Boot Sock Research Articles

Rapid detection of Salmonella enterica in primary production samples by eliminating DNA amplification inhibitors using an improved sample pre-treatment method.

Sensitive detection of pathogens in livestock farms is an integral part of the One Health Action Plan of the European Union (EU). Ensuring this requires on-site testing devices that are compatible with complex matrices such as primary production samples. Among all, faeces are considered the most challenging matrix type that makes it difficult to identify pathogens because of complexity in sample preparation for molecular testing. We have developed a loop-mediated isothermal amplification (LAMP) based veterinary point-of-care (POC) device (VETPOD) and adapted it to detect Salmonella enterica in primary production samples. Three different sampling methods (semi-wet chicken faeces, boot socks collection and dust samples from poultry shed) were iteratively tested to assess their nature of complexity and possibility for adapting them as suitable sampling methods for on-site testing. During the study, the sample preparation method that included a two-step centrifugation combined with washing of the enriched Salmonella cells was found crucial in eliminating amplification inhibitors originating from the faecal matrices. A total of 90 samples were tested that included 60 samples for sensitivity study and 30 samples for relative level of detection (RLOD, a level of detection in comparison to ISO 6579:1 reference method). Overall, the VETPOD had a sensitivity of 90%, 84.62% and 81.82% for boot sock, faecal and dust samples, respectively. The RLOD was 2.23 CFU/25 g which was found to be 1.33 times higher than the ISO 6579:1. Performing with an excellent agreement with ISO 6579:1, the VETPOD proved as a promising alternative to detect Salmonella spp. in primary production and animal husbandry samples.

Combined Quantification and Deep Serotyping for Salmonella Risk Profiling in Broiler Flocks.

Despite a reduction of Salmonella contamination on final poultry products, the level of human salmonellosis cases attributed to poultry has remained unchanged over the last few years. There needs to be improved effort to target serovars which may survive antimicrobial interventions and cause illness, as well as to focus on lessening the amount of contamination entering the processing plant. Advances in molecular enumeration approaches allow for the rapid detection and quantification of Salmonella in pre- and postharvest samples, which can be combined with deep serotyping to properly assess the risk affiliated with a poultry flock. In this study, we collected a total of 160 boot sock samples from 20 broiler farms across four different integrators with different antibiotic management programs. Overall, Salmonella was found in 85% (68/80) of the houses, with each farm having at least one Salmonella-positive house. The average Salmonella quantity across all four complexes was 3.6 log10 CFU/sample. Eleven different serovars were identified through deep serotyping, including all three key performance indicators (KPIs; serovars Enteritidis, Infantis, and Typhimurium) defined by the U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS). There were eight multidrug resistant isolates identified in this study, and seven which were serovar Infantis. We generated risk scores for each flock based on the presence or absence of KPIs, the relative abundance of each serovar as calculated with CRISPR-SeroSeq (serotyping by sequencing the clustered regularly interspaced palindromic repeats), and the quantity of Salmonella organisms detected. The work presented here provides a framework to develop directed processing approaches and highlights the limitations of conventional Salmonella sampling and culturing methods. IMPORTANCE Nearly one in five foodborne Salmonella illnesses are derived from chicken, making it the largest single food category to cause salmonellosis and indicating a need for effective pathogen mitigation. Although industry has successfully reduced Salmonella incidence in poultry products, there has not been a concurrent reduction in human salmonellosis linked to chicken consumption. New efforts are focused on improved control at preharvest, which requires improved Salmonella surveillance. Here, we present a high-resolution surveillance approach that combines quantity and identity of Salmonella in broiler flocks prior to processing which will further support improved Salmonella controls in poultry. We developed a framework for this approach, indicating that it is possible and important to harness deep serotyping and molecular enumeration to inform on-farm management practices and to minimize risk of cross-contamination between flocks at processing. Additionally, this framework could be adapted to Salmonella surveillance in other food animal production systems.

Association of Broiler Litter Microbiome Composition and Campylobacter Isolation.

Infection with Campylobacter species is one of the leading causes of bacterial diarrhea in humans in the US. Chickens, which become colonized on the farm, are important reservoirs of this bacterium. Campylobacter can establish itself in the broiler house via a variety of sources, can survive in the litter of the house, and possibly persist over successive flock cycles. However, the role of the broiler litter microbiome on Campylobacter persistence is not clear. A matched case-control study was conducted to determine whether the broiler litter microbiome composition was associated with Campylobacter isolation within the broiler house. Flocks were classified as cases when either Campylobacter jejuni or Campylobacter coli was isolated in boot sock samples, or as controls otherwise. Case and control flocks were matched at the broiler house level. Composite broiler litter samples were collected and used for DNA extraction and 16S rRNA gene V4 region sequencing. Reads were processed using the DADA2 pipeline to obtain a table of amplicon sequence variants. Alpha diversity and differential bacterial relative abundance were used as predictors of Campylobacter isolation status in conditional logistic regression models adjusting for flock age and sampling season. Beta diversity distances were used as regressors in stratified PERMANOVA with Campylobacter isolation status as predictor, and broiler house as stratum. When Campylobacter was isolated in boot socks, broiler litter microbiome richness and evenness were lower and higher, respectively, without reaching statistical significance. Campylobacter isolation status significantly explained a small proportion of the beta diversity (genus-level Aitchison dissimilarity distance). Clostridium and Anaerostipes were positively associated with Campylobacter isolation status, whereas Bifidobacterium, Anaerosporobacter, and Stenotrophomonas were negatively associated. Our results suggest the presence of bacterial interactions between Campylobacter and the broiler litter microbiome. The negative association of Campylobacter with Bifidobacterium, Anaerosporobacter, and Stenotrophomonas in litter could be potentially exploited as a pre-harvest control strategy.

Integrated Salmonella control in Turkey Production

In recent years, an increase of fattening turkey flocks positive for Salmonella spp. was noted in the European Union. Considering these latest trends, there is a high need to increase knowledge on specific risk factors for fattening turkey flocks in conjunction with successful control measures to combat Salmonella infections on farm. Here, we report a commercial turkey fattening farm that was found positive for Salmonella enterica serovar Typhimurium for two consecutive batches. By means of the Food Safety Program, a risk assessment developed by Elanco (Antwerpen, Belgium) and a farm walk, a Food Safety Index was generated, highlighting specific risk factors. Consequently, an action plan was set up, leading to a favorable increase in the Food Safety Index from 45% to 67%. Among others, vaccinating fattening turkeys was an important control measure. Monitoring of the Salmonella status of the batches was performed by sampling paper coming from the transport boxes at day of arrival and boot sock samples at the age of 14 weeks. Finally neck skin samples were taken at the slaughterhouse. Uptake of the vaccine was evaluated by sampling of the caeca and taking cloacal swabs 24 hours after first and second vaccination, respectively. Results coming from the analysis of the caeca indicate that vaccination at day one was efficient. Analysis of the cloacal swabs taken after second vaccination was inconclusive. Cooperatively, these actions improved the overall biosecurity of the farm as shown by the negative Salmonella stat us of the boot sock and neck skin samples in the third batch.

First isolation and whole-genome sequencing of a Shewanella algae strain from a swine farm in Brazil

BackgroundInfections caused by Shewanella spp. have been increasingly reported worldwide. The advances in genomic sciences have enabled better understanding about the taxonomy and epidemiology of this agent. However, the scarcity of DNA sequencing data is still an obstacle for understanding the genus and its association with infections in humans and animals.ResultsIn this study, we report the first isolation and whole-genome sequencing of a Shewanella algae strain from a swine farm in Brazil using the boot sock method, as well as the resistance profile of this strain to antimicrobials. The isolate was first identified as Shewanella putrefaciens, but after whole-genome sequencing it showed greater similarity with Shewanella algae. The strain showed resistance to 46.7% of the antimicrobials tested, and 26 resistance genes were identified in the genome.ConclusionsThis report supports research made with Shewanella spp. and gives a step forward for understanding its taxonomy and epidemiology. It also highlights the risk of emerging pathogens with high resistance to antimicrobial formulas that are important to public health.

Regional differences in presence of Shiga toxin-producing Escherichia coli virulence-associated genes in the environment in the North West and East Anglian regions of England.

Shiga toxin-producing Escherichia coli is carried in the intestine of ruminant animals, and outbreaks have occurred after contact with ruminant animals or their environment. The presence of STEC virulence genes in the environment was investigated along recreational walking paths in the North West and East Anglia regions of England. In all, 720 boot sock samples from walkers' shoes were collected between April 2013 and July 2014. Multiplex PCR was used to detect E. coli based on the amplification of the uidA gene and investigate STEC-associated virulence genes eaeA, stx1 and stx2. The eaeA virulence gene was detected in 45·5% of the samples, where stx1 and/or stx2 was detected in 12·4% of samples. There was a difference between the two regions sampled, with the North West exhibiting a higher proportion of positive boot socks for stx compared to East Anglia. In univariate analysis, ground conditions, river flow and temperature were associated with positive boot socks. The detection of stx genes in the soil samples suggests that STEC is present in the English countryside and individuals may be at risk for infection after outdoor activities even if there is no direct contact with animals. SIGNIFICANCE AND IMPACT OF THE STUDY: Several outbreaks within the UK have highlighted the danger of contracting Shiga toxin-producing Escherichia coli from contact with areas recently vacated by livestock. This is more likely to occur for STEC infections compared to other zoonotic bacteria given the low infectious dose required. While studies have determined the prevalence of STEC within farms and petting zoos, determining the risk to individuals enjoying recreational outdoor activities that occur near where livestock may be present is less researched. This study describes the prevalence with which stx genes, indicative of STEC bacteria, were found in the environment in the English countryside.

Comparison of Different Invasive and Non-Invasive Methods to Characterize Intestinal Microbiota throughout a Production Cycle of Broiler Chickens.

In the short life of broiler chickens, their intestinal microbiota undergoes many changes. To study underlying biological mechanisms and factors that influence the intestinal microbiota development, longitudinal data from flocks and individual birds is needed. However, post-mortem collection of samples hampers longitudinal data collection. In this study, invasively collected cecal and ileal content, cloacal swabs collected from the same bird, and boot sock samples and cecal droppings from the litter of the broilers’ poultry house, were collected on days 0, 2, 7, 14 and 35 post-hatch. The different sample types were evaluated on their applicability and reliability to characterize the broiler intestinal microbiota. The microbiota of 247 samples was assessed by 16S ribosomal RNA gene amplicon sequencing. Analyses of α and β measures showed a similar development of microbiota composition of cecal droppings compared to cecal content. Furthermore, the composition of cecal content samples was comparable to that of the boot socks until day 14 post-hatch. This study shows that the value of non-invasive sample types varies at different ages and depends on the goal of the microbiota characterization. Specifically, cecal droppings and boot socks may be useful alternatives for cecal samples to determine intestinal microbiota composition longitudinally.

A Quantitative Real-Time PCR Approach for Assessing Campylobacter jejuni and Campylobacter coli Colonization in Broiler Herds

Human campylobacteriosis is a major public health concern in developed countries, with Campylobacter jejuni and Campylobacter coli from poultry recognized as the main source of human infection. Identification of Campylobacter-positive broiler herds before slaughter is essential for implementing measures to avoid carryover of pathogens via the slaughter process into the food chain. However, appropriate methods that have been validated for testing poultry flocks antemortem are lacking for Campylobacter. A quantitative real-time PCR (qPCR) that allows simultaneous detection and quantification of C. jejuni and C. coli was adapted and optimized to be applied on boot socks. The adjusted qPCR serves as an easy, sensitive, and quantitative method for Campylobacter detection in poultry flocks antemortem by analysis of boot socks. An adequate correlation was found between qPCR and culture, as well as between boot socks and cecal samples, which are regarded as the "gold standard." Therefore, boot sock sampling followed by qPCR analysis provides a reliable and simple method for assessing Campylobacter load within a flock prior to slaughter. The approach allows categorization of broiler herds into negative, low, moderate, or high Campylobacter colonization. Based on the results of this new approach, risk assessment models, such as evaluating the possible effect of sorting flocks before slaughter, can be easily implemented. Similarly, targeted identification of highly colonized flocks for improvement of biosecurity measures at the farm level will become feasible, presenting an opportunity to increase food safety.

Estimating sensitivity and specificity of a PCR for boot socks to detect Campylobacter in broiler primary production using Bayesian latent class analysis

The present study compares three different assays for sample collection and detection of Campylobacter spp. in broiler flocks, based on (i) the collection of faecal samples from intestinal organs (caecum), (ii) individual faecal droppings collected from the bedding and (iii) faecal material collected by socks placed on the outside of a pair of boots (boot socks) and used for walking around in the flock. The two first methods are examined for Campylobacter using a culture method (ISO-10272-2:2006), while the boot socks are tested using PCR. The PCR-assay is a genus specific multiplex PCR with primers targeting 16S rDNA in Campylobacter and primers targeting Yersinia ruckerii. Sixty-seven broiler flocks from Austria and 83 broiler flocks from Denmark were included in this prospective study and 89 of these were found to be positive in at least one method (AT: 49 samples, DK: 40 samples) whereas 61 of these were negative in all assays. In Austria samples for the three assays were collected simultaneously, which facilitates a direct comparison of the diagnostic test performance. In Denmark, however, boot socks and faecal droppings were collected three days before slaughter while caecum samples were collected at slaughter.The results were evaluated in the absence of a gold standard using a Bayesian latent class model. Austrian results showed higher sensitivity for PCR detection in sock samples (0.98; Bayesian credible interval (BCI) [0.93–1]) than for culture of faecal droppings (0.86; BCI [0.76–0.91]) or caecal samples (0.92; BCI [0.85–0.97]). The potential impact of Campylobacter introduction within the final three days before slaughter was observed in Denmark, where four flocks were tested negative three days before slaughter, but were detected positive at the slaughterhouse. Therefore the model results for the PCR sensitivity (0.88; BCI [0.83–0.97]) and cultural ISO-method in faecal samples (0.84; BCI [0.76–0.92]) are lower than for caecal samples (0.93; BCI [0.85–0.98]). In our study, PCR detection on boot sock samples is more sensitive than conventional culture. In view of the advantage of rapid results before slaughter and low costs for sampling, especially in combination with existing Salmonella surveillance systems (just another pair of boot socks needed), this method-matrix combination could be a valuable surveillance tool in the broiler primary production.

Prevalence of Salmonella in Neck Skin and Bone of Chickens

Bone-in and boneless parts, such as drumsticks, are used in ground chicken production. In addition, neck skin is used as a source of fat in ground products. Contaminated chicken neck skin and bones containing internalized Salmonella are potential sources of this pathogen in ground chicken. This study determined the prevalence of Salmonella and serotype distribution in drumstick bones and neck skin of postchill chicken carcasses. One week prior to slaughter, chicken houses (n = 26) at nine farms were tested for the presence of Salmonella, using the boot sock method. Chicken flocks from these houses originated from Salmonella-positive breeders. Eight Salmonella-positive chicken flocks and one flock with undetermined Salmonella status were monitored through processing. Three hundred postchill drumsticks and 299 neck skin samples were analyzed for Salmonella prevalence. Skin samples were rinsed and stomached prior to analysis. Bones were extracted from the drumsticks, external surfaces were sterilized, and bones were crushed for analysis. One Salmonella isolate from each positive sample was serogrouped. Half of the isolates representing different sample types were serotyped. Overall, Salmonella was found in 0.8, 21.4, and 80.1% of bone marrow, neck skin, and farms, respectively. Prevalence of Salmonella on rinsed skin samples (2.3%) and stomached skin samples (20.7%) differed significantly (P ≤ 0.05). Serogroups B, C2, D, and E were found at 23.4, 31.9, 11.7, and 29.8%, respectively. Six Salmonella serotypes were identified: Liverpool (37.9%), Kentucky (27.6%), and Typhimurium (27.6%) were isolated most frequently from neck skin; the two bone isolates were Kentucky; and more than 50% of the farm isolates were Kentucky and Ouakam. Salmonella-contaminated neck skin might be a more significant source of this contamination in ground chicken than Salmonella internalized in bones.

Heat loss and moisture retention variations of boot membranes and sock fabrics: A foot manikin study

Heat loss and moisture retention properties of footwear were characterized using a walking thermal manikin foot. The same type of military boot was equipped with different membranes: (i) GORE TEX with IQ TEX, (ii) halve OUTDRY, (iii) full OUTDRY, and (iv) OUTDRY with IQ TEX. In a separate experiment a single boot type was used to evaluate four different sock fabrics: (i) wool/polypropylene, (ii) polypropylene, (iii) polypropylene/polyamide, and (iv) wool/polyamide. Both boot membranes and sock fabrics were assessed under three conditions: (i) standstill no sweating, (ii) walking no sweating, and (iii) walking and sweating. The walking rate was set at 15 step min−1 and the sweat rates were 9 g h−1 and 12 g h−1, for boot membrane and sock fabric measurements, respectively. Moisture retention was assessed by weighing the footwear components before and after each measurement. GORE TEX with IQ TEX resulted in a higher heat loss during walking without sweating compared to the other membranes (p = 0.017). GORE TEX with IQ TEX retained more moisture in the sock compared to the other membranes (p < 0.001) but also retained more moisture in the inlay sole compared to halve OUTDRY (p = 0.015). No differences in heat loss were found among sock fabrics, while wool/polyamide retained more moisture compared to polypropylene/polyamide (p = 0.036). Furthermore, a moisture vapour transmission rate of 61.2 ± 6.6 g m−2 h−1 was calculated for all sweating conditions. Finally, the measurements suggest that no pumping effect takes place in the measured footwear under the present conditions.Relevance to industry: Understanding heat loss and moisture retention of footwear is necessary for optimization of footwear for blister incidence and (thermal) comfort.

Influence of Boot-Sock Systems on Frequency and Severity of Foot Blisters

This study examined the infleunce of three types of boot-sock systems on incidence and severity of foot blisters. Participants were 357 men undergoing U.S. Marine recruit training at Parris Island, South Carolina. Each participant was assigned to one of three treatment groups. The first group wore the standard military boot sock consisting of a wool-cotton-nylon-Spandex combination. The second group wore the standard military boot sock with a thin inner or liner sock consisting of polyester. The third group wore a very thick, dense, prototype outer sock consisting of a wool-polypropylene combination over the same liner sock as the second group. Foot blister incidence was lower among participants who wore the the prototype boot sock and liner than among those wearing the standard military sock (40 vs. 69%, p < 0.001) or among subjects wearing the standard military sock with liner (40 vs. 77%, p < 0.001). Foot blisters and cellulitis severe enough to require medical attention occurred with greater frequency in the standard military sock group compared to both the prototype boot-sock group (24 vs. 11%, p = 0.02) and the standard military sock with liner group (24 vs. 9%, p < 0.01); there was no difference between the latter two groups. Blister reduction was most apparent in the early weeks of recruit training. The standard military sock with a polyester liner reduced the incidence of severe blisters, but the dense sock with the polyester liner reduced the overall incidence of blisters as well as the incidence of severe blisters.