The effects of lipopolysaccharide exposure on social interaction, cytokine expression, and alcohol consumption in male and female mice.

Much recent research has demonstrated a role of inflammatory pathways in depressive-like behavior and excess alcohol consumption. Lipopolysaccharide (LPS) is a cell wall component of gram-negative bacteria that can be used to trigger a strong inflammatory response in rodents in a preclinical research setting to study the mechanisms behind this relationship. In our study, we exposed male and female mice to LPS and assessed depressive-like behavior using the social interaction (SI) test, alcohol consumption in the two-bottle choice procedure, and expression of inflammatory mediators using quantitative PCR. We found that LPS administration decreased SI in female mice but had no significant impact on male mice when assessed 24 h after injection. LPS resulted in increased proinflammatory cytokine expression in both male and female mice; however, some aspects of the cytokine upregulation observed was greater in female mice as compared to males. A separate cohort of male and female mice underwent drinking for 12 days before receiving a saline or LPS injection, which we found to increase alcohol intake in both males and females. We have previously observed a role of the neurokinin-1 receptor (NK1R) in escalated alcohol intake, and in the inflammatory and behavioral response to LPS. The NK1R is the endogenous target of the neuropeptide SP, and this system has wide ranging roles in depression, anxiety, drug/alcohol seeking, pain, and inflammation. Thus, we administered a NK1R antagonist prior to alcohol access. This treatment reduced escalated alcohol consumption in female mice treated with LPS but did not affect drinking in males. Taken together, these results indicate that females are more sensitive to some physiological and behavioral effects of LPS administration, but that LPS escalates alcohol consumption in both sexes. Furthermore, NK1R antagonism can reduce alcohol consumption that is escalated by LPS treatment, in line with our previous findings.

Aryl Coenzyme A Ligases, a Subfamily of the Adenylate-Forming Enzyme Superfamily.

Aryl coenzyme A (CoA) ligases belong to class I of the adenylate-forming enzyme superfamily (ANL superfamily). They catalyze the formation of thioester bonds between aromatic compounds and CoA and occur in nearly all forms of life. These ligases are involved in various metabolic pathways degrading benzene, toluene, ethylbenzene, and xylene (BTEX) or polycyclic aromatic hydrocarbons (PAHs). They are often necessary to produce the central intermediate benzoyl-CoA that occurs in various anaerobic pathways. The substrate specificity is very diverse between enzymes within the same class, while the dependency on Mg2+, ATP, and CoA as well as oxygen insensitivity are characteristics shared by the whole enzyme class. Some organisms employ the same aryl-CoA ligase when growing aerobically and anaerobically, while others induce different enzymes depending on the environmental conditions. Aryl-CoA ligases can be divided into two major groups, benzoate:CoA ligase-like enzymes and phenylacetate:CoA ligase-like enzymes. They are widely distributed between the phylogenetic clades of the ANL superfamily and show closer relationships within the subfamilies than to other aryl-CoA ligases. This, together with residual CoA ligase activity in various other enzymes of the ANL superfamily, leads to the conclusion that CoA ligases might be the ancestral proteins from which all other ANL superfamily enzymes developed.

Will there be any more classical scrapie cases in sheep in Great Britain? A modelling study to predict future cases.

The aim of this study was to apply a back-calculation model to Great Britain (GB) classical scrapie surveillance data, and use this model to estimate how many more cases might be expected, and over what time frame these cases might occur. A back-calculation model was applied to scrapie surveillance data between 2005 and 2019 to estimate the annual rate of decline of classical scrapie. This rate was then extrapolated to predict the number of future cases each year going forward. The model shows that there may be yet further cases of classical scrapie in GB. These will most likely occur in the fallen stock scheme, with approximately a 25% probability of at least 1 further scrapie positive, with a very low probability (~0.2%) of having up to three additional scrapie positives. This highlights the difficulty of completely eliminating all further cases, even in the presence of very effective control measures.

Evaluating the application of the dual path platform VetTB test for badgers (Meles meles) in the test and vaccinate or remove (TVR) wildlife research intervention project in Northern Ireland.

European badgers (Meles meles) are accepted as a wildlife reservoir host for Mycobacterium bovis, which causes bovine tuberculosis (bTB) in the British Isles. The objective of this study was to evaluate the use of Dual Path Platform (DPP) VetTB test (Chembio Diagnostic Systems Inc., Medford, NY, USA) within a Test and Vaccinate or Remove (TVR) wildlife research intervention project. Blood samples were collected from 456 individual badgers, trapped in 2015 and 2016, and tested in the field with DPP VetTB test using whole blood. Additionally, whole blood and serum samples were taken to the laboratory for further DPP VetTB testing and for gamma interferon (IFN-γ) testing. Swabs were taken from the oropharynx and trachea and submitted for bacteriological culture as were swabs from wounds, if present. Field DPP VetTB test positive badgers were euthanised and underwent post-mortem examination and bTB confirmatory testing. The results demonstrated that the test performed as well in the field using whole blood as DPP Vet TB tests in the laboratory using sera or whole blood, and as well as other established tests for M. bovis. Visual assessment of the DPP VetTB test using serum under laboratory conditions showed a high degree of consistency between raters. Using a relative gold standard (parallel interpretation of IFN-γ assay and oropharyngeal/tracheal sample/culture), sensitivity estimates for the DPP VetTB test using sera and whole blood were 0.5 (95%CI 0.34-0.66) and 0.42 (95%CI 0.24-0.66), respectively. Specificity estimates were 0.95 (95%CI 0.93-0.97) for sera and 0.89 (95%CI 0.86-0.92) for whole blood. Parallel interpretation of Band 1 (MPB83) and Band 2 (CFP-10/ESAT-6) of the DPP VetTB test was not superior to interpretation of Band 1 only. The results give confidence in the reliability and reproducibility of the DPP VetTB test for badgers under field conditions and therefore it is considered appropriate for use in a badger bTB control campaign.

Identification of naphthalene carboxylase subunits of the sulfate-reducing culture N47.

Expanding industrialization and the associated usage and production of mineral oil products has caused a worldwide spread of polycyclic aromatic hydrocarbons. These pollutants accumulate and persist under anoxic conditions but little is known about the biochemical reactions catalyzing their anaerobic degradation. Recently, carboxylation of naphthalene was demonstrated for the sulfate-reducing culture N47. Proteogenomic studies on N47 allowed the identification of a gene cluster with products suggested to be involved in the initial reaction of naphthalene degradation. Here, we performed comparative proteomic studies with N47 proteins extracted from naphthalene versus 2-methylnapththalene-grown cells on blue native PAGE. The analysis led to the identification of subunits of the naphthalene carboxylase of N47. Moreover, we show that the identified subunits are encoded in an operon structure within the previously mentioned naphthalene carboxylase gene cluster. These findings were supported by a pull-down experiment revealing in vitro interaction partners of a heterologously produced GST-tagged naphthalene carboxylase subunit. Based on these lines of evidence, naphthalene carboxylase is proposed to be a complex of about 750 kDa. Naphthalene carboxylase can be seen as a prototype of a new enzyme family of UbiD like de-/carboxylases catalyzing the anaerobic activation of non-substituted polycyclic aromatic hydrocarbons.

Evaluation of ELISA and haemagglutination inhibition as screening tests in serosurveillance for H5/H7 avian influenza in commercial chicken flocks.

Avian influenza virus (AIV) subtypes H5 and H7 can infect poultry causing low pathogenicity (LP) AI, but these LPAIVs may mutate to highly pathogenic AIV in chickens or turkeys causing high mortality, hence H5/H7 subtypes demand statutory intervention. Serological surveillance in the European Union provides evidence of H5/H7 AIV exposure in apparently healthy poultry. To identify the most sensitive screening method as the first step in an algorithm to provide evidence of H5/H7 AIV infection, the standard approach of H5/H7 antibody testing by haemagglutination inhibition (HI) was compared with an ELISA, which detects antibodies to all subtypes. Sera (n = 1055) from 74 commercial chicken flocks were tested by both methods. A Bayesian approach served to estimate diagnostic test sensitivities and specificities, without assuming any 'gold standard'. Sensitivity and specificity of the ELISA was 97% and 99.8%, and for H5/H7 HI 43% and 99.8%, respectively, although H5/H7 HI sensitivity varied considerably between infected flocks. ELISA therefore provides superior sensitivity for the screening of chicken flocks as part of an algorithm, which subsequently utilises H5/H7 HI to identify infection by these two subtypes. With the calculated sensitivity and specificity, testing nine sera per flock is sufficient to detect a flock seroprevalence of 30% with 95% probability.

Is there a decline in bovine spongiform encephalopathy cases born after reinforced feed bans? A modelling study in EU member states.

Occasional cases of classical bovine spongiform encephalopathy (BSE) still continue to occur within the European Union (EU) for animals born after reinforced feed bans (BARBs), which should in theory have eliminated all risk of infection. The study aimed to determine (i) whether a common rate of decline of BSE infection was evident across EU member states, i.e. to determine whether control measures have been equally effective in all member states, (ii) whether there was any evidence of spontaneous occurrence of BSE in the data and (iii) the expected date for the last BSE case in UK. It was found that there was no significant difference in the rate of decline of BSE prevalence between member states, with a common rate of decline of 33·9% per annum (95% CI 30·9-37%) in successive annual birth cohorts. Trend analysis indicated an ultimate decline to 0 prevalence, suggesting that spontaneous occurrence does not explain the majority of cases. Projecting forward the trends from the back-calculation model indicated that there was approximately a 50% probability of further cases in the UK, and should the current rate of decline continue, there remains the possibility of further occasional cases up until 2026.

Epidemiological investigations on the potential transmissibility of a rare disease: the case of atypical scrapie in Great Britain.

Multiple cases of atypical scrapie in the same holding and co-existence with classical scrapie have been reported in Great Britain. A two-stage simulation tool was developed by combining a sampling algorithm and a hierarchical Bayesian model to simulate the number of positive cases of atypical scrapie from: (i) random sampling and (ii) using the actual sampled population in Great Britain, being the output probability of detection of flocks with one and more cases. Cluster analysis was conducted to assess the level of geographical over- and under-sampling over the years. The probability of detecting at least two cases of atypical scrapie in the same holding is much lower in simulated random data than in simulated actual data for all scenarios. Sampling bias in the selection of sheep for testing led to multiple sampling from fewer but larger holdings, Scotland, and areas of Wales were under-sampled and the South-West and East of England oversampled. The pattern of atypical scrapie cases observed is unlikely to be explained by a multi-case event epidemiologically linked. The co-existence of classical and atypical scrapie is a rare event with 19 holdings detected in GB and does not suggest an epidemiological link between the two types of disease.

Development of risk-based trading farm scoring system to assist with the control of bovine tuberculosis in cattle in England and Wales.

Identifying and ranking cattle herds with a higher risk of being or becoming infected on known risk factors can help target farm biosecurity, surveillance schemes and reduce spread through animal trading. This paper describes a quantitative approach to develop risk scores, based on the probability of infection in a herd with bovine tuberculosis (bTB), to be used in a risk-based trading (RBT) scheme in England and Wales. To produce a practical scoring system the risk factors included need to be simple and quick to understand, sufficiently informative and derived from centralised national databases to enable verification and assess compliance. A logistic regression identified herd history of bTB, local bTB prevalence, herd size and movements of animals onto farms in batches from high risk areas as being significantly associated with the probability of bTB infection on farm. Risk factors were assigned points using the estimated odds ratios to weight them. The farm risk score was defined as the sum of these individual points yielding a range from 1 to 5 and was calculated for each cattle farm that was trading animals in England and Wales at the start of a year. Within 12 months, of those farms tested, 30.3% of score 5 farms had a breakdown (sensitivity). Of farms scoring 1-4 only 5.4% incurred a breakdown (1-specificity). The use of this risk scoring system within RBT has the potential to reduce infected cattle movements; however, there are cost implications in ensuring that the information underpinning any system is accurate and up to date.

Evaluation of the sensitivity of faecal sampling for detection of monophasic Salmonella Typhimurium and other Salmonella in cattle and pigs.

There has been a rapid rise in the prevalence of cases of monophasic Salmonella Typhimurium (mST) in both humans and farm animals, and it has been found in pigs, cattle and poultry. It is therefore vital to have a good understanding of how to efficiently detect infected farms. The objective of this project was to determine sample type sensitivity in the detection of Salmonella to detect infected groups of animals on both pig (breeder, grower and finisher sites) and cattle (beef and dairy) farms, using data collected from a study investigating farms that were positive for mST, and to explore any variation between different age groups and management practices. A Bayesian approach in the absence of a gold standard was adopted to analyse the individual and pooled faecal sample data collected from each epidemiological group on each of the farms. The sensitivity of pooled sampling depended on the prevalence of infection in the group being sampled, with a higher prevalence leading to higher sensitivity. Pooled sampling was found to be more efficient at detecting positive groups of animals than individual sampling, with the probability of a random sample from a group of animals with 5% prevalence testing positive being equal to 15·5% for immature pigs (3·6% for an individual faecal sample, taking into account the sensitivity and infection prevalence), 7·1% for adult pigs (1·2% for individual sampling), 30% for outdoor cattle (2% for individual sampling) and 34% for indoor cattle (1% for individual sampling). The mean prevalence of each epidemiological group was higher in outdoor farms than indoor for both pigs and cattle (mean within-farm prevalence of 29·4% and 38·7% for outdoor pigs and cattle, respectively, compared to 19·8% and 22·1% for indoor pigs and cattle).

Allelic variants at codon 146 in the PRNP gene show significant differences in the risk for natural scrapie in Cypriot goats.

Previous studies have shown the association between the polymorphisms serine (S) or aspartic acid (D) at codon 146 of the PRNP gene and resistance to scrapie. All goats aged >12 months (a total of 1075 animals) from four herds with the highest prevalence of scrapie in the country were culled and tested, of which 234 (21·7%) were positive by either the rapid test or immunohistochemistry (IHC) for any of the tissues tested. The odds of scrapie infection occurring in NN146 goats was 101 [95% credible interval (CrI) 19-2938] times higher than for non-NN146 or unknown genotypes. IHC applied to lymphoreticular tissue produced the highest sensitivity (94%, 95% CrI 90-97). The presence of putatively resistant non-NN146 alleles in the Cypriot goat population, severely affected by scrapie, provides a potential tool to reduce/eradicate scrapie provided that coordinated nationwide breeding programmes are implemented and maintained over time.

Bayesian analysis of culture and PCR methods for detection of Campylobacter spp. in broiler caecal samples.

The objective of this study was to estimate the sensitivity and specificity of a culture method and a polymerase chain reaction (PCR) method for detection of two Campylobacter species: C. jejuni and C. coli. Data were collected during a 3-year survey of UK broiler flocks, and consisted of parallel sampling of caeca from 436 batches of birds by both PCR and culture. Batches were stratified by season (summer/non-summer) and whether they were the first depopulation of the flock, resulting in four sub-populations. A Bayesian approach in the absence of a gold standard was adopted, and the sensitivity and specificity of the PCR and culture for each Campylobacter subtype was estimated, along with the true C. jejuni and C. coli prevalence in each sub-population. Results indicated that the sensitivity of the culture method was higher than that of PCR in detecting both species when the samples were derived from populations infected with at most one species of Campylobacter. However, from a mixed population, the sensitivity of culture for detecting both C. jejuni or C. coli is reduced while PCR is potentially able to detect both species, although the total probability of correctly identifying at least one species by PCR is similar to that of the culture method.

Estimating the time at which commercial broiler flocks in Great Britain become infected with Campylobacter: a Bayesian approach.

Campylobacter is a common cause of intestinal disease in humans and is often linked to the consumption of contaminated poultry meat. Despite considerable research on the topic there is a large amount of uncertainty associated with Campylobacter epidemiology. A Bayesian model framework was applied to multiple longitudinal datasets on Campylobacter infection in UK broiler flocks to estimate the time at which each flock was first infected with Campylobacter. The model results suggest that the day of first infection ranges from 10 to 45 days; however, over half had a time of infection between 30 and 35 days. When considering only those flocks which were thinned, 48% had an estimated day of infection within 2 days of the day of thinning, thus suggesting an association between thinning and Campylobacter infection. These results demonstrate how knowledge of the time of infection can be correlated to known events to identify potential risk factors for infection.

Estimation of the rate of egg contamination from Salmonella-infected chickens.

Salmonella enterica serovar Enteritidis (S. Enteritidis) is one of the most prevalent causes for human gastroenteritis and is by far the predominant Salmonella serovar among human cases, followed by Salmonella Typhimurium. Contaminated eggs produced by infected laying hens are thought to be the main source of human infection with S. Enteritidis throughout the world. Although previous studies have looked at the proportion of infected eggs from infected flocks, there is still uncertainty over the rate at which infected birds produce contaminated eggs. The aim of this study was to estimate the rate at which infected birds produce contaminated egg shells and egg contents. Data were collected from two studies, consisting of 15 and 20 flocks, respectively. Faecal and environmental sampling and testing of ovaries/caeca from laying hens were carried out in parallel with (i) for the first study, testing 300 individual eggs, contents and shells together and (ii) for the second study, testing 4000 eggs in pools of six, with shells and contents tested separately. Bayesian methods were used to estimate the within-flock prevalence of infection from the faecal and hen post-mortem data, and this was related to the proportion of positive eggs. Results indicated a linear relationship between the rate of contamination of egg contents and the prevalence of infected chickens, but a nonlinear (quadratic) relationship between infection prevalence and the rate of egg shell contamination, with egg shell contamination occurring at a much higher rate than that of egg contents. There was also a significant difference in the rate of egg contamination between serovars, with S. Enteritidis causing a higher rate of contamination of egg contents and a lower rate of contamination of egg shells compared to non-S. Enteritidis serovars. These results will be useful for risk assessments of human exposure to Salmonella-contaminated eggs.

Estimation of the sensitivity of environmental sampling for detection of Salmonella in commercial layer flocks post-introduction of national control programmes.

A key element of national control programmes (NCPs) for Salmonella in commercial laying flocks, introduced across the European Union, is the identification of infected flocks and holdings through statutory sampling. It is therefore important to know the sensitivity of the sampling methods, in order to design effective and efficient surveillance for Salmonella. However, improved Salmonella control in response to the NCP may have influenced key factors that determine the sensitivity of the sampling methods used to detect Salmonella in NCPs. Therefore the aim of this study was to compare estimates of the sensitivity of the sampling methods using data collected before and after the introduction of the NCP, using Bayesian methods. There was a large reduction in the sensitivity of dust in non-cage flocks between the pre-NCP studies (81% of samples positive in positive flocks) and post-NCP studies (10% of samples positive in positive flocks), leading to the conclusion that sampling dust is not recommended for detection of Salmonella in non-cage flocks. However, cage dust (43% of samples positive in positive flocks) was found to be more sensitive than cage faeces (29% of samples positive in positive flocks). To have a high probability of detection, several NCP-style samples need to be used. For confirmation of Salmonella, five NCP faecal samples for cage flocks, and three NCP faecal boot swab samples for non-cage flocks would be required to have the equivalent sensitivity of the EU baseline survey method, which was estimated to have an 87% and 75% sensitivity to detect Salmonella at a 5% within-flock prevalence in cage and non-cage flocks, respectively.

Evaluation of the pooling of swabs for real-time PCR detection of low titre shedding of low pathogenicity avian influenza in turkeys.

The purpose of this study was to determine whether pooling avian influenza (AI)-positive swabs with negative swabs has a detrimental effect on the sensitivity of AI real-time reverse transcription-polymerase chain reactions (rRT-PCRs). Cloacal and buccal swabs were sampled daily from 12 turkeys infected with A/goose/England/07(H2N2). For half the turkeys, each swab was mixed with four swabs from known AI-negative turkeys, and for the other half the swabs were tested individually. Bayesian modelling was used to (i) determine whether pooling the positive swabs compromised the cycle threshold (C(t)) value obtained from the rRT-PCRs, and (ii) estimate the likelihood of detection of an H2N2 infected turkey flock via rRT-PCR for pooled and individually tested swabs (cloacal and buccal) vs. the number of days post-infection of the flock. Results indicated that there was no significant effect of compromising AI rRT-PCR sensitivity by pooling a weak positive swab with negative swabs on the Ct values which were obtained. Pooled sampling was able to widen the detection window compared to individual sampling, for the same number of rRT-PCR tests. This indicates that pooled sampling would be an effective method of reducing the number of tests to be performed to determine flock status during an AI outbreak and for surveillance.

Investigation into sampling strategies in response to potential outbreaks of low pathogenicity notifiable avian influenza initiated in commercial duck holdings in Great Britain.

The aim of this study was to evaluate potential sampling strategies for detection of infected flocks that could be applied during an outbreak of low pathogenicity notifiable avian influenza (LPNAI) initiated in duck holdings, following initial detection. A simulation model of avian influenza virus transmission and spread within and between holdings, respectively, was used to predict the impact on the size and duration of an outbreak of (i) changing the tracing window within which premises that might be the source of infection or that may have been infected by the index premises were sampled and (ii) changing the number of birds sampled in the flock being tested. It has shown that there is potential benefit in increasing the tracing window in terms of reducing the likelihood of a large outbreak. It has also shown that there is comparatively little benefit from increasing the number of birds sampled per flock.

Phylogenetic and molecular characteristics of Eurasian H9 avian influenza viruses and their detection by two different H9-specific RealTime reverse transcriptase polymerase chain reaction tests.

Avian influenza viruses (AIVs) of the H9 haemagglutinin subtype are endemic in many Asian and Middle-East countries, causing mortality and morbidity in poultry. Consequently there is a need for accurate and sensitive detection of Eurasian H9 subtype viruses. Two H9 RealTime reverse transcriptase polymerase chain reaction (RRT-PCR) tests, developed by Monne et al. (2008) and Ben Shabat et al. (2010), were originally validated with a limited number of H9 specimens. In the present study, the two tests have been assessed using 66 diverse H9 isolates and 139 clinical specimens from six H9 poultry outbreaks in four geographically disparate Eurasian countries. The Monne et al. (2008) test was modified and successfully detected all H9 viruses from all three Eurasian H9 lineages. Bayesian analysis of the clinical specimens' results revealed this test to be more sensitive (97%) than the Ben Shabat et al. (2010) test (31%). The latter test detected most H9 isolates of the G1 lineage, but no isolates from other H9 lineages. Mismatches in the primer/probe binding sequences accounted for sensitivity differences between the two H9 RRT-PCRs. Genetic analysis of 34 sequenced H9 haemagglutinin genes showed the South Asian and Middle-East H9 isolates to belong to the H9 G1 lineage, and possessed residues that appear to preferably bind alpha 2,6-linked sialic acid receptors which indicate a potential for human infection. European H9s clustered phylogenetically in a broader geographical group that includes recent North American H9 wild bird isolates and contemporary Asian viruses in the Y439 H9 lineage.

Experimental bovine spongiform encephalopathy: detection of PrP(Sc) in the small intestine relative to exposure dose and age.

European regulations for the control of bovine spongiform encephalopathy (BSE) decree destruction of the intestines from slaughtered cattle, therefore producers have been obliged to import beef casings from countries with a negligible BSE risk. This study applies immunohistochemical and biochemical approaches to investigate the occurrence and distribution of disease-associated prion protein (PrP(Sc)) in the duodenum, jejunum and ileum of cattle orally exposed to a 1 g or 100 g dose of a titrated BSE brainstem homogenate. Samples were derived from animals at various times post exposure. Lymphoid follicles were counted and the frequency of affected follicles recorded. No PrP(Sc) was detected in the duodenum or jejunum of animals exposed to a 1 g dose or in the duodenum of animals receiving a 100 g dose. PrP(Sc) was detected in the lymphoid tissue of the ileum of 1/98 (1.0%) animals receiving the 1 g dose and in the jejunum and ileum of 8/58 (13.8%) and 45/99 (45.5%), respectively, of animals receiving the 100 g dose. The frequency of PrP(Sc)- positive follicles was less than 1.5% per case and biochemical tests appeared less sensitive than immunohistochemistry. The probability of detecting lymphoid follicles in the ileum declined with age and for the 100 g exposure the proportion of positive follicles increased, while the proportion of positive animals decreased with age. Detection of PrP(Sc) in intestinal neural tissue was rare. The results suggest that the jejunum and duodenum of BSE-infected cattle contain considerably less BSE infectivity than the ileum, irrespective of exposure dose. In animals receiving the low exposure dose, as in most natural cases of BSE, the rarity of PrP(Sc) detection compared with high-dose exposure, suggests a very low BSE risk from food products containing the jejunum and duodenum of cattle slaughtered for human consumption.

A comparison of pooled and individual bird sampling for detection of Salmonella in commercial egg laying flocks.

The objective of this study was to determine the sensitivity of culturing pooled samples containing varying numbers of individual droppings for detection of Salmonella in commercial egg-laying flocks relative to the within-flock prevalence. A laboratory experiment was performed to directly measure the effect of diluting positive with negative faeces on the sensitivity of detection, and thus provide priors for a Bayesian model of pooled sampling. Pooled samples made up of different numbers of individual faecal droppings were collected from 20 flocks, and in addition bulked faeces and dust were also sampled using an in-house method that involved collecting 10 dust and 10 faeces samples into jars with buffered peptone water. The results from these flocks were analysed using Bayesian methods for diagnostic test evaluation in the absence of a gold standard, and the sensitivity of each pooled sample type was estimated relative to the within-flock prevalence. The sensitivity of pooled samples depended on the within-flock prevalence, and increased as the prevalence increased. The sensitivity of pooled sampling tended to increase with the number of droppings in the pool, and overall there was a higher proportion of positive samples from the pools of 20, 60 and the in-house faeces pooling method compared to the pools of 10, 5 and the individual droppings. Dust samples were more sensitive than the faeces samples, and so the inclusion of dust in sampling schemes is recommended.