Diagnostic approach for detection and identification of emerging enteric pathogens revisited: the (Ali)arcobacter lanthieri case.

An immunocompetent patient without a history of recent travel or animal exposure developed persistent abdominal bloating and cramps without diarrhoea or fever. Negative additional investigations excluded gastritis, infectious colitis, inflammatory bowel disease and neoplasia, but routine stool culture detected a Campylobacter-like organism. The isolate was obtained with use of a polycarbonate filter technique, emphasizing the importance of culture to support and validate the occurrence of emerging and new bacterial enteric pathogens. The ensuing extensive laboratory examinations proved challenging in identifying this potential pathogen. Phylogenetic marker analysis based on the 16S ribosomal RNA and rpoB gene sequences revealed that the isolate was most closely related to Arcobacter lanthieri and Arcobacter faecis. Subsequent analysis of a draft whole genome sequence assigned the isolate to A. lanthieri. We report the presence of five virulence genes, cadF, ciaB, mviN, hecA and iroE, indicating a possible pathogenic nature of this organism. This case demonstrated the importance of the use of agnostic methods for the detection of emerging pathogens in cases of enteric disease with a wide array of gastrointestinal symptoms.

Yersinia enterocolitica detection in pork products: Evaluation of isolation protocols.

Conventional methods for Yersinia enterocolitica detection in food samples are generally considered inadequate. Problems arise from the presence of the so-called "background flora", coupled to the low contamination level of the pathogen. Since, data on the microbial ecology occurring in competitive microflora are still lacking, MALDI TOF MS was used for strains 'identification after enrichment in PSB or ITC broths, and after plating on selective CIN medium at different incubation times. SYBR Green Real time PCR was used for the Y. enterocolitica strains' detection (4/O:3, 1A/O:5) in experimentally contaminated foods, as well as in naturally contaminated samples. A higher number of different bacterial genera (10 on CIN and 18 on PCA) was recorded after enrichment in PSB, whilst enrichment in ITC led to recovery of 6 and 10 genera on CIN and PCA, respectively. Yersiniaceae was the dominant family on the first day of incubation, but on the second day the percentage of isolation considerably decreased. By testing experimentally contaminated samples, substantial difficulties were encountered. The biotype 1A was always detected, whereas strain 4/O:3 proved to be poorly competitive. Based on the data, the enrichment media PSB and ITC, currently proposed for Y. enterocolitica detection, need to be improved to promote a successful pathogen's recovery.

Assessment of microbial communities on freshly killed wild boar meat by MALDI-TOF MS and 16S rRNA amplicon sequencing.

Wild boars (Sus scrofa) are the most widely distributed large mammals and recent increase in consumption of wild boar meat urges the need of microbiological quality criteria. The aim of the study was to characterize the initial bacterial contamination on freshly-killed wild boar meat using a culture-dependent approach with ISO-methods combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification and 16S rRNA amplicon sequencing. Moreover, the presence of foodborne pathogens was examined using Real-Time-PCR and confirmed by classical isolation. Analysing 22 unrelated wild boar meat samples showed a higher bacterial contamination level compared to pork, with Salmonella present in almost one third of the samples. A great variability of the microbial contamination between the samples was recorded, as well as complementary results between culturing and 16S rRNA amplicon sequencing as frequently isolated genera were not always detected, and vice versa. Furthermore, the foodborne pathogen Salmonella was never detected with 16S rRNA amplicon sequencing, demonstrating the necessity for a cautious approach in the implementation of new analysis techniques in food safety. The present work determines that attention should be paid to the trade of non-inspected meat directly to retail or consumers.

Determination of the microbiological contamination in minced pork by culture dependent and 16S amplicon sequencing analysis.

Routine evaluation of bacterial contamination in minced pork is still mainly performed by the enumeration of indicator bacteria, including total aerobic colony count and E. coli, using standardized isolation methods. However, the bacterial community structure as well as the effect of the storage time and temperature on the aerobic plate count are largely unknown for this matrix. The aim of the study was to characterize the microbial community in minced pork by 16S rRNA amplicon sequencing compared to classical isolation methods combined with identification by Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI TOF MS) and 16S rRNA gene sequencing. Analysis of 14 unrelated samples showed that total aerobic counts determined at 30 °C and 7 °C showed no significant difference, but the richness was higher on PCA at 30 °C for 7 samples, equal in 5, and higher at 7 °C for 2 samples. Members of the genus Pseudomonas, along with the genera Brochothrix and Carnobacterium were commonly identified among both the mesophilic and psychrotrophic population. Comparing to 16S rRNA amplicon sequencing, some contrasting data were obtained. Except for Brochothrix spp. and Pseudomonas spp., that were abundant and always detected, genera obtained with the two methods in the same sample were not always the same. Comparison of different sample preparation techniques and DNA extraction methods demonstrated also in this matrix that different results on the microbial composition and complexity are obtained. Present data illustrate that the combined isolation and identification of isolates using MALDI TOF MS and 16S gene sequencing and overall community profiling using 16S rRNA amplicon sequencing provides complementary results and yields important insights into the complex relationship between microorganisms in a food.

Diversity, not uniformity: slaughter and electrical waterbath stunning procedures in Belgian slaughterhouses.

Data on slaughter and stunning protocols in Belgian poultry abattoirs were collected, and subsequently the survival rate after electrical waterbath stunning in 1,400 animals across 7 selected slaughterhouses was determined. The majority of the abattoirs applied electrical waterbath stunning (72%), whereas the other methods were gas stunning (13%), head-only stunning (6%), and dry stunning (3%). In 6% of the slaughterhouses, the birds were killed without previous stunning, according to religious rites (i.e., ritual or religious slaughter). Although religious slaughter occurred in a substantial number of abattoirs, the customers of the majority of them allowed stunning, provided the animals were not killed by the stunning procedure. Substantial variation both in electrical waterbath devices and technical settings (electrical current type, wave type, voltage, frequency) combinations was observed. These settings did not only differ between slaughterhouses, but also between subsequent visits to the same slaughterhouse. Despite this variation, all systems comprised a constant voltage, multiple bird stunner. The minimum average electrical current that every chicken should receive at a certain frequency, as stated by the European Regulation No 1099/2009, was not achieved for each animal monitored due to the bird's characteristics and the different applied stunning settings, with the exception of all ISA laying hens and broiler breeders in one particular slaughterhouse. The survival rate ranged from 96.0 to 100%, 97.0 to 100%, 68.0%, 72.0 to 85.1%, and 5.6 to 52.4% in 5-wk-old broilers, 6-wk-old broilers, LSL laying hens, ISA laying hens, and broiler breeders, respectively. Monitoring of unconsciousness after passing through the waterbath was not always performed and when it was, there was no uniformity in the applied criteria. It was concluded that a large variation in slaughter and stunning practices exists among slaughterhouses in Belgium. Further research should explore the effect of the different settings on inducing a successful stun and on carcass quality, and assess if the observed variation also exists in other EU member states.

Assessment of factors influencing the within-batch seroprevalence of human enteropathogenic Yersinia spp. of pigs at slaughter age and the analogy with microbiology.

The microbiologically and serologically-based prevalence of human enteropathogenic Yersinia spp. at moment of slaughter varies between pig farms due to different herd-level factors. A face-to-face questionnaire concerning a broad range of farm aspects (e.g., management and housing system, biosecurity, and hygiene measurements) was performed on one hundred farms. Factors influencing the seropositivity of 7047 pigs against human pathogenic Yersinia spp. were determined and compared to the microbiology. At the slaughterhouse, pieces of diafragm of on average 70 slaughter pigs per batch were sampled to determine the level of antibodies against enteropathogenic Yersinia spp. After univariable mixed-effect logistic regressions, variables that were related to the seropositivity (p<0.05) were included in a multivariable model (p<0.1). The factors remaining significantly associated in the latter model were an increasing number of piglet suppliers (zero up to eleven suppliers) (Odds Ratio=1.4), a high density of pig farms in the area (high versus low density) (Odds Ratio=2.3), the use of semislatted floors in the fattening pig unit (semi slatted floor versus fully slatted floor) (Odds Ratio=3.8) and the possibility of snout contact in the fattening pig unit (snout contact or not) (Odds Ratio=0.1). Decreasing the risk of infection with human enteropathogenic Yersinia spp. at moment of slaughter or during rearing is possible by changing farm management factors.

Free-living protozoa in the gastrointestinal tract and feces of pigs: Exploration of an unknown world and towards a protocol for the recovery of free-living protozoa.

Associations with free-living protozoa (FLP) have been implicated in the persistence of foodborne pathogenic bacteria in food-related environments. To date however no information is available on the presence of FLP in the gastrointestinal tract (GIT) of pigs, which represents an important reservoir for zoonotic foodborne bacteria and hence a potential location for associations with FLP. This is at least partly due to the lack of adequate protocols to recover FLP from intestinal content and feces. In the present study different protocols to recover FLP from the porcine GIT and feces were tested. The most effective protocols were then applied to explore the presence of live FLP in the pig GIT and feces. A filtration based protocol was identified as the most suitable method to recover viable FLP from the porcine GIT and feces. Cultivable FLP were recovered from different parts of the GIT, suggesting at least a transient presence of FLP in this habitat. Free-living amoebae species (Acanthamoeba spp., Hyperamoeba sp., Vannella sp., Vermamoeba vermiformis, hartmannellids and vahlkampfiids) but also ciliates (Colpoda sp. and Tetrahymena/Glaucoma lookalike) and flagellates (cercomonads, bodonids and glissomonads) were recovered and cultured from pig intestinal content. Acanthamoeba hatchetti and Filamoeba sinensis were isolated for the first time from pig intestinal content. Despite high gastric acidity, non-cyst forming amoeba species were also detected which suggests survival of their trophozoites in the animal GIT.

Comparative performance of isolation methods using Preston broth, Bolton broth and their modifications for the detection of Campylobacter spp. from naturally contaminated fresh and frozen raw poultry meat.

The performance of different isolation methods was evaluated for the detection of Campylobacter from naturally contaminated raw poultry meat. Therefore, fresh and frozen poultry meat samples were analysed using the standard procedure (ISO 10272-1:2006), enrichment in Preston broth, and enrichment in modified Bolton broth (supplemented with (i) potassium clavulanate (C-BB), (ii) triclosan (T-BB), (iii) polymyxin B (P-BB)). The enrichment cultures were streaked onto both modified charcoal cefoperazone deoxycholate agar (mCCDA) and RAPID'Campylobacter agar (RCA). Moreover, direct plating on mCCDA and RCA was performed to quantify Campylobacter. In total, 33 out of 59 fresh retail meat samples (55.9%) were Campylobacter positive. For both fresh and frozen poultry meat samples, enrichment in Bolton broth (ISO 10272-1:2006) resulted in a higher number of positive samples than enrichment in Preston broth. Supplementation of Bolton broth with potassium clavulanate (C-BB) and triclosan (T-BB) enhanced the Campylobacter recovery from fresh poultry meat compared to non-supplemented Bolton broth, although the use of C-BB was less applicable than T-BB for Campylobacter recovery from frozen samples. Additionally, the use of RCA resulted in a higher isolation rate compared to mCCDA. The present study demonstrates the impact of culture medium on the recovery of Campylobacter from fresh and frozen naturally contaminated poultry meat samples and can support laboratories in choosing the most appropriate culturing method to detect Campylobacter.

Abundance, diversity and community composition of free-living protozoa on vegetable sprouts.

Interactions with free-living protozoa (FLP) have been implicated in the persistence of pathogenic bacteria on food products. In order to assess the potential involvement of FLP in this contamination, detailed knowledge on their occurrence, abundance and diversity on food products is required. In the present study, enrichment and cultivation methods were used to inventory and quantify FLP on eight types of commercial vegetable sprouts (alfalfa, beetroot, cress, green pea, leek, mung bean, red cabbage and rosabi). In parallel, total aerobic bacteria and Escherichia coli counts were performed. The vegetable sprouts harbored diverse communities of FLP, with Tetrahymena (ciliate), Bodo saltans and cercomonads (flagellates), and Acanthamoeba and Vannella (amoebae) as the dominant taxa. Protozoan community composition and abundance significantly differed between the sprout types. Beetroot harbored the most abundant and diverse FLP communities, with many unique species such as Korotnevella sp., Vannella sp., Chilodonella sp., Podophrya sp. and Sphaerophrya sp. In contrast, mung bean sprouts were species-poor and had low FLP numbers. Sampling month and company had no significant influence, suggesting that seasonal and local factors are of minor importance. Likewise, no significant relationship between protozoan community composition and bacterial load was observed.

Contamination of freshly slaughtered pig carcasses with enteropathogenic Yersinia spp.: Distribution, quantification and identification of risk factors.

A cross-sectional survey was undertaken to determine the overall prevalence of enteropathogenic Yersinia spp. in the tonsils, feces and on carcasses of pigs at slaughter. Moreover, factors associated with Yersinia contamination of freshly eviscerated pig carcasses were studied. Yersinia enterocolitica serotype O:3 was isolated from the tonsils and feces of 55.3% and 25.6% of pigs, and Y. pseudotuberculosis from 1.4% and 0.6%, respectively. The pathogens were also recovered from 39.7% of carcass surfaces post-evisceration. The highest prevalence was found at the mandibular region (28.9%), followed by the sternal region (16.4%), pelvic duct (7.8%), and split surface near the sacral vertebrae (6.9%). Regarding the quantification of the pathogen, the median concentration of pathogenic Y. enterocolitica was 4.14l og10 CFU/g in tonsils with countable numbers (n=143) and 2.80 log10 CFU/g for fecal samples with countable numbers (n=26). The quantitative load on the carcass surface was generally low as the majority of the carcass samples (97.0%) had Yersinia concentrations below the detection limit of enumeration (<1.30 log10 CFU/100 cm(2)). The initial presence of Y. enterocolitica in the tonsils and/or feces was significantly associated with carcass contamination at all sampled areas. Other risk factors for carcass contamination are the splitting of the head together with the carcass, and incision of the tonsils during removal of the pluck. Small adaptations in slaughter practices and the training of slaughterhouse personnel to respect basic hygienic instructions may diminish carcass contamination with enteropathogenic Yersinia.

Co-occurrence of free-living protozoa and foodborne pathogens on dishcloths: implications for food safety.

In the present study, the occurrence of free-living protozoa (FLP) and foodborne bacterial pathogens on dishcloths was investigated. Dishcloths form a potentially important source of cross-contamination with FLP and foodborne pathogens in food-related environments. First various protocols for recovering and quantifying FLP from dishcloths were assessed. The stomacher technique is recommended to recover flagellates and amoebae from dishcloths. Ciliates, however, were more efficiently recovered using centrifugation. For enumeration of free-living protozoa on dishcloths, the Most Probable Number method is a convenient method. Enrichment was used to assess FLP diversity on dishcloths (n=38). FLP were found on 89% of the examined dishcloths; 100% of these tested positive for amoebae, 71% for flagellates and 47% for ciliates. Diversity was dominated by amoebae: vahlkampfiids, vannellids, Acanthamoeba spp., Hyperamoeba sp. and Vermamoeba vermiformis were most common. The ciliate genus Colpoda was especially abundant on dishcloths while heterotrophic nanoflagellates mainly belonged to the genus Bodo, the glissomonads and cercomonads. The total number of FLP in used dishcloths ranged from 10 to 10(4) MPN/cm(2). Flagellates were the most abundant group, and ciliates the least abundant. Detergent use was identified as a prime determinant of FLP concentrations on used dishcloths. Bacterial load on dishcloths was high, with a mean total of aerobic bacteria of 7.47 log 10 cfu/cm(2). Escherichia coli was detected in 68% (26/38) of the used dishcloths, with concentrations up to 4 log 10 cfu/cm(2). Foodborne pathogens including Staphylococcus aureus (19/38), Arcobacter butzleri (5/38) and Salmonella enterica subsp. enterica ser. Halle (1/38) were also present. This study showed for the first time that FLP, including some opportunistic pathogens, are a common and diverse group on dishcloths. Moreover, important foodborne pathogens are also regularly recovered. This simultaneous occurrence makes dishcloths a potential risk factor for cross-contamination and a microbial niche for bacteria-FLP interactions.

Presence of Helicobacter suis on pork carcasses.

Helicobacter (H.) suis is a world-wide spread pathogen which not only colonizes the stomach of pigs, but is also the most prevalent gastric non-H. pylori Helicobacter (NHPH) species in humans. H. suis infections are associated with gastric lesions both in pigs and in humans. Recently, the presence of viable H. suis bacteria has been demonstrated in minced pork, suggesting that manipulation or consumption of contaminated pig meat is a possible route of transmission of this zoonotic agent. The main goal of this study was to determine the extent of pork carcass contamination with H. suis at slaughter. In two consecutive studies, the occurrence of H. suis DNA was assessed in scalding water, head and mouth swabs, mesenteric lymph nodes, palatine tonsils and on the chest, shoulder and ham region of pork carcasses from three slaughterhouses using qPCR with ureA gene based H. suis-specific primers. H. suis DNA was detected on carcasses in all slaughterhouses, in 8.3% of all 1083 samples. It was found in all sampled matrices, except for the palatine tonsils and scalding water samples. Contamination levels of dressed pork samples did not exceed 184 genomic equivalents per 100cm(2) (shoulder, ham) or 300cm(2) (chest). All positive PCR products were subjected to sequence analysis of the ureA gene to confirm the identification of H. suis bacteria. Using multilocus sequence typing (MLST) on a selection of the positive samples, 5 unique sequence types (STs) could be assigned. Multiple H. suis strains were present on samples derived from one specific pig herd. Since H. suis DNA was detected in 11% (n: 90) of the mesenteric lymph nodes derived at the slaughterhouse, it was determined whether these organisms can colonize the mesenteric lymph nodes after experimental infection. Despite high-level colonization of the porcine stomachs with the H. suis strain, no H. suis DNA was detected in the mesenteric lymph nodes at four weeks after experimental infection. This might indicate that its presence in these tissues of slaughtered pigs is due to contamination during the slaughter process, but further studies are necessary to confirm this. In conclusion, we demonstrate a relatively high prevalence of H. suis on pork carcasses.

Behavior of Yersinia enterocolitica in the presence of the bacterivorous Acanthamoeba castellanii.

Free-living protozoa play an important role in the ecology and epidemiology of human-pathogenic bacteria. In the present study, the interaction between Yersinia enterocolitica, an important food-borne pathogen, and the free-living amoeba Acanthamoeba castellanii was studied. Several cocultivation assays were set up to assess the resistance of Y. enterocolitica to A. castellanii predation and the impact of environmental factors and bacterial strain-specific characteristics. Results showed that all Y. enterocolitica strains persist in association with A. castellanii for at least 14 days, and associations with A. castellanii enhanced survival of Yersinia under nutrient-rich conditions at 25°C and under nutrient-poor conditions at 37°C. Amoebae cultivated in the supernatant of one Yersinia strain showed temperature- and time-dependent permeabilization. Intraprotozoan survival of Y. enterocolitica depended on nutrient availability and temperature, with up to 2.8 log CFU/ml bacteria displaying intracellular survival at 7°C for at least 4 days in nutrient-rich medium. Transmission electron microscopy was performed to locate the Yersinia cells inside the amoebae. As Yersinia and Acanthamoeba share similar ecological niches, this interaction identifies a role of free-living protozoa in the ecology and epidemiology of Y. enterocolitica.

Assessment of the efficacy of benzalkonium chloride and sodium hypochlorite against Acanthamoeba polyphaga and Tetrahymena spp.

The efficacy of benzalkonium chloride and sodium hypochlorite against Acanthamoeba polyphaga and two Tetrahymena spp. was determined based on the European Standard EN 1276:2009 suspension test. Trophozoite viability was assessed by determination of the membrane integrity using flow cytometry as a fast screening technique. Bovine serum albumin was added to simulate clean (0.3 g/liter) and dirty (3 g/liter) conditions. Benzalkonium chloride caused cell lysis at concentrations above 50 mg/liter under clean and dirty conditions. A concentration of 50 mg of free chlorine per liter had a strong biocidal effect on acanthamoebae and tetrahymenae after 15 min under clean and dirty conditions. Our results suggest that benzalkonium chloride and sodium hypochlorite were effective against the three microorganisms at concentrations commonly applied in the food industry.

Intramuscular inoculation of cattle with Sarcocystis antigen results in focal eosinophilic myositis.

Bovine eosinophilic myositis (BEM) is a subclinical myopathy characterized by multifocal white to grey-green discolorations in skeletal muscles, heart, tongue and oesophagus. These lesions are found at slaughter or during meat cutting and result in considerable economic losses. The etiology and pathogenesis are unclear, although it has been suggested, that Sarcocystis species are involved. To elucidate their role, two calves were repeatedly injected intramuscularly with adjuvanted Sarcocystis antigen. The morphological changes at the injection sites in these calves were histologically and immunohistochemically compared to spontaneous lesions from 44 BEM condemned carcasses sampled in slaughterhouses. Experimental intramuscular injection of Sarcocystis antigen resulted in lesions at the injection sites that were similar to the lesions of natural cases of BEM. They were characterized by massive infiltration of eosinophilic granulocytes, reactive macrophages (MAC387(+) cells), T-cells (CD3(+)) and B-cells (CD20(+)). Both in the experimental and in the natural cases, COX-2 expression was present in endothelial cells adjacent to lesional areas. MHC class II(+) staining was found amongst others in muscle cells surrounding the lesion. These results show that Sarcocystis antigens can induce an inflammatory response in bovine muscle having the characteristics of natural BEM.

Detection and characterization of Salmonella in lairage, on pig carcasses and intestines in five slaughterhouses.

In this study, conducted at five slaughterhouses, individual pigs were sampled and followed up from stunning to cooling down of the carcasses. In this way, Salmonella prevalence and possible risk points were described. At the lairage area, pens were sampled using overshoes. At stunning and bleeding, pigs were individually identified and subsequently swabs were taken of the oral cavity and the carcass after polishing, splitting and forced chilling. Additionally, duodenum, ileum, rectum and mesenteric lymph nodes were extracted and samples were taken of the scalding water. All samples were submitted to Salmonella isolation and Salmonella isolates were serotyped and genotyped by pulsed-field gel electrophoresis (PFGE). Of all samples taken (n = 1953), 14.1% were Salmonella positive. The prevalence of S. in the lairage area varied widely (from 0 to 100%) between the slaughterhouses. Of the sampled pigs (n = 226), 48.2% were positive in at least one sample. Statistical analysis revealed that the contamination of the lairage area was related to a higher amount of positive carcasses after polishing. Furthermore, the contamination of the carcasses after splitting and forced chilling was related to the contamination level of the carcass after polishing. A relation between the outer (carcass) contamination and the inner (gut content and lymph nodes) contamination of a pig could not be established. The predominant serotypes were S. Typhimurium (58.7%) and S. Derby (17.4%). Genotyping revealed 46 different PFGE profiles among the 276 Salmonella isolates. The same genotype at the lairage area as in the oral cavity of the pigs was found in 95%. The results indicate that the lairage area is a primary source of Salmonella in slaughter pigs and that carcass contamination originates from the environment rather than from the pig (inner contamination) itself. It further shows that slaughterhouses vary in their capability of dealing with Salmonella positive pigs. A slaughterhouse specific approach is needed, however, general guidelines should be provided to decrease the contamination level of the lairage area and the slaughter environment.

Effect of coated and non-coated fatty acid supplementation on broiler chickens experimentally infected with Campylobacter jejuni.

The aim of this study was to examine whether and to what extent the supplementation of feed with a coated or non-coated mixture of fatty acids (caprylic and capric acid) affects broiler chickens experimentally infected with Campylobacter jejuni. The study was carried out using 48 chickens divided into four experimental groups. Throughout the whole rearing period (1-42 days), the chickens were fed a diet supplemented with 0.25% caprylic and capric acid (1:1), coated or non-coated. At the age of 14 and 28 days, chickens were orally challenged with C. jejuni. At regular time intervals post-inoculation, the shedding of C. jejuni was assayed using quantitative real-time PCR. Both supplements significantly decreased faecal C. jejuni counts by 1.2-4.1 log(10) CFU/g 4 days post-inoculation; after this time period, the effect of medium-chain fatty acids (MCFA) was less pronounced or absent. Campylobacter jejuni counts in excreta samples were significantly lower in chickens fed coated MCFA than in those fed non-coated MCFA. No effect of MCFA on feed intake or growth of chickens was observed. In conclusion, (i) MCFA are active against C. jejuni and (ii) the encapsulation enhanced the efficacy of the acids. These results allow the recommendation of using MCFA as feed additives in chickens, preferably 2-3 days before slaughter.

Sampling strategy, occurrence and diversity of free-living protozoa in domestic refrigerators.

AIMS: Evaluation of a sampling method to recover free-living protozoa (FLP) from plastic surfaces. Application of the method on different areas inside domestic refrigerators. METHODS AND RESULTS: Plastic coupons seeded with representatives of FLP were swabbed with cotton wools. The recovery efficiency was the highest for Chilomonas paramecium, followed by Tetrahymena pyriformis and the lowest for Acanthamoeba polyphaga. From 43 refrigerators, 19 and 26 were considered FLP positive when sample cultures were incubated at 7°C and 20°C, respectively. The number of FLP-positive cultures was the highest in samples taken from vegetable trays followed by discharge gutters, whereas interior walls were rarely FLP positive. Higher numbers of taxa were observed in enrichment cultures incubated at 20°C instead of 7°C. The combination of microscopy and denaturing gradient gel electrophoresis revealed that discharge gutters occasionally were contaminated with a persistent protozoan population of flagellates (Cercozoa) and amoebae (Tubulinea). The FLP-positive status of refrigerator surfaces was correlated with a high aerobic plate count. CONCLUSIONS: The cotton wool sampling method is useful to sample FLP from plastic surfaces. FLP are part of the microbial communities in domestic refrigerators. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge on the occurrence of FLP in food-related indoor environments is scarce. For the first time, a high protozoan diversity in domestic refrigerators is described.

Arcobacter contamination on pre- and post-chilled bovine carcasses and in minced beef at retail.

AIMS: The present study aimed to assess the Arcobacter contamination on bovine carcasses postevisceration and postcooling in two slaughterhouses and in ready-to-eat minced beef. METHODS AND RESULTS: Carcasses (n = 247) were sampled at four sites in two slaughterhouses and 100 minced beef samples were collected at retail. Isolation was performed by a quantitative and qualitative Arcobacter selective method, and the isolates were identified by multiplex PCR, after which a part of them were characterized by enterobacterial repetitive intergenic consensus (ERIC)-PCR. Although arcobacters were isolated from 37% of the bovine carcasses postevisceration with the chest and the foreleg as most contaminated sites, cooling the carcasses for at least 24 h reduced the incidence of Arcobacter (7%) on the carcass surface significantly. Arcobacter butzleri was the species most frequently isolated, although co-contamination with multiple species also occurred. At retail, arcobacters were present in 9% of the minced beef samples, with Arcobacter butzleri as the dominant species. CONCLUSIONS: Forced air cooling of bovine carcasses for at least 24 h decreased the number of positive carcasses, but did not eliminate all arcobacters. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that maintaining good hygiene practices throughout the food supply chain is crucial to ensure safe food products at the consumer level.

Escherichia coli O157 prevalence in different cattle farm types and identification of potential risk factors.

Although the prevalence of Escherichia coli O157 on cattle farms has been examined extensively, the relationship between this pathogen and farm type has been established only rarely. A large-scale study was designed to determine the prevalence of E. coli O157 in the Flemish region of Belgium on farms of dairy cattle, beef cattle, mixed dairy and beef cattle, and veal calves. The effect of various factors on the occurrence at the pen level also was evaluated. In 2007, 180 farms were randomly selected based on region, farm size, and number of animals purchased and were examined using the overshoe sampling method. When possible, overshoes used in areas containing animals in three different age categories (< 8 months, 8 to 30 months, and > 30 months) were sampled on each farm. In total, 820 different pens were sampled and analyzed for the presence of E. coli O157 by enrichment, immunomagnetic separation, and plating on selective agar. Presumptive E. coli O157 colonies were identified using a multiplex PCR assay for the presence of the rfb(O157) and fliC(H7) genes. The statistical analysis was carried out with Stata SE/10.0 using a generalized linear regression model with a logit link function and a binomial error distribution. The overall farm prevalence of E. coli O157 was 37.8% (68 of 180 farms). The highest prevalence was found on dairy cattle farms (61.2%, 30 of 49 farms). The prevalences on beef, mixed dairy and beef, and veal calf farms were 22.7% (17 of 75 farms), 44.4% (20 of 45 farms), and 9.1% (1 of 11 farms), respectively. A significant positive correlation between age category and E. coli O157 prevalence was found only on mixed dairy and beef farms and dairy farms. No influence of farm size or introduction of new animals was demonstrated.