward.

Despite efforts to eliminate the use of animals in testing and the availability of many accepted alternative methods, animals are still widely used for toxicological research and testing. While research using in vitro and computational models has dramatically increased in recent years, such efforts have not yet measurably impacted animal use for regulatory testing and are not likely to do so for many years or even decades. Until regulatory authorities have accepted test methods that can totally replace animals and these are fully implemented, large numbers of animals will continue to be used and many will continue to experience significant pain and distress. In order to positively impact the welfare of these animals, accepted alternatives must be implemented, and efforts must be directed at eliminating pain and distress and reducing animal numbers. Animal pain and distress can be reduced by earlier predictive humane endpoints, pain-relieving medications, and supportive clinical care, while sequential testing and routine use of integrated testing and decision strategies can reduce animal numbers. Applying advances in science and technology to the development of scientifically sound alternative testing models and strategies can improve animal welfare and further reduce and replace animal use.

Investigating lung responses with functional hyperpolarized xenon-129 MRI in an ex vivo rat model of asthma.

PurposeAsthma is a disease of increasing worldwide importance that calls for new investigative methods. Ex vivo lung tissue is being increasingly used to study functional respiratory parameters independent of confounding systemic considerations but also to reduce animal numbers and associated research costs. In this work, a straightforward laboratory method is advanced to probe dynamic changes in gas inhalation patterns by using an ex vivo small animal ovalbumin (OVA) model of human asthma.MethodsHyperpolarized (hp) 129Xe was actively inhaled by the excised lungs exposed to a constant pressure differential that mimicked negative pleural cavity pressure. The method enabled hp 129Xe MRI of airway responsiveness to intravenous methacholine (MCh) and airway challenge reversal through salbutamol.ResultsSignificant differences were demonstrated between control and OVA challenged animals on global lung hp 129Xe gas inhalation with P < 0.05 at MCh dosages above 460 μg. Spatial mapping of the regional hp gas distribution revealed an approximately three‐fold increase in heterogeneity for the asthma model organs.ConclusionThe experimental results from this proof of concept work suggest that the ex vivo hp noble gas imaging arrangement and the applied image analysis methodology may be useful as an adjunct to current diagnostic techniques. Magn Reson Med 76:1224–1235, 2016. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Assessment of reproductive toxicity of multiwalled carbon nanotubes and their putative effects on population ecology of mouselike rodents

An assessment of the reproductive toxicity of multiwalled carbon nanotubes (MWCNTs) for male laboratory mice according to a set of morphophysiological, biochemical, and histological criteria is reported. Oral administration of MWCNTs at doses of 0.3, 3, and 30 mg/kg evoked a dose-dependent decrease in the level of free testosterone in blood plasma, as well as a 15–40% decrease in male fertilization capacity. Mathematical modeling allowed for the assumption that a decrease in the parameter named above can induce changes in the population dynamics of mouselike rodents, namely, a reduction in the peak numbers of animals and an increase of the oscillation period by ∼30%. The results can be used for developing environmental standards of MWCNT intake by mammals, as well for the development of new products for controlling rodent populations.

Optimizing research animal necropsy and histology practices.

Reduction in animal numbers is an important tenet of laboratory animal research. Research animals should undergo complete necropsy at the close of a study to yield valuable information about the effects and safety of experimental manipulations. Proper and thorough tissue collection, storage and assessment are paramount in attaining the most efficient use of animals in research.

One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT).

BackgroundThe pronuclear injection (PI) is the simplest and widely used method to generate transgenic (Tg) mice. Unfortunately, PI-based Tg mice show uncertain transgene expression due to random transgene insertion in the genome, usually with multiple copies. Thus, typically at least three or more Tg lines are produced by injecting over 200 zygotes and the best line/s among them are selected through laborious screening steps. Recently, we developed technologies using Cre-loxP system that allow targeted insertion of single-copy transgene into a predetermined locus through PI. We termed the method as PI-based Targeted Transgenesis (PITT). A similar method using PhiC31-attP/B system was reported subsequently.ResultsHere, we developed an improved-PITT (i-PITT) method by combining Cre-loxP, PhiC31-attP/B and FLP-FRT systems directly under C57BL/6N inbred strain, unlike the mixed strain used in previous reports. The targeted Tg efficiency in the i-PITT typically ranged from 10 to 30%, with 47 and 62% in two of the sessions, which is by-far the best Tg rate reported. Furthermore, the system could generate multiple Tg mice simultaneously. We demonstrate that injection of up to three different Tg cassettes in a single injection session into as less as 181 zygotes resulted in production of all three separate Tg DNA containing targeted Tg mice.ConclusionsThe i-PITT system offers several advantages compared to previous methods: multiplexing capability (i-PITT is the only targeted-transgenic method that is proven to generate multiple different transgenic lines simultaneously), very high efficiency of targeted-transgenesis (up to 62%), significantly reduces animal numbers in mouse-transgenesis and the system is developed under C57BL/6N strain, the most commonly used pure genetic background. Further, the i-PITT system is freely accessible to scientific community.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1432-5) contains supplementary material, which is available to authorized users.

Safety testing of veterinary vaccines using magnetic resonance imaging in pigs.

Safety testing of veterinary vaccines requires the use of a large number of animals to investigate possible local and systemic reactions. This includes amongst others the pathological examination of the injection site in frequent intervals. For this a selected killing of animals in frequent intervals is inevitable. To reduce the number of animals needed for this kind of safety testing, magnetic resonance imaging (MRI) was used to detect and quantify possible inflammatory reactions after vaccination in vivo. Sixty four pigs were subdivided into 4 experimental groups (n=16); two groups consisting of 12 weeks old pigs and 2 of 6 month old pigs at vaccination day. The pigs were vaccinated with four licensed products (each group receiving one vaccine) and examined up to 6 times using MRI during a period of 5 weeks. The MRI images were evaluated semi-automatically comparing the volumes of altered signal intensities at the vaccination side (VS) with the volumes of the signal intensities at the control side (CS). A paired t-Test was used to identify significant differences (p<0.05) between VS and CS. The results show that MRI allows a 3D-quantification of the extent of local reactions in vivo, scanning the same animals at several points of time after vaccination. MRI is a suitable alternative method for non-invasive safety testing of injectable medicines and can therefore be used as an alternative method to reduce animal numbers for safety testing purposes.

Abstract 102: A longitudinal, orthotopic mouse imaging model using modified hNIS-A549 human lung adenocarcinoma cells and SPECT/CT imaging

Abstract The human sodium iodide symporter (hNIS; SLC5A5) is a membrane glycoprotein that is a transporter for iodide uptake and also concentrates the radiotracer technetium 99mTc-pertechnetate (99mTc). In this study, we investigated the use of hNIS as an imaging reporter gene expressed in lung cancer cells in nude mice that can be quantified using single-photon emission computed tomography (SPECT) imaging. The radionuclide 99mTc was selected for its rapid clearance from the body and its short physical half-life (i.e., 6 hours). These characteristics allowed for the development a highly sensitive spatial and temporal tumor-imaging model. Human lung adenocarcinoma A549 cells were genetically modified using plasmid and lentiviral vectors to stably express hNIS. Using these modified cells, we monitored xenograft and orthotopic tumor growth using SPECT/CT for a total of 47 days. hNIS expression was quantified using qPCR and tumor progression was monitored by SPECT voxel analysis and validated using H&amp;E staining of pulmonary tumors. hNIS-lentiviral vector tumors exhibited significant 99mTc uptake over hNIS-plasmid vector cells and A549 unmodified control cells in vitro and in vivo. Furthermore, this mouse study achieved a 100% orthotopic tumor take rate. Thus, a highly sensitive preclinical imaging mouse lung cancer model was developed. This model can be imaged longitudinally, which is made possible through the stable incorporation of hNIS into the tumor cell, allowing for 99mTc uptake and SPECT/CT analysis of tumor burden. In future studies, we will investigate whether chemotherapeutic regimens can effectively reduce the growth of hNIS-expressing A549 tumors over time using this preclinical in vivo model. This novel lung cancer animal model could significantly reduce animal numbers, improve the quality of data by avoiding inter-animal variation, and allow for longitudinal analysis and efficacy of experimental chemotherapeutics. Citation Format: Amber Alane McBride, Dominique Nicole Price, Jeffrey P. Norenberg, Debra A. MacKenzie, Todd Anthony Thompson, Pavan Muttil. A longitudinal, orthotopic mouse imaging model using modified hNIS-A549 human lung adenocarcinoma cells and SPECT/CT imaging. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 102. doi:10.1158/1538-7445.AM2014-102

A sheep model for cancellous bone healing.

Appropriate well-characterized bone defect animal models remain essential for preclinical research. This pilot study demonstrates a relevant animal model for cancellous bone defect healing. Three different defect diameters (8, 11, 14 mm) of fixed depth (25 mm) were compared in both skeletally immature (18-month-old) and aged sheep (5-year-old). In each animal, four defects were surgically created and placed in the cancellous bone of the medial distal femoral and proximal tibial epiphyses bilaterally. Animals were euthanized at 4 weeks post-operatively to assess early healing and any biological response. Defect sites were graded radiographically, and new bone formation quantified using μCT and histomorphometry. Fibrous tissue was found within the central region in most of the defects with woven bone normally forming near the periphery of the defect. Bone volume fraction [bone volume (BV)/TV] significantly decreased with an increasing defect diameter. Actual BV, however, increased with defect diameter. Bone ingrowth was lower for all defect diameters in the aged group. This pilot study proposes that the surgical creation of 11 mm diameter defects in the proximal tibial and distal femoral epiphyses of aged sheep is a suitable large animal model to study early healing of cancellous bone defects. The refined model allows for the placement of four separate bone defects per animal and encourages a reduction in animal numbers required for preclinical research.

Further progress on steps to reduce animal numbers used on regulatory toxicology studies utilising microsampling and sample-sparing techniques

Further progress on steps to reduce animal numbers used on regulatory toxicology studies utilising microsampling and sample-sparing techniques

Strategic focus on 3R principles reveals major reductions in the use of animals in pharmaceutical toxicity testing.

The principles of the 3Rs, Replacement, Reduction and Refinement, are being increasingly incorporated into legislations, guidelines and practice of animal experiments in order to safeguard animal welfare. In the present study we have studied the systematic application of 3R principles to toxicological research in the pharmaceutical industry, with particular focus on achieving reductions in animal numbers used in regulatory and investigatory in vivo studies. The work also details major factors influencing these reductions including the conception of ideas, cross-departmental working and acceptance into the work process. Data from 36 reduction projects were collected retrospectively from work between 2006 and 2010. Substantial reduction in animal use was achieved by different strategies, including improved study design, method development and project coordination. Major animal savings were shown in both regulatory and investigative safety studies. If a similar (i.e. 53%) reduction had been achieved simultaneously within the twelve largest pharmaceutical companies, the equivalent reduction world-wide would be about 150,000 rats annually. The results point at the importance of a strong 3R culture, with scientific engagement, collaboration and a responsive management being vital components. A strong commitment in leadership for the 3R is recommended to be translated into cross-department and inter-profession involvement in projects for innovation, validation and implementation. Synergies between all the three Rs are observed and conclude that in silico-, in vitro- and in vivo-methods all hold the potential for applying the reduction R and should be consequently coordinated at a strategic level.

A Novel Rodent Hybrid Model of Renal Ischemia Reperfusion Injury/Autotransplantation.

Introduction: Ischaemia reperfusion injury (IRI) is major cause of acute kidney injury and allograft dysfunction. No evidence-based protective or restorative treatments are in widespread clinical use. This reflects the limitations of current in-vivo models, which cannot mimic the severity of IRI observed clinically, without excessive post-procedure animal deaths from acute renal failure. Stem cell-based therapies may protect against IRI and aid regeneration of damaged tissues. However, systemic drug delivery methods lead to side-effects that limit dosing and produce sub-optimal drug concentrations in the key tissues. Serum markers commonly tested lack the sensitivity to demonstrate quantitative changes in renal function in response to injury +/- therapeutic intervention. Methods: In a novel rat model of severe left renal IRI the left renal artery is clamped and transected. The kidney is perfused via intra-renal artery infusion of therapeutic agents or vehicle. After arterial anastomosis, clamps removed to provide 120 mins of warm ischaemia. The right kidney remains to prevent death from acute renal failure. Animals are recovered for a 2 or 6 week period before terminal GFR studies by inulin clearance. Each ureter is cannulated to allow the GFR of kidney to be calculated separately. Kidneys are analysed for histology, and molecular markers of damage. Results: Compared to sham operations, saline treated animals suffer a long-term reduction in GFR of around 40%, with associated histological, and molecular markers of injury. Anastomotic patency rates approach 100%. Despite the injury severity, post-operative animal losses are <5%. Conclusion: Using this model, we are able to inflict a severe, consistent renal injury. Intra-renal artery infusion allows a high local concentration of therapy without systemic distribution, minimising total drug amounts administered, reducing side effects. This mimics the route most likely employed in clinical transplantation, where the renal artery is accessible. Inulin clearance most accurately characterizes GFR, allowing full assessment of therapeutic intervention. This model is a useful tool for screening potential therapeutic agents, prior to testing in a more complex uraemic or transplant model. This reduces animal numbers needed to test drugs for clinical transplantation and allows for refinement of dosing schedules.

Refining Housing, Husbandry and Care for Animals Used in Studies Involving Biotelemetry.

Simple SummaryBiotelemetry, the remote detection and measurement of an animal function or activity, is widely used in animal research. Biotelemetry devices transmit physiological or behavioural data and may be surgically implanted into animals, or externally attached. This can help to reduce animal numbers and improve welfare, e.g., if animals can be group housed and move freely instead of being tethered to a recording device. However, biotelemetry can also cause pain and distress to animals due to surgery, attachment, single housing and long term laboratory housing. This article explains how welfare and science can be improved by avoiding or minimising these harms.Biotelemetry can contribute towards reducing animal numbers and suffering in disciplines including physiology, pharmacology and behavioural research. However, the technique can also cause harm to animals, making biotelemetry a ‘refinement that needs refining’. Current welfare issues relating to the housing and husbandry of animals used in biotelemetry studies are single vs. group housing, provision of environmental enrichment, long term laboratory housing and use of telemetered data to help assess welfare. Animals may be singly housed because more than one device transmits on the same wavelength; due to concerns regarding damage to surgical sites; because they are wearing exteriorised jackets; or if monitoring systems can only record from individually housed animals. Much of this can be overcome by thoughtful experimental design and surgery refinements. Similarly, if biotelemetry studies preclude certain enrichment items, husbandry refinement protocols can be adapted to permit some environmental stimulation. Nevertheless, long-term laboratory housing raises welfare concerns and maximum durations should be defined. Telemetered data can be used to help assess welfare, helping to determine endpoints and refine future studies. The above measures will help to improve data quality as well as welfare, because experimental confounds due to physiological and psychological stress will be minimised.

The development of response surface pathway design to reduce animal numbers in toxicity studies.

BackgroundThis study describes the development of Response Surface Pathway (RSP) design, assesses its performance and effectiveness in estimating LD50, and compares RSP with Up and Down Procedures (UDPs) and Random Walk (RW) design.MethodsA basic 4-level RSP design was used on 36 male ICR mice given intraperitoneal doses of Yessotoxin. Simulations were performed to optimise the design. A k-adjustment factor was introduced to ensure coverage of the dose window and calculate the dose steps. Instead of using equal numbers of mice on all levels, the number of mice was increased at each design level. Additionally, the binomial outcome variable was changed to multinomial. The performance of the RSP designs and a comparison of UDPs and RW were assessed by simulations. The optimised 4-level RSP design was used on 24 female NMRI mice given Azaspiracid-1 intraperitoneally.ResultsThe in vivo experiment with basic 4-level RSP design estimated the LD50 of Yessotoxin to be 463 μg/kgBW (95% CI: 383–535). By inclusion of the k-adjustment factor with equal or increasing numbers of mice on increasing dose levels, the estimate changed to 481 μg/kgBW (95% CI: 362–566) and 447 μg/kgBW (95% CI: 378–504 μg/kgBW), respectively. The optimised 4-level RSP estimated the LD50 to be 473 μg/kgBW (95% CI: 442–517). A similar increase in power was demonstrated using the optimised RSP design on real Azaspiracid-1 data. The simulations showed that the inclusion of the k-adjustment factor, reduction in sample size by increasing the number of mice on higher design levels and incorporation of a multinomial outcome gave estimates of the LD50 that were as good as those with the basic RSP design. Furthermore, optimised RSP design performed on just three levels reduced the number of animals from 36 to 15 without loss of information, when compared with the 4-level designs. Simulated comparison of the RSP design with UDPs and RW design demonstrated the superiority of RSP.ConclusionOptimised RSP design reduces the number of animals needed. The design converges rapidly on the area of interest and is at least as efficient as both the UDPs and RW design.

Practical Advice on Calculating Confidence Intervals for Radioprotection Effects and Reducing Animal Numbers in Radiation Countermeasure Experiments

The dose of a substance that causes death in P% of a population is called an LDP, where LD stands for lethal dose. In radiation research, a common LDP of interest is the radiation dose that kills 50% of the population by a specified time, i.e., lethal dose 50 or LD50. When comparing LD50 between two populations, relative potency is the parameter of interest. In radiation research, this is commonly known as the dose reduction factor (DRF). Unfortunately, statistical inference on dose reduction factor is seldom reported. We illustrate how to calculate confidence intervals for dose reduction factor, which may then be used for statistical inference. Further, most dose reduction factor experiments use hundreds, rather than tens of animals. Through better dosing strategies and the use of a recently available sample size formula, we also show how animal numbers may be reduced while maintaining high statistical power. The illustrations center on realistic examples comparing LD50 values between a radiation countermeasure group and a radiation-only control. We also provide easy-to-use spreadsheets for sample size calculations and confidence interval calculations, as well as SAS® and R code for the latter.

Models of respiratory disease symposium

The symposium brought together representatives from the pharmaceutical industry and academia who are actively involved with establishing animal models and also in vitro translational assays of respiratory disease. It was the second of its kind, (the first one was held at the Glaxo SmithKline (GSK) Stevenage, UK site in April 2009) exchanging information on the difficult challenge of establishing predictive animal models of respiratory diseases. The organising group, which was led by Dr Kathy Abbott Banner and Prof. Richard Knowles, were originally drawn from the respiratory representatives of the European Federation of Pharmaceutical Industries and Associations (EFPIA) group of companies involved in a bid for EU funds to support work on severe asthma, but have spread the network to include many companies outside this group. The objectives of the symposium were to share knowledge in an open and collaborative atmosphere, and subsequently reach a consensus on best practice for animal models of respiratory disease. It is hoped that this will lead to decreased unnecessary duplication of animal studies, and thus a reduction in animal numbers. The symposium was held on September 6th and 7th 2012 at Novartis site in Horsham, UK. There were ~120 participants from 16 different pharmaceutical companies/contract research organisations and 10 academic institutions based in Europe, U.S.A and Australia. The NC3Rs and UBIOPRED were also represented. Key opinion leaders (Prof. Stephen Holgate, Prof. Dave Singh, Prof. Sebastian Johnston and Dr Paul Mercer) gave plenary lectures. In addition there were oral and poster presentations on COPD (P25-P31), asthma (P1-18, P40), exacerbations (P19-P24, P41), fibrosis (P38, P39) and pulmonary arterial hypertension (P37) and workshops on asthma, COPD and exacerbations. In addition there were posters on inhaled delivery systems (P35, P36). As the majority of the abstracts received were on asthma, COPD and exacerbations, this meeting summary will focus on the highlights from those. Several pharmaceutical companies provided sponsorship of the symposium.

Substantive reduction of animal numbers on regulatory toxicology studies by using microsampling or sample-sparing techniques

Substantive reduction of animal numbers on regulatory toxicology studies by using microsampling or sample-sparing techniques

Opportunities and strategies to further reduce animal use for Leptospira vaccine potency testing

Hamsters are routinely infected with virulent Leptospira for two purposes in the regulation of biologics: the performance of Codified potency tests and maintenance of challenge culture for the Codified potency tests. Options for reducing animal use in these processes were explored in a plenary lecture at the “International Workshop on Alternative Methods for Leptospira Vaccine Potency Testing: State of the Science and the Way Forward” held at the Center for Veterinary Biologics in September 2012. The use of validated in vitro potency assays such as those developed by the U.S. Department of Agriculture for Leptospira (L.) canicola, Leptospira grippotyphosa, Leptospira pomona, and Leptospira icterohaemorrhagiae rather than the Codified hamster vaccination–challenge assay was encouraged. Alternatives such as reduced animal numbers in the hamster vaccination–challenge testing were considered for problematic situations. Specifically, the merits of sharing challenge controls, reducing group sizes, and eliminating animals for concurrent challenge dose titration were assessed. Options for maintaining virulent, stable cultures without serial passage through hamsters or with decreased hamster use were also discussed. The maintenance of virulent Leptospira without the use of live animals is especially difficult since a reliable means to maintain virulence after multiple in vitro passages has not yet been identified.

A Lung Segmental Model of Chronic Pseudomonas Infection in Sheep

BackgroundChronic lung infection with Pseudomonas aeruginosa is a major contributor to morbidity, mortality and premature death in cystic fibrosis. A new paradigm for managing such infections is needed, as are relevant and translatable animal models to identify and test concepts. We sought to improve on limitations associated with existing models of infection in small animals through developing a lung segmental model of chronic Pseudomonas infection in sheep.Methodology/Principal FindingsUsing local lung instillation of P. aeruginosa suspended in agar beads we were able to demonstrate that such infection led to the development of a suppurative, necrotising and pyogranulomatous pneumonia centred on the instilled beads. No overt evidence of organ or systemic compromise was apparent in any animal during the course of infection. Infection persisted in the lungs of individual animals for as long as 66 days after initial instillation. Quantitative microbiology applied to bronchoalveolar lavage fluid derived from infected segments proved an insensitive index of the presence of significant infection in lung tissue (>104 cfu/g).Conclusions/SignificanceThe agar bead model of chronic P. aeruginosa lung infection in sheep is a relevant platform to investigate both the pathobiology of such infections as well as novel approaches to their diagnosis and therapy. Particular ethical benefits relate to the model in terms of refining existing approaches by compromising a smaller proportion of the lung with infection and facilitating longitudinal assessment by bronchoscopy, and also potentially reducing animal numbers through facilitating within-animal comparisons of differential therapeutic approaches.