Search Results (19)

Salmonella spp. is one of the most important foodborne pathogens worldwide. Given the fact that poultry and poultry products are the main source of human infection, Salmonella control in these farms is of utmost importance. To better control this pathogen in farms, boot swabs are used to sample farm environments but the analysis of these swabs is mainly based on culture-dependent methods. In the present study, a novel loop-mediated isothermal amplification (LAMP) method was developed for the rapid screening of Salmonella spp. in boot swab samples from broiler flock environments. Four different DNA extraction protocols were evaluated in depth, including a simple thermal lysis, a chelex-based protocol and two thermal lysis protocols followed by the purification of magnetic beads made of silica (“glass milk”) in order to determine the most suitable alternative for potential on-site, farm analyses. The methodology evaluation included a blind interlaboratory assay and as a proof-of-concept, a naked-eye colorimetric assay was also included. Following the final methodology, it was possible to reach an LoD₅₀ of 1.8 CFU/25 g of the samples, with a high relative sensitivity (95.7%), specificity (100%) and accuracy (96.6%) along with Cohen’s kappa of concordance with respect to the ISO standard 6579-1:2017 of 0.9, with an RLOD of 1.3. In addition to this, due to the relevance of certain serotypes with the genus Salmonella spp., a serotype LAMP panel for the specific identification of S. Typhimurium, S. Enteritidis, S. Infantis, S. Hadar and S. Virchow was also developed. Even though some degree of cross-reactivity among the primers developed was observed, all the serotypes could be accurately identified based on their melt curve analysis profile. Taken together, in the present study, a rapid Salmonella spp. screening method, suitable for farm applications, was developed, along with a serotyping panel that could be used in a laboratory setup for the identification of the most relevant serotypes of the genus, taking advantage of real-time amplification followed by melt curve analysis. Full article

Salmonella Mbandaka is one of the most globally widespread serovars, occurring in many sources and included among twenty serovars that contribute to human salmonellosis in Europe. In Poland, it has been noted in non-human sources since 1996, being found firstly in feeds and later in waterfowl and chicken. Over the years, it gained epidemiological importance, being isolated from a wide range of animal species, including livestock. Generally, it is characterized by sensitivity to most antimicrobials and the ability to form biofilms. The occurrence of cephalosporin-resistant Salmonella in non-human sources is an extremely rare phenomenon in Poland. In this report, we characterized the full genome of the ESBL-producing S. Mbandaka strain isolated from a broiler farm environment (boot swab sample) in Poland in 2022. The isolate was serotyped as S. Mbandaka according to the White–Kaufmann–Le Minor scheme. Antimicrobial susceptibility testing performed with the microbroth dilution method showed its resistance to ampicillin, cefotaxime, ceftazidime, ciprofloxacin, and nalidixic acid. The whole-genome sequence was reconstructed using short and long reads and assembled into the complete chromosome and three plasmids: IncI1 pST113 (89,439 bp), Col(pHAD28) (2699 bp), and Col440 (2495 bp). The strain belonged to sequence type ST413. Plasmid analysis showed bla_CTX-M-8 mobilization on IncI1(alpha) surrounded with insertion sequences. The analyzed genome content draws attention to the possibility of the horizontal spread of the resistance genes. To the best of our knowledge, this is the first report of bla_CTX-M-8-positive Salmonella in Poland. Full article

The food chain acts as an entry point for antibiotic resistance to reach humans and environment. Because of the importance of the poultry sector, we investigated the prevalence and evolution of antibiotic resistance in Escherichia coli isolates from a series of 14,500 breeding hens and their farm environment during the rearing period. Samples included meconium from one-day-old breeders and fecal samples and boot swabs from the breeding sheds of pullets and adult hens. All E. coli isolates from one-day-old chicks, 77% from feces and 61% from boot swabs, were resistant to at least one antibiotic. Cefotaxime and multi-drug resistance in fecal isolates decreased during the rearing period from 41.2% and 80.8% in one-day-old chicks to 3.8% and 33.8% in adults. All genes studied were detected in E. coli from feces and boot swabs, the most common being bla_TEM (75%), bla_SHV (72%), and qnrB (67%). bla_CMY-2 was detected in 100% of one-day-old breeders. The combination of at least one cephalosporin and one quinolone resistance gene was detected in 68.7% of fecal and boot swab isolates. Our results highlight the need to monitor the prevalence of antibiotic resistance on farms and to take appropriate measures to reduce the risk to public and environmental health. Full article

The rapid emergence of antimicrobial resistance is a global concern, and high levels of resistance have been detected in chicken populations worldwide. The purpose of this study was to determine the prevalence of antimicrobial resistance in Escherichia coli and Salmonella spp. isolated from healthy chickens in Timor-Leste. Through a cross-sectional study, cloacal swabs and boot swabs were collected from 25 live bird markets and two layer farms respectively. E. coli and Salmonella spp. from these samples were tested for susceptibility to six antimicrobials using a disk diffusion test, and a subset was tested for susceptibility to 27 antimicrobials using broth-based microdilution. E. coli and Salmonella spp. isolates showed the highest resistance towards either tetracycline or ampicillin on the disk diffusion test. E. coli from layer farms (odds ratio:5.2; 95%CI 2.0–13.1) and broilers (odds ratio:18.1; 95%CI 5.3–61.2) were more likely to be multi-drug resistant than those from local chickens. Based on the broth-based microdilution test, resistance to antimicrobials in the Timor-Leste Antimicrobial Guidelines for humans were low, except for resistance to ciprofloxacin in Salmonella spp. (47.1%). Colistin resistance in E. coli was 6.6%. Although this study shows that antimicrobial resistance in chickens was generally low in Timor-Leste, there should be ongoing monitoring in commercial chickens as industry growth might be accompanied with increased antimicrobial use. Full article

Finalyse, a T4 bacteriophage, is a pre-harvest intervention that utilizes a combination of bacteriophages to reduce incoming Escherichia coli O157:H7 prevalence by destroying the bacteria on the hides of harvest-ready cattle entering commercial abattoirs. The objective of this study was to evaluate the efficacy of Finalyse, as a pre-harvest intervention, on the reduction in pathogens, specifically E. coli O157:H7, on the cattle hides and lairage environment to overall reduce incoming pathogen loads. Over 5 sampling events, a total of 300 composite hide samples were taken using 25 mL pre-hydrated Buffered Peptone Water (BPW) swabs, collected before and after the hide wash intervention, throughout the beginning, middle, and end of the production day (n = 10 swabs/sampling point/timepoint). A total of 171 boot swab samples were also simultaneously taken at the end of the production day by walking from the front to the back of the pen in a pre-determined ‘Z’ pattern to monitor the pen floor environment from 3 different locations in the lairage area. The prevalence of pathogens was analyzed using the BAX^® System Real-Time PCR Assay. There were no significant reductions observed for Salmonella and/or any Shiga toxin-producing E. coli (STEC) on the hides after the bacteriophage application (p > 0.05). Escherichia coli O157:H7 and O111 hide prevalence was very low throughout the study; therefore, no further analysis was conducted. However, boot swab monitoring showed a significant reduction in E. coli O157:H7, O26, and O45 in the pen floor environment (p < 0.05). While using Finalyse as a pre-harvest intervention in the lairage areas of commercial beef processing facilities, this bacteriophage failed to reduce E. coli O157:H7 on the hides of beef cattle, as prevalence was low; however, some STECs were reduced in the lairage environment, where the bacteriophage was applied. Overall, an absolute conclusion was not formed on the effectiveness of Finalyse and its ability to reduce E. coli O157:H7 on the hides of beef cattle, as prevalence on the hides was low. Full article

Salmonellosis is the second most commonly reported foodborne gastrointestinal infection in humans in the European Union (EU). Most outbreaks are caused by Salmonella Enteritidis, present in contaminated food products, particularly in egg and egg products. In recent years, an increase in the prevalence of Salmonella in laying hen flocks in the EU has been observed. For the effective control of infection, adequate detection is key. In laying hen flocks, the occurrence of Salmonella in the EU is monitored by the culture of environmental samples (dust, faeces, and boot swabs). The performance of sampling procedures described in the literature for the detection of Salmonella in laying hens was reviewed. In total, 924 abstracts were screened, resulting in the selection of 87 abstracts and 18 publications for qualitative and quantitative analyses, respectively. Sample sizes and sampling locations of faecal material and dust were variable and poorly described. Microbiological culture methods used to detect Salmonella were variably described in the literature and were often incomplete. Overall, the available literature indicates higher sensitivity of environmental versus individual hen matrices and points to differences in sensitivity between environmental matrices. For non-cage housing systems, boot swabs are the preferred samples, while for cage housing systems dust might be a more reliable sample. Full article

The nutritional benefits of rye (and therefore rye bran) are mainly due to its high content of fermentable dietary fiber, the non-starch polysaccharides (NSP). Microorganisms in the large intestine are able to convert these into short-chain fatty acids (SCFA), including butyrate. Butyrate strengthens the epithelial barrier function in the colon by nourishing the enterocytes and inhibiting the spread of Salmonella in the intestinal tract. Therefore, the aim of this study was to test under field conditions whether a diet with rye or rye bran as the main ingredient for gilts, sows, and weaned piglets is associated with a lower Salmonella prevalence. Depending on the age groups, between 20–30% rye or between 15–20% rye bran was used in the experimental group. A total of n = 1983 boot swabs, n = 356 fecal samples, and n = 1909 serum samples were examined. The results of this study show that rye or rye bran at the levels used had no apparent effect on the number of positive Salmonella samples. However, the Salmonella OD values in the experimental groups were significantly lower than in the control group. This suggests that the use of rye leads to a lower incidence of infection, but this effect could not be proven from swabs. Full article

This study aimed to investigate the prevalence of Mycobacterium avium subsp. paratuberculosis (MAP) in small ruminants in Austria by testing 22,019 serum samples with ELISA for the presence of specific antibodies. Furthermore, detailed investigations in five MAP-infected goat herds were carried out by ELISA, qPCR and bacterial culture. The found animal-level apparent MAP seroprevalence was 2.0% for goats and 0.7% for sheep (calculated true prevalence 3.5% and 1.2%, respectively). Herd-level apparent MAP seroprevalence was 11.1% for goat herds and 8.9% for sheep flocks. Significant risk factors for seropositivity in goat herds were: herd size, animal trading, farmed as a dairy herd, Animal Health Service membership and cohabitation with farmed game. For sheep flocks, seroprevalence was significantly higher in flocks with animal trading and where cattle or goats were kept in the flock, respectively. The overall apparent within-herd MAP seroprevalence in the five goat farms investigated was 21.8% (11.7%–28.0%, calculated true seroprevalence 38.6%) and an overall rate of MAP shedding of 12.3% was detected (5.0%–24.7%). It was possible to identify MAP by culture using boot swab samples in each herd. The results indicated a moderate MAP infection rate in small ruminants in Austria. Full article

Campylobacteriosis outbreaks have repeatedly been associated with the consumption of raw milk. This study aimed to explore the variation in the prevalence and concentration of Campylobacter spp. in cows’ milk and feces, the farm environment and on the teat skin over an entire year at a small German dairy farm. Bi-weekly samples were collected from the environment (boot socks), teats, raw milk, milk filters, milking clusters and feces collected from the recta of dairy cows. Samples were analyzed for Campylobacter spp., E. coli, the total aerobic plate count and for Pseudomonas spp. The prevalence of Campylobacter spp. was found to be the highest in feces (77.1%), completely absent in milking equipment and low in raw milk (0.4%). The mean concentration of Campylobacter spp. was 2.43 log₁₀ colony-forming units (CFU)/g in feces and 1.26 log₁₀ CFU/teat swab. Only a single milk filter at the end of the milk pipeline and one individual cow’s raw milk sample were positive on the same day, with a concentration of 2.74 log₁₀ CFU/filter and 2.37 log₁₀ CFU/mL for the raw milk. On the same day, nine teat swab samples tested positive for Campylobacter spp. This study highlights the persistence of Campylobacter spp. for at least one year in the intestine of individual cows and within the general farm environment and demonstrates that fecal cross-contamination of the teats can occur even when the contamination of raw milk is a rare event. Full article

Pre-weaned dairy calves are very susceptible to disease in the first months of life due to having a naïve immune system and because of the numerous physiological stressors they face. Hygiene management is a key element in minimizing enteric disease risk in calves by reducing their exposure to pathogens. Samples of milk, concentrate feed and drinking water, boot swabs of bedding and swabs of feed equipment were collected from 66 dairy farms as part of a survey of calf rearing practice and housing design. All the samples were cultured to determine total viable counts (TVC), total coliforms (TCC) and Escherichia coli as indicators of hygiene. Target ranges for levels of TVC, TCC and E. coli were defined from the literature and the sample results compared against them. The TVC targets in milk, MR and water were <4.0 log₁₀ CFU/mL. TCC and E. coli targets of <1.1 log₁₀ CFU/mL (the detection limit) were used for milk, MR, concentrate feed and feeding equipment. For water, the TCC and E. coli targets were <1.0 log₁₀ CFU/100 mL. The targets used for bedding boot swabs were <6.3 log₁₀ TVC CFU/mL and <5.7 log₁₀ TCC or E. coli CFU/mL. Farm management factors were included as fixed effects in a generalized linear mixed model to determine the probability of samples being within each hygiene indicator target range. Milk replacer samples obtained from automatic feeders were more likely to be within the TVC target range (0.63 probability) than those prepared manually (0.34) or milk samples taken from the bulk tank (0.23). Concentrate feed samples taken from buckets in single-calf pens were more likely to have E. coli detected (0.89) than samples taken from group pen troughs (0.97). A very small proportion of water samples were within the indicator targets (TVC 9.8%, TCC 6.0%, E. coli 10.2%). Water from self-fill drinkers had a lower likelihood of being within the TVC target (0.03) than manually filled buckets (0.14), and water samples from single pens were more likely to be within TCC target ranges (0.12) than those from group pens (0.03). However, all self-fill drinkers were located in group pens so these results are likely confounded. Where milk feeders were cleaned after every feed, there was a greater likelihood of being within the TVC target range (0.47, compared with 0.23 when not cleaned after every feed). Detection of coliforms in milk replacer mixing utensils was linked with reduced probability of TVC (0.17, compared with 0.43 when coliforms were not detected) and TCC (0.38, compared with 0.62), which was within target in feeders. Key factors related to increased probability of bedding samples being within TCC target range were use of group calf pens (0.96) rather than single-calf pens (0.80), use of solid floors (0.96, compared with 0.76 for permeable floors) and increased space allowance of calves (0.94 for pens with ≥2 m²/calf, compared with 0.79 for pens with <2 m²/calf). Bedding TVC was more likely to be within the target range in group (0.84) rather than in single pens (0.66). The results show that hygiene levels in the calf rearing environment vary across farms and that management and housing design impact hygiene. Full article

Salmonellosis is the second most reported gastrointestinal infection in humans after campylobacteriosis and a common cause of foodborne outbreaks in the European Union (EU). In addition to consumption of contaminated animal-based foods, such as poultry, beef and eggs, pork is an important source of human salmonellosis outbreaks; therefore, Salmonella (S.) control should start in the early stages of pig production. To be able to implement effective control measures to reduce the risk of pigs being infected by Salmonella, it is important to identify the serovars circulating on farm within the different stages of production, including as early as sow and piglet breeding. The aim of the present study was to assess the Salmonella status of sow farms either producing their own finishers or delivering piglets to fattening farms with a known high serological prevalence identified within the QS Salmonella monitoring system. Overall, 97 (92.4%) of 105 investigated piglet-producing farms across Germany tested positive in at least one sample. Salmonella was detected in 38.2% of the sock and 27.1% of the environmental swab samples. S. Typhimurium was the most frequent serovar. In conclusion, sock and environmental swab samples are well suited for non-invasive Salmonella detection in different production units in farrowing farms. To establish a holistic Salmonella control program, all age classes of pig production should be sampled to enable intervention and implementation of countermeasures at an early stage if necessary. Full article

Pigs are a reservoir of hepatitis E virus (HEV), which causes hepatitis in humans. To study the epidemiology of HEV in pig farms, sampling methods are currently used that cause discomfort to pigs, such as rectal sampling. In line with the 3Rs principle, we aimed to evaluate non-invasive methods to detect pens with HEV-shedding pigs. Twenty-eight pens of one farm were sampled cross-sectionally. Individual rectal swabs (IRS) were collected to determine prevalence within pens. Four pen-level samples were compared: a pool of IRS per pen (P), boot socks (BS), oral fluid (OF) and pooled faecal droppings (FD). Each sample was tested by RT-PCR and the sensitivity and specificity of each method was determined by Bayesian latent class analysis. According to IRS, 19/28 pens were HEV positive. BS had a sensitivity of 95% and detected HEV in pens with 10% of pigs shedding; however, specificity was below 30%. FD were comparably accurate to P, with a sensitivity and specificity of 94% and 86%, respectively. BS sampling is thus advised to detect early shedding of HEV or pen contamination, and FD to determine the duration of shedding. This study demonstrates that non-invasive sampling can replace rectal swabs in research on HEV in pigs. Full article

One of the pillars of Johne’s disease control is to break the cycle of transmission from infectious adult animals to young susceptible animals. Many control programmes utilise a risk assessment and management plan (RAMP) to identify specific risks for transmission of infection on individual farms and then recommend tailored biosecurity measures to mitigate the risk. It is important that infected farms in particular, practice effective biocontainment in the calving area and pre-weaned calf area. The objectives of this study were: (1) to determine what proportion of MAP-infected farms (PCR-positive) enrolled in a control programme had detectable MAP in their calf environment; (2) to compare RAMP scores and apparent within-herd prevalence (aWHP) of PCR-positive and PCR-negative farms; (3) to evaluate the correlation between RAMP scores, aWHP and levels of contamination based on PCR test Ct value. A novel environmental sampling protocol combining manure samples and boot swab samples was conducted in the calving area and pre-weaned calf area on 28 dairy farms with PCR-confirmed MAP infection. All samples were tested with PCR. Logistic regression modelling was used to evaluate the association between RAMP score and aWHP and PCR outcome. Overall, 46% of farms had positive PCR samples in either the calving area, pre-weaned calf area or both. The calving area was positive in 36% of farms and the pre-weaned calf area was positive in 21% of farms. There was a moderate, negative correlation (r = −0.45; p = 0.08) observed between RAMP scores and Ct value at the sample-level that slightly missed the required significance level. No significant association was identified between RAMP scores or aWHP and PCR test outcome (positive or negative). It was concluded that environmental sampling of the calving area and pre-weaned calf area has potential applications in the assessment of transmission risk on infected farms and could be used to monitor the efficacy of biosecurity measures over time. Further research conducted on a larger scale is required to support these results. Full article

In piglet production, the beginning of pork production, Salmonella prevalence requires greater attention as having an impact on the subsequent production steps. The aim of this study was to investigate Salmonella prevalence in three sow herds with attached piglet rearing units. Salmonella prevalence was investigated either directly by boot swabs and feces or indirectly by serum samples taken during gilt integration, the peripartal period, and piglet rearing. Boot swabs and feces were analyzed by real-time PCR and subsequent microbiology. Results indicated that high biosecurity measures in sow husbandry do not necessarily result in a low Salmonella prevalence. Furthermore, the sow herds’ Salmonella prevalence should not be used to infer the situation in the associated piglet rearing. The proportion of positive boot swabs was 10.5, 3.6, and 21.3% for sows (gilts and peripartal) with an inverse situation in piglet rearing with 50.0, 63.3, and 5.8% positive swabs for farms A, B, and C, respectively. Boot swabs are suitable as a direct sampling method to gain an overview of Salmonella prevalence in both sows and piglets. Indirect serum antibody testing can be useful, although it should be evaluated considering age-dependent levels of antibody titres. Full article

On-farm environmental sampling is an effective method for herd-level diagnosis of Mycobacterium avium ssp. paratuberculosis (MAP) infection and between-herd prevalence estimation. So far, no prevalence study enrolling important livestock-farming regions has been conducted. As the structure of dairy farming differs between main livestock-farming regions in Germany, our objective was to assess the between-herd prevalence of paratuberculosis for these regions in a standardized approach. Methods: In total, 457 randomly selected dairy farms from three regions of Germany (North: 183, East: 170, South: 104) were sampled between 2017 and 2019. Environmental samples (boot-swabs, aggregate feces and/or liquid manure samples) were cultured and analyzed using an IS900-qPCR for MAP determination. Of the 457 selected farms, 94 had at least one MAP-positive environmental sample with significant differences between regions regarding the apparent (North: 12.0%, East: 40.6%, South: 2.9%) or corrected true (North: 14.8%, East: 50.1%, South: 3.6%) between-herd prevalence. In conclusion, regional differences of between-herd prevalence of paratuberculosis are substantial in Germany, indicating the need for control approaches with different aims. Taking into account regional MAP prevalence, MAP-control programs should focus on on-farm prevalence reduction or on mitigating the risk of between-herd transmission, depending on region. Full article