Using SNP addresses for Salmonella Typhimurium DT104 in routine veterinary outbreak detection.

SNP addresses are a pathogen typing method based on whole-genome sequences (WGSs), assigning groups at seven different levels of genetic similarity. Public health surveillance uses it for several gastro-intestinal infections; this work trialled its use in veterinary surveillance for salmonella outbreak detection. Comparisons were made between temporal and spatio-temporal cluster detection models that either defined cases by their SNP address or by phage type, using historical data sets. Clusters of SNP incidents were effectively detected by both methods, but spatio-temporal models consistently detected these clusters earlier than the corresponding temporal models. Unlike phage type, SNP addresses appeared spatially and temporally limited, which facilitated the differentiation of novel, stable, or expanding clusters in spatio-temporal models. Furthermore, these models flagged spatio-temporal clusters containing only two to three cases at first detection, compared with a median of seven cases in phage-type models. The large number of SNP addresses will require automated methods to implement these detection models routinely. Further work is required to explore how temporal changes and different host species may impact the sensitivity and specificity of cluster detection. In conclusion, given validation with more sequencing data, SNP addresses are likely to be a valuable addition to early warning systems in veterinary surveillance.

Abattoir based survey of Salmonella in finishing pigs in the United Kingdom 2006-2007.

All European Union (EU) Member States (MSs) were required to conduct a baseline survey from October 2006 to September 2007 to estimate the prevalence of Salmonella infection in finisher pigs at slaughter. In the United Kingdom (UK), samples for microbiological culture were collected from the ileo-caecal lymph nodes, from carcass swabs and from caecal contents. Meat juice samples were also collected for testing in an enzyme-linked immunosorbent assay (MJE) to detect a serological response. Salmonella was isolated from 22% (CI95% 19-25%) of ileo-caecal lymph nodes, 15% (CI95% 12-18%) of carcass swabs and 22% (CI95% 19-26%) of caecal contents. A quarter (25%, CI95% 22-29%) of MJE samples were positive for Salmonella antibodies (cut-off, S/P≥0.25). The most frequently identified serovars were Salmonella typhimurium (57%) and Salmonella derby (26%). The results were used to investigate some factors associated with Salmonella infection in slaughter pigs. The weight of the carcass was significant, with a lower probability of obtaining a positive meat juice result from pigs with a weight greater than 75 kg (p=0.03). The weight of the lymph node sample was significantly associated with Salmonella status, with a heavier sample of lymph nodes being more likely to be positive (OR=2.16 CI95% 1.07-4.39). 'Carcass weight' in two of the classes - 75-79 kg and 80-84 kg - (OR=0.44 CI95% 0.28-0.70; OR=0.64 CI95% 0.49-0.85) and 'fewer-than-1500-pigs' scheduled for slaughter on the day of sampling' (OR=0.41 CI95% 0.24-0.71) were also associated with a reduced risk of isolating Salmonella from lymph node. The model for carcass swabs showed a positive association with 'time-elapsed-since-the-start-of-the-line' indicating a higher risk of contamination as the day progressed (p<0.01). This model also showed positive association between isolation of Salmonella from a carcass swab and the occurrence of Salmonella in caecal contents from the same pig (OR=2.22 CI95% 1.38-3.59) and a negative association with time in singeing units (OR=0.88 CI95% 0.79-0.98).

A longitudinal field trial assesing the impact of feeding waste milk containing antibiotic residues on the prevalence of ESBL-producing Escherichia coli in calves.

A longitudinal field trial was carried out on a farm known to harbour cefotaximase (CTX-M)-positive Escherichia coli, in order to assess the impact of feeding waste milk containing antibiotic residues (WM+AR) on the prevalence of these bacteria in the faeces of calves. Fifty calves were alternately assigned to one of two groups at birth and fed either milk replacer (control group) or WM+AR (treatment group). Faecal samples were collected from all calves daily for the first week after enrolment, twice weekly until weaning, then weekly for a further six weeks. Environmental samples from the calf housing were collected weekly. WM+AR and powdered milk samples were examined for antibiotic residues and CTX-M-positive E. coli. Total E. coli and CTX-M-positive E. coli in faecal samples were enumerated using selective media. Regression analyses were performed on the bacterial count data using a population-averaged approach based on generalised estimating equations (GEE) to account for repeated measurements on individual calves over time. Cefquinome, a fourth generation cephalosporin, was detected in 87% of WM+AR samples at a mean concentration of 0.746 mg/l. All environmental sampling locations yielded CTX-M-positive E. coli. Significantly more pen floor samples were positive in the treatment group. Calves in the treatment group shed greater numbers of CTX-M-positive E. coli than calves in the control group throughout the study, and shedding decreased at a slower rate in the treatment group. CTX-M-positive E. coli persisted in a larger number of calves fed WM+AR compared with calves fed milk replacer where the prevalence in the treatment group declined significantly slower over time. There was no difference between calves fed WM+AR or calves fed milk replacer in the proportion of E. coli isolates that were CTX-M-positive. These findings indicate that feeding WM+AR increased the amount of resistant bacteria shed in the faeces. Shedding of CTX-M-positive E. coli persisted for longer in calves fed WM+AR, and persisted after weaning.

Risk factors for antimicrobial resistance in Escherichia coli found in GB turkey flocks.

Four models are presented investigating risks present on Great Britain (GB) turkey farms in breeding and fattening flocks for ciprofloxacin and cephalosporin resistance. Risk factors for ciprofloxacin resistance in fattening flocks were sourcing of feed from national compounders, antimicrobial use in the flock and evidence of mice. Disinfection of floors and walls at depopulation, older flocks and division of the flock with partitions reduced the risk. In breeding farms holding over 10,000 birds, administration of fluoroquinolones within the last year and horses on the neighbouring farm all increased the risk, whereas replenishing foot dips more than once a week reduced the risk. For cephalosporin-resistant Escherichia coli on fattening farms, being an independent farm, having a watercourse near the poultry houses, dividing the flock with partitions and providing staff with gloves reduced the risk. Factors that increased the risk included if staff worked with other livestock and if there were pigs on neighbouring farms. This work suggests that good hygiene and biosecurity, rodent control and responsible use of antimicrobials on turkey farms might help minimise the prevalence of fluoroquinolone and cephalosporin resistance in E coli, and restrict the spread of resistance genes to other organisms.

Evaluation of methods for measuring coverage and representativeness of an early-warning disease surveillance system.

Early-warning surveillance provides an essential component of the evidence required to protect animal health. Assessing the proportion of the population included in surveillance systems (coverage) provides a measure of the effectiveness of early-warning surveillance, and contributes to ensuring that these systems are efficient. This paper describes an investigation of methods used for assessing the coverage and representativeness of the 'FarmFile' early-warning surveillance system. This system uses information collected with samples submitted to diagnostic laboratories by private veterinary practitioners in England and Wales. Available data on pig holdings and veterinary practices in four English counties, selected to represent a range of diverse population characteristics, were supplemented using surveys of veterinary practices. Coverage assessments were based on submissions made to FarmFile in 2009. The proportion of holdings covered varied from 5-62 per cent in Devon and Cumbria, and 16-97 per cent in Norfolk and East Riding of Yorkshire. The results suggest that while the proportion of individual pigs covered by the current early-warning surveillance system is high, small and breeding-only holdings in some regions may be poorly covered. Coverage assessments vary depending on the methods used for their assessment, and multiple assessment methods can provide a 'range' within which coverage lies.

A survey of antimicrobial usage on dairy farms and waste milk feeding practices in England and Wales.

The cause for the high prevalence of cefotaximase-producing Escherichia coli reported in dairy calves is unknown but may be partly due to the selective pressure of antimicrobial residues in waste milk (milk unfit for human consumption) fed to the calves. Antimicrobial use and waste milk feeding practices were investigated in 557 dairy farms in 2010/2011 that responded to a randomised stratified postal survey. The mean number of cases of mastitis per herd in the previous year was 47, and 93 per cent of respondents used antibiotic intra-mammary tubes to treat mastitis. The most frequently used lactating cow antibiotic tubes contained dihydrostreptomycin, neomycin, novobiocin, and procaine penicillin (37 per cent), and cefquinome (29 per cent). Ninety-six per cent of respondents used antibiotic tubes at the cessation of lactation ('drying off'). The most frequently used dry cow antibiotic tube (43 per cent) contained cefalonium. Frequently used injectable antibiotics included tylosin (27 per cent), dihydrostreptomycin and procaine penicillin (20 per cent) and ceftiofur (13 per cent). Eighty-three per cent of respondents (413) fed waste milk to calves. Of these 413, 87 per cent fed waste milk from cows with mastitis, and only one-third discarded the first milk after antibiotic treatment. This survey has shown that on more than 90 per cent of the farms that feed waste milk to calves, waste milk can contain milk from cows undergoing antibiotic treatment. On some farms, this includes treatment with third- and fourth-generation cephalosporins. Further work is underway to investigate the presence of these antimicrobials in waste milk.

Risk factors associated with extended spectrum beta-lactamase Escherichia coli (CTX-M) on dairy farms in North West England and North Wales.

This study investigated the potential spread of CTX-M-14 Escherichia coli from a known ESBL E. coli positive farm and risk factors for the presence of CTX-M E. coli on dairy farms. Between November 2009 and March 2010, 65 farms in North West England and North Wales were visited and animals sampled for E. coli producing CTX-M ESBLs. Seventeen of these were known to have received animals from a known ESBL E. coli positive 'index' farm since 2005 (linked farms). The prevalence of CTX-M E. coli in the population of linked farms was 58.8% (10/17; CI(95%) 32.9-81.6%) and in the randomly selected control population was 35.4% (17/48; CI(95%) 22.2-50.5%). There was no significant (p>0.05) linkage for the detection of any CTX-M E. coli or specifically a CTX-M-14 E. coli to the index farm. Group 1 (CTX-M-15, CTX-M-55, CTX-M-1, CTX-M-32), group 2 (CTX-M-2) and group 9 (CTX-M-14, CTX-M-14 B, CTX-M-27) CTX-M E. coli were identified on the study farms. Molecular analysis revealed that three plasmids from linked farms had similar sizes (95 kbp), replicon type (IncK) and backbone genes as that from the index farm. Logistic regression analysis revealed that farms that had used a 3rd or 4th generation cephalosporin (ceftiofur, cefoperazone and cefquinome) in livestock in the last 12 months were nearly 4 times more likely to have ESBL E. coli present (p=0.037; OR=3.93). There was no significant association between presence of CTX-M E. coli and the use of any 1st or 2nd generation cephalosporins. Several other risk factors for the presence of CTX-M E. coli were identified, such as storage of slurry in a pit, operating an open herd policy and infrequent cleaning of calf feeding equipment.

Investigation of the presence of ESBL-producing Escherichia coli in the North Wales and West Midlands areas of the UK in 2007 to 2008 using scanning surveillance.

Between November 5, 2007 and November 4, 2008, faecal samples from cattle and sheep submitted for diagnostic purposes to the Aberystwyth and Shrewsbury Veterinary Laboratories Agency (VLA) (now AHVLA) regional laboratories (covering North Wales and the West Midlands) were screened for the presence of Escherichia coli that produces CTX-M extended-spectrum ß-lactamase (ESBL) using the selective medium CHROMagar CTX. Samples from 113 farms were tested and eight ESBL-positive farms identified. Of these, six farms were identified via submissions of cattle faeces and two from sheep. Gene sequencing revealed both group 1 and group 9 CTX-M enzymes corresponding to CTX-M-14, CTX-M-14B (group 9) and CTX-M-15/28 (group 1). Analysis of these isolates by nanoarray revealed that some were carrying a range of virulence genes including ireA, iroN and prfB, which have been associated with extraintestinal pathogenic E coli, and were multidrug resistant. Geographical analysis with choropleth maps suggested that these CTX-M genes are relatively widespread in the North Wales and West Midlands study area. This work was carried out concurrently with the running of a VLA ESBL surveillance system, which has subsequently identified many more CTX-M positive farms in the UK.

Survey of Salmonella prevalence on commercial turkey breeding and fattening farms in the UK in 2006 to 2007.

A total of 29 breeding turkey holdings and 317 fattening turkey holdings were sampled between October 2006 and September 2007 in order to establish the baseline prevalence of Salmonella in turkeys in the UK. The weighted holding level Salmonella prevalence was found to be 20.1 per cent (95 per cent confidence interval [CI] 8.6 to 40.3 per cent) in breeding turkeys and 37.7 per cent (95 per cent CI 33.4 to 42.3 per cent) in fattening turkeys. For breeding turkeys, a weighted flock-level prevalence, as more than one flock per holding was sampled, was estimated at 7.1 per cent (95 per cent CI 3.2 to 14.8 per cent). A total of 13 different serovars were identified in the survey. The most frequent serovar in both turkey flock classes was Salmonella Kottbus, which was found on two breeding holdings and 63 of the fattening holdings giving weighted prevalences of 10.4 per cent (95 per cent CI 2.6 to 34.1 per cent) and 23.0 per cent (95 per cent CI 19.3 to 27.3 per cent), respectively. On breeding holdings, a single isolate of Salmonella Typhimurium, identified as DT12 (weighted prevalence 3.5 per cent [95 per cent CI 0.7 to 15.8 per cent] [holding], 0.7 per cent [95 per cent CI 0.1 to 3.7 per cent] [flock)], was found. On fattening holdings, there were 55 isolates of S Typhimurium from 16 holdings, giving a weighted prevalence of this serovar of 5.4 per cent (95 per cent CI 3.6 to 8.0 per cent). There were no isolates of Salmonella serovars Enteritidis, Hadar, Infantis or Virchow.

Salmonella serovars and antimicrobial resistance patterns on a sample of high seroprevalence pig farms in England and Wales (2003-2008).

Following the introduction of a national abattoir-based monitoring programme for Salmonella in pigs, advisory visits were made to pig farms in England and Wales with high Salmonella seroprevalence assessed by muscle tissue fluid (meat juice) ELISA. Samples (n = 15 790), including pooled pen floor faeces (n = 12 136), were collected for Salmonella culture from 296 farms, between October 2003 and February 2008. Salmonella was isolated from 4489 (28%) of all samples collected, including 3301 (27%) of pooled pen floor faecal samples, from 270 (91%) of farms visited. Salmonella Typhimurium and S. Derby were the most prevalent serovars, representing 64% and 16% of isolates serotyped, respectively. The main phage types of S. Typhimurium identified were U288 and DT193. Antimicrobial resistance (AMR) was seen in 92% of isolates tested, with the highest frequencies of resistance occurring to tetracyclines (T), sulphonamide compounds (SU), ampicillin (AM), sulphamethoxazole/trimethoprim (SXT), streptomycin (S) and chloramphenicol (C). Fifty-nine AMR patterns were observed, the most frequent of these being T, AM, SXT, C, S, SU, seen in 35% of isolates tested. Multi-drug resistance was commonly found, with 67% of isolates submitted for AMR testing showing resistance to between four and nine antimicrobials.

Investigation of risk factors for Salmonella on commercial egg-laying farms in Great Britain, 2004-2005.

In 2004/05, all European Union member states were required to carry out standardised prevalence surveys to establish the baseline prevalence of Salmonella in commercial laying flocks. As part of the survey in Great Britain, additional data were collected from 380 of the enrolled laying hen holdings to investigate risk factors for Salmonella at farm level. Stratified, simple random sampling was used to select holdings from which dust and boot swab samples were collected and tested for Salmonella using a modification of ISO 6579:2002. Using a multivariable logistic model weighted to account for the survey design, several factors significantly associated with Salmonella and Salmonella Enteritidis status were identified. Larger holdings (>or=30,000 birds) were found to be at higher risk of Salmonella (odds ratio [OR] 4.79, P=0.025), while vaccination (OR 0.28, P=0.013), providing foot dips with brushes (OR 0.27, P=0.042), washing and disinfecting the house at depopulation (OR 0.19, P=0.003), having a clean car park away from house (OR 0.14, P=0.001), using an independent (OR 0.19, P=0.007) or other non-company (OR 0.40, P=0.049) source of feed, being over 1 km from the nearest neighbouring farm (OR 0.45, P=0.021) and the presence of cats and dogs on the farm (OR 0.26, P=0.002) or on contiguous farms (OR 0.44, P=0.030) reduced the risk of any Salmonella serovars being present. Factors found to be associated specifically with an increased risk of S Enteritidis infection included holding size (OR 14.88, P=0.001) and frequent sightings of rats (OR 8.17, P<0.001) or mice (OR 5.78, P=0.006). Non-caged systems (OR 0.14, P=0.002), vaccination (OR 0.08, P=0.001), the use of a non-company feed source (OR 0.11, P=0.003), running the site as all-in/all-out (OR 0.06, P<0.001) and the presence of cats and dogs on the farm (OR 0.14, P=0.002) were associated with a reduced risk.

Investigation of risk factors for Salmonella on fattening-turkey farms.

A cross-sectional study into risk factors for Salmonella was undertaken using data gathered from 252 fattening turkey flocks in the UK. The data was derived from the EU baseline survey conducted during 2006 and 2007, in addition to a voluntary questionnaire. Multivariate logistic regression models identified significant risk factors for Salmonella spp. and Salmonella Typhimurium. A decreased risk of Salmonella spp. infection was associated with a history of intestinal illness in the sampled flock (OR 0.17), the use of wood shavings as litter (OR 0.21), use of disinfectant in the cleaning process (OR 0.25), incineration of dead birds on farm (OR 0.29), seasonal production (OR 0.31), farm staff also working with cattle (OR 0.31), and the presence of pigs on neighbouring farms (OR 0.38). The risk of isolating Salmonella spp. varied according to the company from which the poults were sourced. A reduced risk of S. Typhimurium infection was associated with the use of wax blocks to control rodents (OR 0.09), using mains water (OR 0.19) and having a Salmonella test programme (OR 0.23). An increased risk of S. Typhimurium infection was associated with storage of items around the turkey house (OR 5.20), evidence of mice (OR 4.71) and a soil surface surrounding the turkey house (OR 2.70). This study therefore identifies a number of important practical measures which can be implemented by farmers and veterinarians within the turkey industry to assist in the control of salmonellosis at the farm level.

Survey of the prevalence of Salmonella on commercial broiler farms in the United Kingdom, 2005/06.

Between October 2005 and September 2006, all European Union member states were required to carry out standardised surveys of the prevalence of Salmonella in broiler flock holdings to establish baseline data from which to derive national targets for disease reduction. In the uk 382 holdings were sampled, 41 of which were positive for Salmonella, giving an estimated weighted prevalence of 10.7 per cent (95 per cent confidence interval [ci] 8.1 to 13.1 per cent). The serotype most frequently isolated was Salmonella Ohio, with a weighted prevalence of 2.2 per cent (95 per cent ci 1.2 to 3.7 per cent), followed by Salmonella Kedougou at 1.7 per cent (95 per cent ci 0.9 to 3.2 per cent). There were no isolates of Salmonella Enteritidis and only a single isolation of Salmonella Typhimurium (0.2 per cent, 95 per cent ci 0.0 to 1.6 per cent). Of the three other serotypes given top priority by the eu owing to their public health significance, Salmonella Virchow was isolated from one holding, but Salmonella Hadar and Salmonella Infantis were not detected on any of the holdings.

Risk-based surveillance for H5N1 avian influenza virus in wild birds in Great Britain.

Recent outbreaks of the H5N1 strain of avian influenza in Europe have highlighted the need for continuous surveillance and early detection to reduce the likelihood of a major outbreak in the commercial poultry industry. In Great Britain (gb), one possible route by which H5N1 could be introduced into domestic poultry is through migratory wild birds from Europe and Asia. Extensive monitoring data on the 24 wild bird species considered most likely to introduce the virus into GB, and analyses of local poultry populations, were used to develop a risk profile to identify the areas where H5N1 is most likely to enter and spread to commercial poultry. The results indicate that surveillance would be best focused on areas of Norfolk, Suffolk, Lancashire, Lincolnshire, south-west England and the Welsh borders, with areas of lower priority in Anglesey, south-west Wales, north-east Aberdeenshire and the Firth of Forth area of Scotland. These areas have significant poultry populations including a large number of free-range flocks, and a high abundance of the 24 wild bird species.

Survey of the prevalence of Salmonella species on commercial laying farms in the United Kingdom.

A survey of salmonella infection on 454 commercial layer flock holdings in the uk was carried out between October 2004 and September 2005. Fifty-four (11.7 per cent, 95 per cent confidence interval 9.3 to 14.0 per cent) were salmonella positive. The most common serovar identified was Salmonella Enteritidis at a prevalence of 5.8 per cent, and 70 per cent of these isolates were phage types 4, 6, 7 and 35. Salmonella Typhimurium was the second most prevalent serovar, found in 1.8 per cent of the farms. Of the three other serovars given top priority by the eu because of their public health significance, Salmonella Virchow and Salmonella Infantis were each isolated from one holding, but Salmonella Hadar was not isolated from any of the holdings. Analysis of antimicrobial resistance patterns revealed that over 76 per cent of the isolates were sensitive to all of the 16 drugs tested, and all the isolates were sensitive to ciprofloxacin, gentamicin, ceftazidime, apramycin, amikacin, amoxicillin/clavulanic acid, neomycin and cefotaxime.

Transmission dynamics of lymphatic filariasis: vector-specific density dependence in the development of Wuchereria bancrofti infective larvae in mosquitoes.

The principles of meta-analysis developed in a previous study were extended to investigate the process of Wuchereria bancrofti (Cobbold) (Filarioidea: Onchocercidae) infection in mosquito (Diptera: Culicidae) hosts, focusing specifically on the functional forms and strength of density dependence in the development of ingested microfilariae (mf) to infective (third instar) larvae (L3). Mathematical models describing observed mf-L3 functional responses for each of the major three parasite-transmitting vector genera, Aedes, Culex and Anopheles mosquitoes, were fitted to paired mf-L3 data collated from all available studies in the published literature. Model parameters were estimated and compared by deriving and applying a data synthetic framework, based on applying a non-linear weighted regression model for fitting mathematical models to multistudy data. The results confirm previous findings of the existence of significant between-genera differences in the mf-L3 development relationship, particularly with regard to the occurrence of limitation in Culex mosquitoes and facilitation in Aedes and Anopheles mosquitoes. New and unexpected findings regarding L3 development from ingested mf were discovered as follows: (1) for Culex, overcompensation in L3 development at higher intensities of mf (or a peaked mf-L3 functional response) was detected; (2) for Aedes mosquitoes, facilitation (with an apparent asymptotic constraint on L3 development at high mf densities) was shown to be the major process governing L3 development, and (3) for Anopheles, a stronger facilitation type of response with no apparent saturation in L3 development appears to govern L3 output from ingested mf. These results yield major new insights regarding filarial vector infection dynamics and their potential impacts on parasite control, and demonstrate the efficacy of employing a data synthetic approach to reveal and estimate parasitic infection processes in host populations.

Transmission dynamics of lymphatic filariasis: density-dependence in the uptake of Wuchereria bancrofti microfilariae by vector mosquitoes.

Gaining a better understanding of parasite infection dynamics in the vector mosquito (Diptera: Culicidae) population is central to improving knowledge regarding the transmission, persistence and hence control of lymphatic filariasis. Here, we use data on mosquito feeding experiments collated from the published literature to examine the available evidence regarding the functional form of the first component of this parasite-vector relationship for Wuchereria bancrofti (Filarioidea: Onchocercidae) causing Bancroftian filariasis, i.e. the rate of microfilariae (mf) uptake from the blood of infected humans by the feeding mosquito vector. Using a simple logarithmic regression model for describing the observed relationships between the mean numbers of mf ingested per mosquito and parasite load in humans in each study, and a linear mixed-effects meta-analytical framework for synthesizing the observed regressions across studies, we show here for the first time clear evidence for the existence of density-dependence in this process for all the three major filariasis transmitting mosquito vectors. An important finding of this study is that this regulation of mf uptake also varies significantly between the vector genera, being weakest in Culex, comparatively stronger in Aedes and most severe and occurring at significantly lower human mf loads in Anopheles mosquitoes. The analysis of the corresponding mf uptake prevalence data has further highlighted how density-dependence in mf uptake may influence the observed distributions of mf in vector populations. These results show that whereas strong regulation of mf uptake, especially when it leads to saturation in uptake at low human parasite intensities, can lead to static distributions of mf per mosquito with host parasite intensity, a weaker regulation of mf ingestion can give rise to changes in both mean mf loads and in the frequency distribution of parasites/mosquito with increasing human parasite intensity. These findings highlight the importance of considering local vector infection dynamics when attempting to predict the impacts of community-based filariasis control. They also emphasize the value of developing and applying robust meta-analytic methods for estimating functional relationships regarding parasitic infection from population ecological data.