Occurrence of mycotoxins in feedstuffs of dairy cows and estimation of total dietary intakes.

A survey was conducted to determine the occurrence of mycotoxins in feedstuffs of dairy cows in the Netherlands and to estimate total dietary intakes of these compounds. Twenty-four dairy farms were visited twice and samples taken of all diet ingredients. Feed intake data were collected by means of questionnaires. A total of 169 feed samples were collected and analyzed for 20 mycotoxins using a liquid chromatography tandem mass spectrometry multimethod. Silage and compound feed were the main diet ingredients, representing on average 67 and 23% of dry matter intake, respectively. Deoxynivalenol (DON), zearalenone, roquefortine C, and mycophenolic acid were the mycotoxins with the highest incidence. The incidence of DON in silage, compound feed, and feed commodity samples was 38 to 54%. The incidence of zearalenone in silage, compound feed, and feed commodity samples was 17 to 38%. The DON and zearalenone had a low incidence in forage samples and were not detected in ensiled by-product samples. Roquefortine C and mycophenolic acid were only detected in silage and ensiled by-product samples (incidence 7 to 19%). Fumonisins B(1) and B(2) were detected in 2 compound feed samples and one feed commodity sample. Aflatoxins B(1), B(2), G(1), and G(2), ochratoxin A, T-2 and HT-2 toxin, 3-acetyl-DON, 15-acetyl-DON, diacetoxyscirpenol, sterigmatocystin, fusarenon-X, ergotamine, and penicillinic acid were not detected in any of the samples. Average concentrations of DON, zearalenone, roquefortine C, and mycophenolic acid in complete diets were 273, 28, 114, and 54 microg/kg, respectively. Maximum concentrations were 969, 203, 2,211, and 1,840 microg/kg, respectively. Calculated average daily intakes of these mycotoxins were 5.0, 0.5, 2.0, and 0.9 mg/animal, respectively, and maximum daily intakes 19.3, 3.5, 38.9, and 32.3 mg/animal, respectively. Corn silage was the major source of all 4 of these mycotoxins in the diet. Extremely high concentrations of roquefortine C and mycophenolic acid (up to 45 and 25 mg/kg, respectively) were detected in visibly molded areas in surface layers of corn silage. These areas appeared to be the main source of roquefortine C and mycophenolic acid in the diet. Because carry-over of DON, zearale-none, roquefortine C, and mycophenolic acid into milk is negligible, their occurrence in feedstuffs is not considered of significant concern with respect to the safety of dairy products for consumers. Potential implications for animal health are discussed.

Occurrence of mycotoxins in maize, grass and wheat silage for dairy cattle in the Netherlands.

The occurrence of mycotoxins in 140 maize silages, 120 grass silages and 30 wheat silages produced in the Netherlands between 2002 and 2004 was determined using a liquid chromatography coupled with tandem mass spectrometry detection (LC-MS/MS) multi-method. Deoxynivalenol (DON) was detected above the limit of quantification (LOQ) of 250 µg kg⁻¹ in 72% of maize and 10% of wheat silages. Average DON concentrations were 854 and 621 µg kg⁻¹, respectively, and maximum concentrations 3142 and 1165 µg kg⁻¹, respectively. Zearalenone was detected above the LOQ of 25 µg kg⁻¹ in 49% of maize and 6% of grass silages. Average zearalenone concentrations were 174 and 93 µg kg⁻¹, respectively, and maximum concentrations 943 and 308 µg kg⁻¹, respectively. The incidences and average concentrations of DON and zearalenone in maize silage were highest in 2004. The incidence of other mycotoxins was low: fumonisin B1 and 15-acetyl-DON were detected in 1.4 and 5% of maize silages, respectively, and roquefortin C in 0.8% of grass silages. None of the silages contained aflatoxins, ochratoxin A, T2-toxin, HT2-toxin, sterigmatocystin, diacetoxyscirpenol, fusarenon-X, ergotamine, penicillinic acid, or mycophenolic acid. This study demonstrates that maize silage is an important source of DON and zearalenone in the diet of dairy cattle. Since the carryover of these mycotoxins into milk is negligible, their occurrence in feed is not considered to be of significant concern with respect to the safety of dairy products for consumers. Potential implications for animal health are discussed.

Short communication: Quantification of the transmission of microorganisms to milk via dirt attached to the exterior of teats.

Pathogens and spoilage microorganisms can be transmitted to milk via dirt (e.g., feces, bedding material, soil, or a combination of these) attached to the exterior of the cows' teats. To determine the relevance of this pathway and to perform quantitative microbial risk analysis of the microbial contamination of farm tank milk (FTM), it is important to know the amount of dirt transmitted to milk via the exterior of teats. In this study at 11 randomly selected Dutch farms the amount of dirt transmitted to milk via the exterior of teats is determined using spores of mesophilic aerobic bacteria as a marker for transmitted dirt. The amount of transmitted dirt to milk varied among farms from approximately 3 to 300 mg/L, with an average of 59 mg/L. The usefulness of the data for microbial risk analyses is briefly illustrated using the contamination of FTM with spores of butyric acid bacteria as a case study. In a similar way the data can be used to identify measures to control the contamination of FTM with other microorganisms or chemical residues.

Minimizing the level of butyric acid bacteria spores in farm tank milk.

A year-long survey of 24 dairy farms was conducted to determine the effects of farm management on the concentrations of butyric acid bacteria (BAB) spores in farm tank milk (FTM). The results were used to validate a control strategy derived from model simulations. The BAB spore concentrations were measured in samples of FTM, feces, bedding material, mixed corn and grass silage fed to cows in the barn, and soil. In addition, a questionnaire was used to gather farm management information such as bedding material used and teat cleaning method applied. The average BAB spore concentration in FTM was 2.7 log10 spores/L, and 33% of the FTM samples exceeded a concentration of 3 log10 spores/L. Control of the average spore concentration in mixed silage fed was the only aspect of farm management that was significantly related to the concentration of BAB spores in FTM. Farms that fed mixed silage with the lowest average BAB spore concentrations (3.4 log10 spores/g) produced FTM with the lowest average concentration (2.1 log10 spores/L). The efficiency of farm management in controlling the BAB spore concentration in FTM depended to a large extent on the ability of farmers to prevent incidents with elevated BAB spore concentrations in mixed silage (>5 log10 spores/g) and not on the average BAB spore concentration in mixed silage across the year. The survey showed that farmers should aim for a concentration in mixed silage of less than 3 log10 spores/g and should prevent the concentration from exceeding 5 log10 spores/g to ensure a concentration in FTM of less than 3 log10 spores/L. These results correspond with the previously reported model simulations.

Minimizing the level of Bacillus cereus spores in farm tank milk.

In a year-long survey on 24 Dutch farms, Bacillus cereus spore concentrations were measured in farm tank milk (FTM), feces, bedding material, mixed grass and corn silage, and soil from the pasture. The aim of this study was to determine, in practice, factors affecting the concentration of B. cereus spores in FTM throughout the year. In addition, the results of the survey were used in combination with a previously published modeling study to determine requirements for a strategy to control B. cereus spore concentrations in FTM below the MSL of 3 log10 spores/L. The B. cereus spore concentration in FTM was 1.2 +/- 0.05 log10 spores/L and in none of samples was the concentration above the MSL. The spore concentration in soil (4.9 +/- 0.04 log10 spores/g) was more than 100-fold higher than the concentration in feces (2.2 +/- 0.05 log10 spores/g), bedding material (2.8 +/- 0.07 log10 spores/g), and mixed silage (2.4 +/- 0.07 log10 spores/g). The spore concentration in FTM increased between July and September compared with the rest of the year (0.5 +/- 0.02 log10 spores/L difference). In this period, comparable increases of the concentrations in feces (0.4 +/- 0.03 log10 spores/g), bedding material (0.5 +/- 0.05 log10 spores/g), and mixed silage (0.4 +/- 0.05 log10 spores/g) were found. The increased B. cereus spore concentration in FTM was not related to the grazing of cows. Significant correlations were found between the spore concentrations in FTM and feces (r = 0.51) and in feces and mixed silage (r = 0.43) when the cows grazed. The increased concentrations during summer could be explained by an increased growth of B. cereus due to the higher temperatures. We concluded that year-round B. cereus spores were predominantly transmitted from feeds, via feces, to FTM. Farmers should take measures that minimize the transmission of spores via this route by ensuring low initial contamination levels in the feeds (<3 log10 spores/g) and by preventing growth of B. cereus in the farm environment. In addition, because of the extremely high B. cereus spore concentrations in soil, the contamination of teats with soil needs to be prevented.

Concentrations of butyric acid bacteria spores in silage and relationships with aerobic deterioration.

Germination and growth of spores of butyric acid bacteria (BAB) may cause severe defects in semihard cheeses. Silage is the main source of BAB spores in cheese milk. The objectives of the study were to determine the significance of grass silages and corn silages as sources of BAB spores and to investigate the relationships between high concentrations of BAB spores in corn silage and aerobic deterioration. In the first survey, samples were taken from various locations in silos containing grass and corn silages and from mixed silages in the ration offered to the cows on 21 farms. We demonstrated that the quantity of BAB spores consumed by cows was determined by a small fraction of silage with a high concentration of spores (above 5 log10 BAB/g). High concentrations were most often found in corn silage within areas with visible molds (69% of the samples). Areas with visible molds in grass silage and surface layers of corn silage contained, respectively, 21 and 19% of the cases of concentrations above 5 log10 BAB spores/g. Based on these results, we concluded that currently in the Netherlands, corn silage is a more important source of BAB than is grass silage. In a second survey, 8 corn silages were divided into 16 sections and each section was studied in detail. High concentrations of BAB spores were found in only the top 50 cm of these 8 silages. Elevated concentrations of BAB spores were associated with different signs of aerobic deterioration. In 13% of the sections in corn silage with more than 5 log10 yeasts and molds/g, more than 5 log10 BAB spores/g were found. Sections with a temperature of more than 5 degrees C above ambient temperature contained, in 21% of the cases, more than 5 log10 BAB spores/g. Concentrations above 5 log10 BAB spores/g were measured in 50% of the sections with a pH above 4.4. All sections with a pH above 4.4 also showed a temperature that was more than 5 degrees C above ambient temperature and a concentration of yeasts and molds above 5 log10 cfu/g. Based on these results, we postulated that high concentrations of BAB spores in corn silage are the result of oxygen penetration into the silage, resulting in aerobic deterioration and the formation of anaerobic niches with an increased pH just below the surface. Growth of BAB in these anaerobic niches with an increased pH caused the locally high concentrations of BAB in corn silage.

Predictive modeling of Bacillus cereus spores in farm tank milk during grazing and housing periods.

The shelf life of pasteurized dairy products depends partly on the concentration of Bacillus cereus spores in raw milk. Based on a translation of contamination pathways into chains of unit-operations, 2 simulation models were developed to quantitatively identify factors that have the greatest effect on the spore concentration in milk. In addition, the models can be used to determine the reduction in concentration that could be achieved via measures at the farm level. One model predicts the concentration when soil is the source of spores, most relevant during grazing of cows. The other model predicts the concentration when feed is the main source of spores, most relevant during housing of cows. It was estimated that when teats are contaminated with soil, 33% of the farm tank milk (FTM) contains more than 3 log(10) spores/L of milk. When feed is the main source, this is only 2%. Based on the predicted spore concentrations in FTM, we calculated that the average spore concentration in raw milk stored at the dairy processor during the grazing period is 3.5 log(10) spores/L of milk and during the housing period is 2.1 log(10) spores/L. It was estimated that during the grazing period a 99% reduction could be achieved if all farms minimize the soil contamination of teats and teat cleaning is optimized. During housing, reduction of the concentration by 60% should be feasible by ensuring spore concentrations in feed below 3 log(10) spores/g and a pH of the ration offered to the cows below 5. Implementation of these measures at the farm level ensures that the concentration of B. cereus spores in raw milk never exceeds 3 log(10) spores/L.

Improving farm management by modeling the contamination of farm tank milk with butyric acid bacteria.

Control of contamination of farm tank milk (FTM) with the spore-forming butyric acid bacteria (BAB) is important to prevent the late-blowing defect in semi-hard cheeses. The risk of late blowing can be decreased via control of the contamination level of FTM with BAB. A modeling approach was applied to identify an effective control strategy at the farm level. The simulation model developed was based on a translation of the contamination pathway into a chain of unit operations. Using various simulations, the effects of factors related to feed quality, feed management, cattlehouse hygiene, and milking practices on the contamination level of FTM were evaluated. Contamination level of silage was found to be the most important factor. When silage contains on average less than 3 log10 BAB/g, a basic pretreatment of udder teats before milking (approximately 75% removal of attached spores) is sufficient to assure an FTM contamination level below 1 BAB/mL. When silage contains more than 5 log10 BAB/g, it should not be fed, because it then becomes almost impossible to assure an FTM contamination level below 1 BAB/mL. Measures aimed at improving cattlehouse hygiene, the contamination via soil, and the contamination level of other feeds contribute only marginally to the control of the contamination of FTM with BAB. Application of the modeling methodology could be beneficial for the control of the contamination of FTM with other microorganisms such as Bacillus cereus.

Bacterial spores in silage and raw milk.

Spore-forming bacteria can survive food-processing treatments. In the dairy industry, Bacillus and Clostridium species determine the shelf-life of a variety of heat-treated milk products, mainly if the level of post-process contamination is low. In order to minimize problems caused by bacterial spores in foods and food production processes a chain management approach, from raw materials, ingredients and environmental sources to final product storage conditions, is most effective. Silage is considered to be a significant source of contamination of raw milk with spores. PCR-RAPD fingerprinting and heat resistance studies of populations of aerobic spore-formers isolated from grass and maize silage and from raw milk confirmed this assumption. Prevention of outgrowth of aerobic spores in silage will contribute to reduction of the total spore load of raw milk. Therefore, it is important that the silage fermentation process is controlled. Application of cultures of lactic acid bacteria or chemical additives can aid silage fermentation and improve aerobic stability.

Prediction of the adherence, growth and release of microorganisms in production chains.

The aim of this study was to develop a mathematical model that describes the bacterial contamination of food as a result of adherence, growth and release of bacteria in process equipment. The model developed can be applied to control the bacterial quality of food products produced in process chains in which the final contamination of the product is governed by growth and heat-induced destruction of bacteria. To set up the model, experiments were carried out with a plate heat exchanger using milk inoculated with Streptococcus thermophilus. The growth rate of S. thermophilus in milk could be described accurately by the modified expanded model of Ratkowsky. The observed increase in the concentration of S. thermophilus in milk at the outlet of the plate heat exchanger could be described quantitatively by the model. To predict the contamination of the product, the model was integrated into NIZO-PCS (Process Chain Simulator). The results of computer simulations were validated by a number of measurements in a cheese factory. It turned out that the agreement between the measured and calculated concentrations of S. thermophilus was sufficient for the model to be used for predictions in industrial production chains.

Stepwise quantitative risk assessment as a tool for characterization of microbiological food safety.

This paper describes a system for the microbiological quantitative risk assessment for food products and their production processes. The system applies a stepwise risk assessment, allowing the main problems to be addressed before focusing on less important problems. First, risks are assessed broadly, using order of magnitude estimates. Characteristic numbers are used to quantitatively characterize microbial behaviour during the production process. These numbers help to highlight the major risk-determining phenomena, and to find negligible aspects. Second, the risk-determining phenomena are studied in more detail. Both general and/or specific models can be used for this and varying situations can be simulated to quantitatively describe the risk-determining phenomena. Third, even more detailed studies can be performed where necessary, for instance by using stochastic variables. The system for quantitative risk assessment has been implemented as a decision supporting expert system called SIEFE: Stepwise and Interactive Evaluation of Food safety by an Expert System. SIEFE performs bacterial risk assessments in a structured manner, using various information sources. Because all steps are transparent, every step can easily be scrutinized. In the current study the effectiveness of SIEFE is shown for a cheese spread. With this product, quantitative data concerning the major risk-determining factors were not completely available to carry out a full detailed assessment. However, this did not necessarily hamper adequate risk estimation. Using ranges of values instead helped identifying the quantitatively most important parameters and the magnitude of their impact. This example shows that SIEFE provides quantitative insights into production processes and their risk-determining factors to both risk assessors and decision makers, and highlights critical gaps in knowledge.

Validation of predictive models describing the growth of Listeria monocytogenes.

In this study, predictions for growth rate of Listeria on food products were evaluated by both general applicable models and specific growth models. Literature values, obtained from a large number of publications, for growth rates in/on a variety of foods were compared by graphical and mathematical analysis with predictions given by various models. Apart for the great advantage of being generally applicable, the general models performed best. However, only small differences between the various models were observed. Model predictions were accurate within a factor of about two to four, depending on the type of product. The predictions should therefore not be considered as absolute; it is important to understand the limitations of the performance of models. All results and all assumptions should be criticised, but in many cases the accuracy will be sufficient to use these types of models as a tool in management decisions.

[Isolation, identification and characterization of Bacillus cereus in the dairy industry]. / Isolatie, identificatie en karakterisering van Bacillus cereus in de zuivelindustrie.

In order to determine the major contamination sources of milk with (psychrotrophic) Bacillus cereus, the incidence of vegetative cells and spores of B. cereus on dairy farms, at two dairy processing plants and in pasteurized milk in household refrigerators was investigated. On dairy farms the major contamination sources were soil and faeces. In winter, when the cows were housed, used bedding probably also participates in this contamination route. The udder will be contaminated, finally resulting in the presence of B. cereus in raw milk. The organism could be detected in 35% of the raw milk samples analyzed. During processing, an increase in the percentage of positive samples was observed. These results suggest that B. cereus can be introduced via sources other than raw milk; equipment may play an important role in this. Biochemical and molecular typing showed that selection of strains takes place in the milk production chain. It was demonstrated that some types were found in the raw milk, during processing and in the end products, indicating that raw milk is an important source of contamination. Other types could only be detected after the pasteurization step in the production process supporting the assumption that additional contamination occurs during processing. If stored under proper conditions, maximum storage temperature 7 degrees C, and consumed within the expiration date, the levels of B. cereus in pasteurized milk will, in general, not exceed 10(5) per ml and cause no problems for healthy adults.

Occurrence and survival of verocytotoxin-producing Escherichia coli O157 in raw cow's milk in The Netherlands.

From May through November 1997, 1,011 samples of raw milk from bulk storage tanks were examined for the presence of verocytotoxin-producing Escherichia coli of serogroup O157 (O157 VTEC) by immunomagnetic separation following selective enrichment. The samples originated from 1,011 different dairy herds located throughout the Netherlands. O157 VTEC was not isolated from any of the milk samples examined. Additionally, survival of O157 VTEC in raw and UHT-sterilized cow's milk at 7 and 15 degrees C was studied, both in the absence and presence of an activated lactoperoxidase-thiocyanate-hydrogen peroxide system (LPS). Results indicated that the O157 VTEC strain tested was able to grow in raw milk at 7 degrees C as well as at 15 degrees C. Naturally occurring amounts of thiocyanate and hydrogen peroxide in the raw milk tested were not sufficient to activate the LPS. Although the LPS exhibited an antimicrobial activity against O157 VTEC in LPS-activated sterilized milk, O157 VTEC populations were not (or not as obviously) reduced in LPS-activated raw milk. Possibly background microflora were more sensitive to the LPS than the O157 VTEC test strain. It was concluded that raw milk contaminated with O157 VTEC will remain a hazard if kept at 7 or 15 degrees C. Effective pasteurization and avoiding postpasteurization contamination are necessary to ensure the safety of milk.

Optimization of haemolysis in enhanced haemolysis agar (EHA)--a selective medium for the isolation of Listeria monocytogenes.

The presence of Listeria monocytogenes in enrichment media can be masked by faster growth of other Listeria spp. Therefore, enhanced haemolysis agar (EHA) is a good alternative for another isolation media, because the presence of a few L. monocytogenes colonies can be detected in a majority of colonies of other listeriae on the basis of haemolysis. In this study the haemolysis reaction in EHA was optimized. In a collaborative study using reference samples, no significant differences in counts on EHA, Palcam and Oxford agar were shown.

Isolation and characterisation of Bacillus cereus from pasteurised milk in household refrigerators in The Netherlands.

The incidence and some characteristics (carbohydrate metabolism, growth profiles, haemolysin production and enterotoxin production) of Bacillus cereus, in pasteurised, low-fat (1.5%) milk, in household refrigerators in the Netherlands was investigated. In 247 (74%) of the 334 milk samples analyzed, the mesophilic aerobic counts were between 50 and 5000 per millilitre. B. cereus could be isolated from 133 (40%) of the samples. In general the B. cereus counts were low; numbers of less than five per millilitre were observed in 258 (77%) of the samples. As expected, both the mesophilic aerobic counts and levels of B. cereus increased with increasing storage temperatures in the refrigerator and prolonged storage times. In total, 143 presumptive B. cereus colonies were isolated. According to the ISO confirmation tests and the carbohydrate patterns (API 50 CHB) 134 (94%) of these isolates were confirmed to be B. cereus. Of these 134 isolates 20% fermented lactose and 53% of the 106 strains tested were able to grow at 7 degrees C. These percentages are much higher than expected for strains isolated from non-dairy products, suggesting that strains can adapt to environmental conditions in milk. All 106 strains tested, produced haemolysin, 27% showed the discontinuous haemolytic pattern characteristic for haemolysin BL, possibly a virulence factor. Of the 37 B. cereus isolates tested for enterotoxin production 27 (73%), 28 (76%) and 26 (70%) were found to be enterotoxigenic (as determined by the Western immunoblot technique, polymerase chain reaction (PCR) and Vero cell assays, respectively). Isolates unable to ferment lactose, produced less enterotoxin in comparison with those able to utilize lactose. Although only a few outbreaks of food poisoning caused by B. cereus in milk (products) have been reported, most strains isolated from these products are able to produce enterotoxins and may represent a health hazard.

Discrimination between Bacillus cereus and Bacillus thuringiensis using specific DNA probes based on variable regions of 16S rRNA.

Identification of Bacillus cereus and differentiation between B. cereus and closely related species are currently based on biochemical tests. The main problem is to discriminate between B. cereus and B. thuringiensis. Sequencing part of the 16S rRNA showed that several B. cereus isolates present in food and involved in food poisoning, confirmed according to the classical biochemical methods, were in fact B. thuringiensis. As this organism is the most commonly used microbial insecticide worldwide, the results of this study emphasize the need for accurate identification methods and for careful screening of strains for use as insecticides. Therefore, specific DNA probes based on the variable region VI of 16S rRNA of B. cereus and B. thuringiensis were designed. The probes were used in hybridization experiments with the variable region amplified using the polymerase chain reaction. In this way, a rapid and sensitive method was developed to distinguish B. cereus and B. thuringiensis.

Listeria species in domestic environments.

Using a direct isolation method Listeria spp. were detected in 101 (47.4%) of 213 houses investigated. L. monocytogenes was present in 45 houses (21.1%). Listeria spp. occurred at all sampling sites. Dish-cloths (37%) and surface samples round the drain in the bathroom (27.2%) were most frequently contaminated. Highest numbers (c. 10(4) c.f.u./object) were found in dish-cloths and washing-up brushes. Lower levels (up to 10(3) c.f.u./object) were obtained from kitchen sinks, refrigerator vegetable compartment samples and tooth brushes. In total, 132 isolations of Listeria spp. were made from 871 samples. L. innocua (53%) and L. monocytogenes (41%) were the predominant species in the positive samples. Other Listeria spp. were found in only 6% of the positive samples.

Confirmation and identification of Listeria spp.

All confirmation and identification methods used in this study can be used for the screening of suspected colonies on isolation media for Listeria spp. In traditional enrichment procedures the Microscreen Listeria latex test gives fast results. The DNA probes (Accuprobe and Gene-Trak) are very specific in detecting Listeria monocytogenes. For identification of Listeria spp. both tests (API and Micro-ID) performed equally well. Preference may be given to the API test, since differentiation of L. monocytogenes from L. innocua is based on the absence of arylamidase, through which tests for haemolytic activity and/or CAMP reactions can be omitted. However, the use of Enhanced Haemolysis Agar as isolation medium makes further testing essentially superfluous, since L. monocytogenes strains can be differentiated from L. innocua.