Heavy metal content and element analysis of infant formula and milk powder samples purchased on the Tanzanian market: International branded versus black market products.

Milk powder is a food for malnourished African children and for healthy infants of women with HIV/AIDS. High demand and low purchasing power has resulted in a huge informal, black market in Sub-Saharan Africa. Forty-three milk powder batches were analyzed for 43 chemical elements using ICP-MS One sample (2.3%) was contaminated at a lead concentration of 240 µg/kg dry weight exceeding the European threshold (130 µg/kg dry weight). Macroelement contents revealed a trend decreasing in concentration through skimmed, full cream products to infant formulae. Concentration ranges by dry weight differed in respect of uncertainty intervals of  ±10%. Median Ca, K and P concentrations declined from 11.14 g/kg to 3.21 g/kg, 14.11 g/kg to 4.95 g/kg and 9.12 g/kg to 2.75 g/kg dry mass, respectively. Milk powder samples obtained from the Tanzanian black market were comparable in respect of nutritional and chemical content to international branded full cream products.

Urban prevalence of Listeria spp. and Listeria monocytogenes in public lavatories and on shoe soles of facility patrons in the European capital city Vienna.

The aim of this study was to determine the prevalence of Listeria spp. and Listeria monocytogenes (L. monocytogenes) in urban public lavatories and on shoe soles of facility patrons in a European capital city. More than 91% of all municipal public lavatories in Vienna close to public hubs were included in this study. Overall, 373 swab samples of public lavatories and shoes of facility patrons were enriched, according to ISO 11290-1. Listeria monocytogenes isolates were subtyped using pulsed-field gel electrophoresis. A total of 24 samples were positive for Listeria spp., yielding an overall prevalence of 6.4% (24/373). Listeria monocytogenes was found in 2.1% (8/373) of all samples. Swabs from lavatories in parks, container lavatories and lavatories at markets had the highest prevalences of 20.7% (6/29), 20% (2/10) and 12.5% (1/8) Listeria spp., respectively. These detection rates were statistically significantly higher than those associated with lavatories in shopping centres (P = 0.003, P = 0.002, P = 0.02) and at public transport locations (P = 0.0004, P = 0.005, P = 0.02). Shoes sampled at Christmas markets showed the highest Listeria spp. and L. monocytogenes prevalences of 80% (4/5) and 40% (2/5), respectively. With regard to shoe type, Listeria spp. detection rates were 14.3% (3/21; winter boots), 13.3% (2/15; hiking boots), sport shoes (5.9%; 2/34) and brogues (5.1%; 4/79). No Listeria spp. were found on shoe soles that had smooth treads (0/76), while Listeria spp. were detected on 19.5% (8/41) of medium depth tread shoe types and on 9.4% (3/32) of deep tread shoes. These data suggest that soil environment is still one of the most important reservoirs for the foodborne pathogen L. monocytogenes.

Population diversity of Listeria monocytogenes in quargel (acid curd cheese) lots recalled during the multinational listeriosis outbreak 2009/2010.

It has been possible to determine the genotype diversity of Listeria monocytogenes in the actual cheese lots of acid curd cheese that caused a multinational outbreak between 2009 and 2010. Following product recall in January 2010 all lots were investigated. A total of 422 L. monocytogenes isolates were characterized by genotyping. In a first approach the PCR serogroups were defined by multiplex-PCR assays. Subsequently, the isolates were subtyped by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Sequence types were assigned by submitting the DNA sequences to the Listeria MLST database at the Institute Pasteur. The serogroup PCR resulted in a homogeneous 1/2a - 3a (genetic linage II) cluster. The generated PFGE patterns divided the strains into two clusters (type 1 and 2) diverging at a homogeneity level of 74%. PFGE-type 2 was predominant, accounting for 98.3% (n = 415/422) of the isolates and was isolated during the whole period of acid curd cheese processing (01.12.2009-13.01.2010). 1.7% of all tested L. monocytogenes isolates (n = 7/422) belonged to PFGE-type 1 and were isolated from 28% of all cheese lots (n = 5/18) produced between the time span of 08.12.2009 to 13.01.2010. Furthermore, PFGE-type 1 and 2 showed the same PFGE patterns as the human outbreak strains (clone 1 and clone 2).

Assessing in-house monitoring efficiency by tracing contamination rates in cheese lots recalled during an outbreak of listeriosis in Austria.

A cluster of 34 cases of listeriosis was traced to consumption of quargel cheese, a sour milk specialty, in Austria, Germany and Czech Republic between 2009 and 2010. After recall from the retail market all soft cheese batches (n = 18) were sent for investigation and ISO 11290 based microbiological analysis revealed all red smear-ripened batches (16/18) to be positive for Listeria monocytogenes whereas mold ripened cheeses were negative. The 16 positive batches were grouped into three categories: those having exceeded shelf-life (G1), those around shelf-life (± 4 days, G2) and those within shelf-life (G3). Tracing the contamination levels as measured after recall (CLR) to the theoretical contamination level after processing (CL0) was considered to provide an estimate as to whether the in-house monitoring system would have been capable of unraveling the contamination scenario. Growth simulations starting from various hypothetical initial contamination levels of cheese at the plant and considering the potential variability in growth of L. monocytogenes due to model parameters and storage conditions suggested that a very low initial contamination level (e.g., <1 CFU/g or 5 CFU/100g) could justify the levels of L. monocytogenes enumerated in recalled samples of G1 and G2 lots. This in turn, may have resulted in low detection probability using ISO 11290:1996. In lots of G3 group, however, high initial contamination levels or temperature abuse at retail are inferred, based on simulated outputs.

Demonstration of the effective performance of a combined enrichment/real-time PCR method targeting the prfA gene of Listeria monocytogenes by testing fresh naturally contaminated acid curd cheese.

AIMS: A rapid real-time PCR-based method for the detection of Listeria monocytogenes was applied to the examination of 44 Quargel cheese samples from a recent outbreak in Austria and compared to the standard method according to ISO-16140. METHODS AND RESULTS: The combined enrichment/real-time PCR method amplifying the prfA locus was performed according to [Rossmanith et al.(2006) Res Microbiol, 157, 763-771]. Qualitative and quantitative examination of the samples was performed according to the standard method ISO-11290. Comparison of the combined enrichment/real-time PCR method with ISO-11290 resulted in 100% relative accuracy, 100% relative sensitivity and 100% relative specificity. CONCLUSIONS: A previously published study describing the validation of the method, including samples after storage at -80 degrees C, resulted in lower performance values. In contrast, the samples were stored at +4 degrees C in this study. The results of this study indicate an effect of storage, thus masking the true performance of the method. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study are discussed together with the previously published data to demonstrate the excellent qualities of this rapid (< or = 30 h) method when applied to fresh specimens stored at +4 degrees C.

Performance testing of six chromogenic ALOA-type media for the detection of Listeria monocytogenes.

AIMS: The aim of the study was to test the performance of commercially available chromogenic plating media for detection and enumeration of the food-borne pathogen Listeria monocytogenes. A wide range of chromogenic media similar to Agar Listeria according to Ottaviani and Agosti (ALOA) were compared using PALCAM agar, according to van Netten et al. METHODS AND RESULTS: Six chromogenic media similar to ALOA were challenged for inclusivity and exclusivity. Additionally, the ability of chromogenic agars to facilitate growth of stressed L. monocytogenes strains and mixed cultures with competitive non-Listeria strains was estimated. Finally, we tested the detection and enumeration of L. monocytogenes in artificially inoculated and naturally contaminated food samples. The results of this study indicated that chromogenic media are a good supplementation to PALCAM agar. A single application is not advisable, as the specificity of chromogenic agars is frequently insufficient (50.0-88.9%), particularly in food samples with a complex microflora. CONCLUSIONS: The competitive flora of food samples is able to overgrow low numbers of L. monocytogenes, especially in half-Fraser enrichment. This might lead to the underestimation of L. monocytogenes positive samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Although many evaluation studies of chromogenic agar have been published recently, harmonized validation strategies are lacking. This survey provides a new concept for stepwise testing of plating media.

Survey on the Listeria contamination of ready-to-eat food products and household environments in Vienna, Austria.

Qualitative and quantitative contamination of ready-to-eat food-stuffs with the pathogen Listeria monocytogenes was studied in 1586 samples collected from 103 supermarkets (n = 946) and 61 households (n = 640) in Vienna, Austria. Seventeen groups of ready-to-eat foods were classified into three risk categories for contamination (CP1-CP3). Three to four samples were randomly collected at the retail level from each CP. Regarding the households, the sampling procedure was started with food items of CP1, and if not available, was continued with sampling of food items of CP2 and finally of CP3. Additionally, 184 environmental samples (swabs from the kitchen area, dust samples from the vacuum cleaner) and faecal samples (household members and pet animals) were included. One-hundred and twenty-four (13.1%) and 45 (4.8%) samples out of 946 food samples collected from food retailers tested positive for Listeria spp. and L. monocytogenes, respectively, with five smoked fish samples exceeding the tolerated limit of 100 CFU/g food. Food-stuffs associated with the highest risk of contamination were twice as frequently contaminated with L. monocytogenes as food-stuffs associated with a medium risk of contamination. Products showing the highest contamination rate were fish and seafood (19.4%), followed by raw meat sausages (6.3%), soft cheese (5.5%) and cooked meat products/patés (4.5%). The overall contamination rate of foods collected at the household level was more than two times lower. Only 5.6% and 1.7% of 640 food-stuffs analysed tested positive for Listeria spp. and L. monocytogenes, respectively. However, CP1 foods were rarely collected. Pulsed-field gel electrophoresis (PFGE) typing of the collected L. monocytogenes isolates revealed a high degree of diversity between the isolates, with some exceptions. PFGE typing of isolates harvested from green-veined cheese revealed a match among strains, although the manufacturer seemed to be distinguishable. Typing of household strains revealed an epidemiological link within one family. In this case, food-stuffs and the kitchen environment were contaminated by an indistinguishable isolate. In addition, the same isolate was collected from a pooled faecal sample of the household members suggesting that consumption of even low contaminated food items (<100 CFU/g) results in Listeria shedding after the passage through the gut.

Outbreak of clinical listeriosis in sheep: evaluation from possible contamination routes from feed to raw produce and humans.

We report the results of clinical and microbiological investigations on Listeria monocytogenes infections in a flock of 55 sheep and describe the implications for the safety of the raw milk and raw-milk cheeses produced in the on-farm dairy. The outbreak was caused by feeding grass silage, which was contaminated with 5 log10 CFU L. monocytogenes/g. Clinically, although having been fed from the same batch of silage, abortive (nine ewes), encephalitic (one ewe) and septicaemic (four ewes) forms of listeriosis were observed during the outbreak phase. As the starting point of feeding the contaminated silage was known we could calculate an incubation period of 18+/-2 and 26 days for the abortive and the encephalitic form of listeriosis, respectively. Pathologically, the septicaemic cases suffered from Listeria accumulation at comparable numbers in visceral organs but not in the brain. Only a single ewe developed central nervous symptoms and a rhomb-encephalitis was immunohistologically confirmed. In this case the infection proceeded from the nasal mucosa into the brain, with no infections of the liver, spleen and other visceral organs. Sampling of the cheese production chain, the farm environment and the persons living at the farm revealed the exposure of a farm-worker to an isolate genetically indistinguishable from the outbreak clone, obviously through the consumption of faecally contaminated bovine raw milk. The cheese under processing was free of Listeria because, as a result of intensive consultations, the farmer ensured a proper acidification of the cheese. The epidemiological findings suggest that food safety matters should be assessed in any case where infection of food-producing animals with potential human pathogens is observed.

Novel approach for assessing performance of PCR cyclers used for diagnostic testing.

As part of a large international project for validation and standardization of PCR, the influence of thermocyclers on PCR was tested. Six brand-new, Peltier technology-driven 96-well thermocyclers were subjected to a novel and stringent in-tube (not block) physical testing. The temperature was directly monitored in PCR tubes containing 50 microl of distilled water at 13 different block positions. The certified temperature accuracy of the measurement system was +/-0.3 degrees C. Finally, the results of the physical testing were compared to those of an amplification efficiency study running an in-house PCR assay. The cyclers did not perform within the manufacturer's specification. Premature timing, under- and overshooting, and spatial variation of heat transfer were found to be the critical factors. The physical testing allowed us to distinguish accurate from less-accurate (2/6) cyclers. The lack of thermal homogeneities became most evident at the denaturation level during the first 15 s. At the time point zero, the accurate cyclers showed temperature deviations of 0.5 to 1.5 degrees C, whereas less-accurate cyclers failed to reach the set temperature by 13 to 20 degrees C. Consequently, the two less-accurate cyclers could not gain positive PCR results by running an in-house PCR assay. However, by modifying the original temperature protocol by increasing the denaturation temperature and time, the amplification efficiency of these two cyclers could be improved significantly. The results have implication for laboratories using diagnostic PCR testing.

Clinical and histopathological aspects of naturally occurring mastitis caused by Listeria monocytogenes in cattle and ewes.

Listeria monocytogenes was isolated from the milk of two cows and two sheep with mastitis in one quarter and one udder half. The animals were observed over a period of 2-12 months. Clinical examination of the udder, bacteriological examinations and determination of somatic cell counts of milk samples were performed monthly. All four cases suffered from a subclinical mastitis characterized by an elevated somatic cell count (0.8-10.1 x 10(6) cells/ml), a persistent shedding of Listeria and by a normal appearance of the milk. The animals did not show any systemic reaction, but all animals developed an atrophy of the infected mammary gland. Histological examinations revealed a chronic interstitial mastitis with diffuse infiltration of lymphocytes, plasma cells and macrophages. All internal organs showed no abnormalities, no Listeria could be isolated. Listeria could however be isolated from the affected mammary parenchyma and from the mammary lymph node. The results of the bacteriological examination could be confirmed by means of PCR. Using PFGE, all the isolates from the same animal were identical. Immunohistochemical examination of the ovine mammary glands achieved a very strong immunoreactivity for CD5 cells. The mode of infection and the reaction of the immune system's defense of the ovine udders are discussed.