Transmission Scenarios of Listeria monocytogenes on Small Ruminant On-Farm Dairies.

Listeria monocytogenes can cause severe foodborne infections in humans and invasive diseases in different animal species, especially in small ruminants. Infection of sheep and goats can occur via contaminated feed or through the teat canal. Both infection pathways result in direct (e.g., raw milk from an infected udder or fresh cheese produced from such milk) or indirect exposure of consumers. The majority of dairy farmers produces a high-risk product, namely fresh cheese made from raw ewe's and goat's milk. This, and the fact that L. monocytogenes has an extraordinary viability, poses a significant challenge to on-farm dairies. Yet, surprisingly, almost no scientific studies have been conducted dealing with the hygiene and food safety aspects of directly marketed dairy products. L. monocytogenes prevalence studies on small ruminant on-farm dairies are especially limited. Therefore, it was our aim to focus on three main transmission scenarios of this important major foodborne pathogen: (i) the impact of caprine and ovine listerial mastitis; (ii) the significance of clinical listeriosis and outbreak scenarios; and (iii) the impact of farm management and feeding practices.

Asymptomatic Carriage of Listeria monocytogenes by Animals and Humans and Its Impact on the Food Chain.

Humans and animals can become asymptomatic carriers of Listeria monocytogenes and introduce the pathogen into their environment with their feces. In turn, this environmental contamination can become the source of food- and feed-borne illnesses in humans and animals, with the food production chain representing a continuum between the farm environment and human populations that are susceptible to listeriosis. Here, we update a review from 2012 and summarize the current knowledge on the asymptomatic carrier statuses in humans and animals. The data on fecal shedding by species with an impact on the food chain are summarized, and the ways by which asymptomatic carriers contribute to the risk of listeriosis in humans and animals are reviewed.

Monitoring by a Sensitive Liquid-Based Sampling Strategy Reveals a Considerable Reduction of Listeria monocytogenes in Smeared Cheese Production over 10 Years of Testing in Austria.

Most Austrian dairies and cheese manufacturers participated in a Listeria monitoring program, which was established after the first reports of dairy product-associated listeriosis outbreaks more than thirty years ago. Within the Listeria monitoring program, up to 800 mL of product-associated liquids such as cheese smear or brine are processed in a semi-quantitative approach to increase epidemiological sensitivity. A sampling strategy within cheese production, which detects environmental contamination before it results in problematic food contamination, has benefits for food safety management. The liquid-based sampling strategy was implemented by both industrial cheese makers and small-scale dairies located in the mountainous region of Western Austria. This report considers more than 12,000 Listeria spp. examinations of liquid-based samples in the 2009 to 2018 timeframe. Overall, the occurrence of L. monocytogenes in smear liquid samples was 1.29% and 1.55% (n = 5043 and n = 7194 tested samples) for small and industrial cheese enterprises, respectively. The liquid-based sampling strategy for Listeria monitoring at the plant level appears to be superior to solid surface monitoring. Cheese smear liquids seem to have good utility as an index of the contamination of cheese up to that point in production. A modelling or validation process should be performed for the new semi-quantitative approach to estimate the true impact of the method in terms of reducing Listeria contamination at the cheese plant level.

Temporal analysis of the Listeria monocytogenes population structure in floor drains during reconstruction and expansion of a meat processing plant.

Due to a higher probability for violation of hygiene measures, reconstruction work is a substantial food safety challenge for food business operators (FBOs). Here, we monitored a Listeria monocytogenes contamination scenario during a timely enduring reconstruction period that aimed at an expansion of the main building of a leading meat processing facility. Reconstruction took place while food production was ongoing. We used a longitudinal sampling scheme targeting 40 floor water drains distributed over the food processing environment (FPE) over a five year period. The population structure of L. monocytogenes was determined by PCR-serogrouping, pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). While the first sampling deciphered a baseline of contamination (45%), intensified sanitation measures decreased L. monocytogenes prevalence before commencement of work (5%). The reconstruction activities increased the prevalence of L. monocytogenes in the FPE (20.5%) and changed the population structure to a higher proportion of disease-associated genotypes (61%). During the first sampling ST121 was prevalent throughout the FPE, even in the packaging area. After the second and third sampling, following increased application of hypochlorite during sanitation, ST121 was only present in the raw material preparation area. A resilient flora was detected during three sampling events (ST8, ST9 and ST37) which might have not been exposed to daily cleaning in the floor drains. After the accomplishment of reconstruction work, the L. monocytogenes population structure shifted to the condition initially found (45% and 20.5% during the first and sixth sampling event). This paper indicates that reconstruction phases are high risk episodes for food safety in FPEs. Special precautions must be taken to avoid cross-contamination of products since reconstruction is usually ongoing for extended periods of time.

A Fast and Easy ATP-Based Approach Enables MIC Testing for Non-resuscitating VBNC Pathogens.

Many bacteria enter the viable but non-culturable (VBNC) state to maximize resources and increase their tolerance to harmful conditions to cope with environmental stress, which has been described for a plethora of important human and foodborne pathogens. VBNC pathogens can potentially present a serious risk to human health as they are invisible to routine microbiological culture-based methods. Of high importance is the increased tolerance to antibiotics or disinfectant measures while in the VBNC state. The greatest remaining challenge for such investigations is the lack of an appropriate, cost-effective multi-species screening method due to experimental constraints. In this study, we investigated if de novo ATP production of cells in the VBNC state is a suitable indicator for overall cell viability that can be utilized to determine the minimum ATP inhibitory concentration (MAIC) of antibiotics and other antimicrobials. To validate this approach, heat-stress time-kill experiments were performed with both culturable and VBNC cells. We developed a comprehensive experimental setup and demonstrated the applicability of this VBNC-MIC assay for testing the tolerance of 12 strains of 4 important bacterial species (Escherichia coli, Bacillus cereus, Pseudomonas aeruginosa, and Listeria monocytogenes) in the VBNC state to eight important antimicrobials including four different antibiotics. We confirmed that bacteria in the VBNC state were resistant to all tested antibiotics (ampicillin, imipenem, ciprofloxacin, and gentamicin) and additionally insensitive to disinfectants (benzalkonium chloride and trioctylmethylammonium chloride) and preservatives (bronopol and sodium azide). These data emphasize the need for further research regarding the characteristics of bacterial pathogens in the VBNC state and present the advantages and high-throughput capabilities of ATP determinations to investigate tolerance of VBNC pathogens to antimicrobials. The presented method should be helpful in order to identify appropriate countermeasures, treatments, or disinfectants when confronted with bacterial pathogens in the VBNC state.

Don't Shut the Stable Door after the Phage Has Bolted-The Importance of Bacteriophage Inactivation in Food Environments.

In recent years, a new potential measure against foodborne pathogenic bacteria was rediscovered-bacteriophages. However, despite all their advantages, in connection to their widespread application in the food industry, negative consequences such as an uncontrolled phage spread as well as a development of phage resistant bacteria can occur. These problems are mostly a result of long-term persistence of phages in the food production environment. As this topic has been neglected so far, this article reviews the current knowledge regarding the effectiveness of disinfectant strategies for phage inactivation and removal. For this purpose, the main commercial phage products, as well as their application fields are first discussed in terms of applicable inactivation strategies and legal regulations. Secondly, an overview of the effectiveness of disinfectants for bacteriophage inactivation in general and commercial phages in particular is given. Finally, this review outlines a possible strategy for users of commercial phage products in order to improve the effectiveness of phage inactivation and removal after application.

A Novel Method for Sampling and Long-Term Monitoring of Microbes That Uses Stickers of Plain Paper.

Detection of pathogens is crucial in food production areas. While it is well established, swabbing as a state-of-the-art sampling method offers several drawbacks with respect to yield, standardization, overall handling, and long-term monitoring. This led us to develop and evaluate a method that is easier to use at a lower cost and that should be at least as sensitive. After evaluating sundry promising materials, we tested text-marking paper stickers for their suitability to take up and release Listeria monocytogenes with their nonsticky paper side over a 14-day time period using quantitative PCR. The recovery rate was similar to that in previous studies using conventional swabs, and we also confirmed the feasibility of pooling besides resilience to cleansing and disinfection. In a proof-of-concept experiment that sampled several locations, such as door handles, the occurrences of L. monocytogenes and Escherichia coli were determined. The results suggest that the presented sticker system might offer a promising cost-effective alternative sampling system with improved handling characteristics.IMPORTANCE As a ubiquitous bacterium, Listeria monocytogenes has a propensity to enter food production areas inadvertently via fomites such as door handles and switches. While the bacterium might not be in direct contact with the food products, knowing the microbial status of the surroundings is essential for risk assessment. Our investigation into a novel quantitative PCR (qPCR)-based sampling system with the highest sensitivity and ability to monitor over long periods of time, yet based on paper, proved to be cost-effective and reasonably convenient to handle.

Essential role of polymerases for assay performance - Impact of polymerase replacement in a well-established assay.

The quantitative real-time polymerase chain reaction (qPCR) is one of the most commonly molecular methods used today. It is central to numerous assays that have since been developed and described around its optimization. The Listeria monocytogenes prfA qPCR assay has been studied in great detail and due to its comprehensive knowledge, excellent performance (sensitivity of one single copy), and internal amplification control, it represents a suitable test platform for qPCR examinations. In this study, we compared ten different polymerases (or ready-to-use mastermixes) as possible (economic) alternatives to our gold standard Platinum Taq polymerase. We sought to determine the reproducibility of these assays under modified conditions, which are realistic because published assays are frequently used with substituted polymerases. Surprisingly, there was no amplification at all with some of the tested polymerases, even although the internal amplification control worked well. Since adaptation of the thermal profile and of MgCl2 concentration could restore amplification, simple replacement of the polymerase can destroy a well-established assay leading up to >106-fold less analytical sensitivity. Further, validation using Poisson and PCR-Stop analyses revealed limits to some assay-polymerase combinations and emphasize the importance of validation.

Induction of the viable but non-culturable state in bacterial pathogens by household cleaners and inorganic salts.

Effective monitoring of microbial pathogens is essential for a successful preventive food safety and hygiene strategy. However, as most monitoring strategies are growth-based, these tests fail to detect pathogenic bacteria that have entered the viable but non-culturable (VBNC) state. The present study reports the induction of the VBNC state in five human pathogens by commercially available household cleaners in combination with inorganic salts. We determined that non-ionic surfactants, a common ingredient in household cleaners, can induce the VBNC state, when combined with salts. A screening study with 630 surfactant/salt combinations indicates a correlation between the hydrophobicity of the surfactant and VBNC induction in L. monocytogenes, E. coli, S. enterica serovar Typhimurium, S. aureus and toxin-producing enteropathogenic E. coli. Cells that were exposed to combinations of surfactants and salts for 5 min and up to 1 h lost their culturability on standard growth media while retaining their ATP production, fermentation of sugars and membrane integrity, which suggests intact and active metabolism. Screening also revealed major differences between Gram-negative and Gram-positive bacteria; the latter being more susceptible to VBNC induction. Combinations of such detergents and salts are found in many different environments and reflect realistic conditions in industrial and domestic surroundings. VBNC cells present in industrial environments, food-processing plants and even our daily routine represent a serious health risk due to possible resuscitation, unknown spreading, production of toxins and especially their invisibility to routine detection methods, which rely on culturability of cells and fail to detect VBNC pathogens.

Listeria monocytogenes Isolated from Illegally Imported Food Products into the European Union Harbor Different Virulence Factor Variants.

Unregulated international flow of foods poses a danger to human health, as it may be contaminated with pathogens. Recent studies have investigated neglected routes of pathogen transmission and reported the occurrence of Listeria monocytogenes in food illegally imported into the European Union (EU), either confiscated at four international airports or sold illegally on the Romanian black market. In this study we investigated the genotype diversity and the amino acid sequence variability of three main virulence factors of 57 L. monocytogenes isolates. These isolates were derived from 1474 food samples illegally imported into the EU and originated from 17 different countries. Multilocus sequence typing revealed 16 different sequence types (STs) indicating moderate genotype diversity. The most prevalent STs were ST2, ST9, and ST121. The pulsed-field gel electrophoresis (PFGE) analysis resulted in 34 unique pulsotypes. PFGE types assigned to the most prevalent STs (ST2, ST9, and ST121) were highly related in their genetic fingerprint. Internalin A (InlA) was present in 20 variants, including six truncated InlA variants, all harbored by isolates of ST9 and ST121. We detected eight ST-specific listeriolysin O (LLO) variants, and among them, one truncated form. The actin-assembly-inducing protein ActA was present in 15 different ST-specific variants, including four ActA variants with an internal truncation. In conclusion, this study shows that L. monocytogenes, isolated from illegally imported food, have moderate genotype diversity, but diverse virulence factors variants, mainly of InlA.

The impact of shelf life on exposure as revealed from quality control data associated with the quargel outbreak.

A cluster of 34 human cases of listeriosis was traced to consumption of contaminated quargel cheese, a sour milk specialty sold in Austria, Germany and Czech Republic. Here, we try to assess how many portions were consumed by the Austrian population at a certain contamination level (CL). In total, 1623 cheese lots were produced during the outbreak period resulting in >3 million portions of cheese delivered to the market. From 650 sets of quality control data provided by the food business operator, we reconstructed the contamination scenario over time and identified 84 lots that were found to be positive. With regard to another sixteen lots, a CL was found ranging from one to 3,84 log10 CFU L. monocytogenes/g, measured in product stored between one to 23 days after production. However the number of storage days at home before consumption is unknown. To resolve this issue, we modelled the theoretical CL of the product if consumed either 20, 30, 40 or 50 days post production. We found that 10 lots (approx. 27,350 portions) would have been contaminated at CLs higher than 3 log10 CFU L. monocytogenes/g if all cheese had been consumed after 20 days of storage. This number shifts to 20 lots (approx. 54,700 portions) after 30 days of storage. If all cheese had been consumed at the end of shelf life (50 days of storage), theoretically 242,5 lots would have exceeded a CL of 6 log10 CFU L. monocytogenes/g. We concluded that the extended shelf life given to the product was a driver of the outbreak scenario. It is stunning to note that so few cases were reported in spite of consumers' massive exposure to L. monocytogenes. We hypothesized that a low pathogenicity of both quargel outbreak clones (QOC1 and QOC2) could have contributed to this discrepancy. Our hypothesis was falsified since both strains QOC1 and QOC2 are fully virulent in an oral infection mouse model, showing even higher pathogenicity than the reference strain EGDe.

A Systematic Investigation of Parameters Influencing Droplet Rain in the Listeria monocytogenes prfA Assay - Reduction of Ambiguous Results in ddPCR.

The droplet digital polymerase chain reaction (ddPCR) determines DNA amounts based upon the pattern of positive and negative droplets, according to Poisson distribution, without the use of external standards. However, division into positive and negative droplets is often not clear because a part of the droplets has intermediate fluorescence values, appearing as "rain" in the plot. Despite the droplet rain, absolute quantification with ddPCR is possible, as shown previously for the prfA assay in quantifying Listeria monocytogenes. Nevertheless, reducing the rain, and thus ambiguous results, promotes the accuracy and credibility of ddPCR. In this study, we extensively investigated chemical and physical parameters for optimizing the prfA assay for ddPCR. While differences in the concentration of all chemicals and the dye, quencher and supplier of the probe did not alter the droplet pattern, changes in the PCR cycling program, such as prolonged times and increased cycle numbers, improved the assay.

Influence of Environmental Factors on Phage-Bacteria Interaction and on the Efficacy and Infectivity of Phage P100.

When using bacteriophages to control food-borne bacteria in food production plants and processed food, it is crucial to consider that environmental conditions influence their stability. These conditions can also affect the physiological state of bacteria and consequently host-virus interaction and the effectiveness of the phage ability to reduce bacteria numbers. In this study we investigated the stability, binding, and replication capability of phage P100 and its efficacy to control Listeria monocytogenes under conditions typically encountered in dairy plants. The influences of SDS, Lutensol AO 7, salt, smear water, and different temperatures were investigated. Results indicate that phage P100 is stable and able to bind to the host under most conditions tested. Replication was dependent upon the growth of L. monocytogenes and efficacy was higher when bacterial growth was reduced by certain environmental conditions. In long-term experiments at different temperatures phages were initially able to reduce bacteria up to seven log10 units after 2 weeks at 4°C. However, thereafter, re-growth and development of phage-resistant L. monocytogenes isolates were encountered.

Evaluation of the performance of quantitative detection of the Listeria monocytogenes prfA locus with droplet digital PCR.

Fast and reliable pathogen detection is an important issue for human health. Since conventional microbiological methods are rather slow, there is growing interest in detection and quantification using molecular methods. The droplet digital polymerase chain reaction (ddPCR) is a relatively new PCR method for absolute and accurate quantification without external standards. Using the Listeria monocytogenes specific prfA assay, we focused on the questions of whether the assay was directly transferable to ddPCR and whether ddPCR was suitable for samples derived from heterogeneous matrices, such as foodstuffs that often included inhibitors and a non-target bacterial background flora. Although the prfA assay showed suboptimal cluster formation, use of ddPCR for quantification of L. monocytogenes from pure bacterial cultures, artificially contaminated cheese, and naturally contaminated foodstuff was satisfactory over a relatively broad dynamic range. Moreover, results demonstrated the outstanding detection limit of one copy. However, while poorer DNA quality, such as resulting from longer storage, can impair ddPCR, internal amplification control (IAC) of prfA by ddPCR, that is integrated in the genome of L. monocytogenes ΔprfA, showed even slightly better quantification over a broader dynamic range. Graphical Abstract Evaluating the absolute quantification potential of ddPCR targeting Listeria monocytogenes prfA.

Prevalence of major foodborne pathogens in food confiscated from air passenger luggage.

The EU has issued several directives and regulations pertaining to the importation of animals and products of animal origin (POAO) and veterinary controls on importation. Unfortunately, little information is available concerning associated risks and no attempts have been made to collect baseline data on the actual prevalence of zoonotic agents in POAO carried by travellers. To meet these challenges the EU recently introduced and financed a research project "PROMISE". Its main objectives were to assess the risks involved when foodborne pathogens are introduced to the EU via uncontrolled imports. With special permission of the Austrian health authorities, spot-checks were made of the luggage of 61,355 passengers from 240 flights from non-EU countries arriving at the Vienna International Airport (VIE airport). Over a period of eight months (August 2012 through March 2013) 1473 POAO items were confiscated. A total of 600 samples were suitable for Salmonella spp., Campylobacter spp., verotoxigenic Escherichia coli and Listeria monocytogenes prevalence analysis. Foodborne pathogens could be detected in 5% (30/600) of all samples. The highest prevalence was attributed to L. monocytogenes, at 2.5%, followed by VTEC and Salmonella spp. at 1.3% and 1.2%, respectively. Campylobacter spp. was not present in any of the 600 samples. Multi-locus sequence typing (MLST) of L. monocytogenes revealed that current sequence types (ST) corresponded to the worldwide most present clonal complexes 1, 2, 3, 5, 9, and 121. Generally, L. monocytogenes ST9 was the predominant allelic profile, which was mainly isolated from Turkish meat products.

Genome sequencing of Listeria monocytogenes "Quargel" listeriosis outbreak strains reveals two different strains with distinct in vitro virulence potential.

A large listeriosis outbreak occurred in Austria, Germany and the Czech Republic in 2009 and 2010. The outbreak was traced back to a traditional Austrian curd cheese called "Quargel" which was contaminated with two distinct serovar 1/2a Listeria monocytogenes strains (QOC1 and QOC2). In this study we sequenced and analysed the genomes of both outbreak strains in order to investigate the extent of genetic diversity between the two strains belonging to MLST sequence types 398 (QOC2) and 403 (QOC1). Both genomes are highly similar, but also display distinct properties: The QOC1 genome is approximately 74 kbp larger than the QOC2 genome. In addition, the strains harbour 93 (QOC1) and 45 (QOC2) genes encoding strain-specific proteins. A 21 kbp region showing highest similarity to plasmid pLMIV encoding three putative internalins is integrated in the QOC1 genome. In contrast to QOC1, strain QOC2 harbours a vip homologue, which encodes a LPXTG surface protein involved in cell invasion. In accordance, in vitro virulence assays revealed distinct differences in invasion efficiency and intracellular proliferation within different cell types. The higher virulence potential of QOC1 in non-phagocytic cells may be explained by the presence of additional internalins in the pLMIV-like region, whereas the higher invasion capability of QOC2 into phagocytic cells may be due to the presence of a vip homologue. In addition, both strains show differences in stress-related gene content. Strain QOC1 encodes a so-called stress survival islet 1, whereas strain QOC2 harbours a homologue of the uncharacterized LMOf2365_0481 gene. Consistently, QOC1 shows higher resistance to acidic, alkaline and gastric stress. In conclusion, our results show that strain QOC1 and QOC2 are distinct and did not recently evolve from a common ancestor.

Microbiological quality of milk in Tanzania: from Maasai stable to African consumer table.

In Tanzania, pastoralists such as the Maasai and small urban farmers are responsible for the country's milk production, and 95% of the national milk supply is sold without regulation. This study was conducted using hygiene checklists and milk sampling to investigate milk quality and safety at various steps throughout the milk production chain. In regions of Dar es Salaam and Lake Victoria, 196 milk samples were collected: 109 samples of raw milk, 41 samples of packed or open served heat-treated products, and 46 samples of fermented products. Samples were taken from (i) the production level (pastoralists and urban farmers), (ii) the collection level (middlemen and depots), (iii) processors (dairies), and (iv) retailers (kiosks). Samples were analyzed for hygiene criteria (total bacteria, total coliforms, Escherichia coli, and coagulase-positive staphylococci) and foodborne pathogens such as Salmonella, enterohemorrhagic E. coli O157:H7, and Listeria monocytogenes. Adequate heating of milk for drinking was determined via heat labile alkaline phosphatase and lactoperoxidase analysis. Total bacterial counts indicated that only 67% (73 of 109) of raw milk samples and 46% (19 of 41) of heat-treated samples met national Tanzanian standards. Bulk milk samples taken from the traditional milking vessels of Maasai pastoralists had the lowest total bacterial counts: ≥ 1 × 10(2) CFU/ml. Foodborne pathogens such as E. coli O157:H7 and Salmonella were isolated from 10.1% (11 of 109) of raw milk samples but were not detected in heat-treated or fermented products, and 83% of heat-treated milk samples were lactoperoxidase negative, indicating overpasteurization. Coliforms were detected in 41% (17 of 41) of processed milk samples, thus indicating a high rate of recontamination. A progressive decrease in microbial quality along the milk production chain was attributed to departures from traditional methods, inadequate milk containers, long transport distances, lack of cooling, and lack of a basic understanding of hygiene.

Fluctuation in contamination dynamics of L. monocytogenes in quargel (acid curd cheese) lots recalled during the multinational listeriosis outbreak 2009/2010.

For the first time it has been possible to determine the contamination level of Listeria monocytogenes in the very cheese lots of acid curd cheese that caused a multinational outbreak between 2009/2010. The listeriosis outbreak accounted for 34 clinical cases and eight deaths. The cheese, which was distributed in Austria, Germany, the Czech Republic, Poland and Slovakia, was recalled on the 23rd January 2010. All recalled lots were immediately investigated after call back from the retail market. The company manufactured two different cheese types, (i) red smear ripened--and (ii) mold coated/white veined--acid curd cheese. Depending on the lot production dates, cheese samples (n=1045) were analyzed at three different time points: (i) beginning to mid shelf-life (lot nos. 15-18; production period 5.1.2010-13.1.2010); (ii) end of shelf-life (lot nos. 9-18; production period 21.12.2009-13.1.2010) and, (iii) ≤46days after the expiry date (lot nos. 1-18; production period 1.12.2009-13.1.2010). Qualitative and quantitative examinations of cheese samples were performed according to ISO 11290-1&2. Examination of the samples, according to ISO 11290-1, resulted in 16 L. monocytogenes positive (red smear type) and two negative lots (mold coated type). These results were confirmed by a combined enrichment/real-time PCR method. The contamination values obtained by quantitative ISO 11290-2 varied from ≤log 2 cell forming units (CFU)/g to log 8.1CFU/g. Three out of sixteen L. monocytogenes positive lots revealed a contamination level of ≤log 2CFU/g at the beginning of their shelf-life when stored at 4°C. Nevertheless, by increasing the storage life and/or the storage temperature (15, 22°C) the contamination level could be raised to between log 5 and log 6CFU/g. Our data indicate that 81.3% (13/16) of the recalled red smear quargel cheese lots were highly contaminated with L. monocytogenes. All this implies that the main contamination of the quargel cheese took place during the red smear process and that quargel cheese can easily support growth of L. monocytogenes.

Important vectors for Listeria monocytogenes transmission at farm dairies manufacturing fresh sheep and goat cheese from raw milk.

The aim of this study was to determine the transmission routs of Listeria spp. in dairy farms manufacturing fresh cheese made from ovine and caprine raw milk and to evaluate the impact of Listeria monocytogenes mastitis on raw milk contamination. Overall, 5,799 samples, including 835 environmental samples, 230 milk and milk product samples, and 4,734 aseptic half-udder foremilk samples were collected from 53 dairy farms in the dairy intensive area of Lower Austria. Farms were selected for the study because raw milk was processed to cheese that was sold directly to consumers. A total of 153 samples were positive for Listeria spp., yielding an overall prevalence of 2.6%; L. monocytogenes was found in 0.9% of the samples. Bulk tank milk, cheese, and half-udder samples were negative for Listeria spp. Because none of the sheep and goats tested positive from udder samples, L. monocytogenes mastitis was excluded as a significant source of raw milk contamination. L. monocytogenes was detected at 30.2% of all inspected farms. Swab samples from working boots and fecal samples had a significantly higher overall prevalence (P < 0.001) of L. monocytogenes (15.7 and 13.0%, respectively) than did swab samples from the milk processing environment (7.9%). A significant correlation was found between the prevalence of L. monocytogenes in the animal and in the milk processing environment and the silage feeding practices. Isolation of L. monocytogenes was three to seven times more likely from farms where silage was fed to animals throughout the year than from farms where silage was not fed to the animals.

Melamine milk powder and infant formula sold in East Africa.

This is the first study proving the existence of melamine in milk powder and infant formula exported to the African market. A total of 49 milk powder batches were collected in Dar-es-Salaam (Tanzania, East Africa), the center of international trade in East Africa, which serves as a commercial bottleneck and shipment hub for sub-Saharan, Central, and East Africa. Two categories of samples were collected between October and December 2008, immediately after the melamine contamination of Chinese products became public: (i) market brands of all international companies supplying the East African market and (ii) illegally sold products from informal channels. Melamine concentration was determined with the AgraQuant Melamine Sensitive Assay. Despite the national import prohibition of Chinese milk products and unlabeled milk powder in Tanzania, 11% (22 of 200) of inspected microretailers sold milk powder on the local black market. Manufacturers could be identified for only 55% (27) of the 49 investigated batches. Six percent (3 of 49) of all samples and 11% (3 of 27) of all international brand name products tested revealed melamine concentrations up to 5.5 mg/kg of milk powder. This amount represents about twice the tolerable daily intake as suggested by the U.S Food and Drug Administration. Based on our study, we can assume that the number of affected children in Africa is substantial.