The occurrence of fumonisin B1 in maize-containing foods in The Netherlands.

Seventy-eight maize-containing foods obtained from retail stores in The Netherlands were analysed for fumonisin B1 contamination. Thirty-six per cent of the samples were contaminated with fumonisin B1 in the range of 8 micrograms kg-1 (limit of detection) to 1430 micrograms/kg-1. Forty-six per cent of the minimally treated maize samples (n = 39; maize for bread production, maize for popcorn, maize flour and polenta) were contaminated with fumonisin B1 in the range of 8-380 micrograms kg-1. Twenty-six per cent of the maize-containing processed foods (n = 39; tostada, canned maize, maize starch, maize bread, popped maize, flour mixes, maize chips and cornflakes) were contaminated with fumonisin B1 in the range of 8-1430 micrograms/kg-1. This survey shows that maize-containing foods in The Netherlands frequently can be contaminated with fumonisin B1.

Fumonisin B1 in maize for food production imported in The Netherlands.

Sixty-two samples of maize imported in The Netherlands and intended for human consumption were screened for the presence and concentration of fumonisin B1. Sixty-one of those samples contained fumonisin B1 with concentrations ranging from 30 to 3350 micrograms kg-1, 11 maize samples contained > 1000 micrograms kg-1. The average fumonisin B1 concentration was 640 micrograms kg-1 for the positive samples and 620 micrograms kg-1 for all samples. Medians were 600 micrograms kg-1 and 550 micrograms kg-1 for positive and all samples, respectively. The results obtained were comparable to results from other studies in maize from various countries.

Assessment of human exposure to fumonisin B1.

Fumonisin B1 is currently regarded as the most significant mycotoxin produced by Fusarium spp. It has carcinogenic properties and may play a role in the etiology of human esophageal cancer. The human population is exposed to fumonisin B1 primarily by intake of fumonisin B1-contaminated maize. Maize consumed in the Netherlands is imported from all parts of the world. Since processing will not affect the overall toxic effect, the fumonisin B1 intake is directly related to the quantity of maize consumed. Literature results concerning the occurrence of fumonisin B1 in a total of 349 samples of maize from 18 countries worldwide demonstrated the presence of this mycotoxin in 93% of the samples. The median fumonisin B1 contamination of all samples was 420 ng of fumonisin B1 per g of maize, and the average contamination level was 1,359 ng of fumonisin B1 per g of maize. Human intake of fumonisin B1 was estimated based on the maize consumption of all people in the Netherlands in 1992. A probability distribution was derived to allow estimation of the exposure of the population to fumonisin B1 intake in relation to maize intake. It showed that among those in the group considered to be at risk, people with gluten intolerance such as people with celiac or Dühring's disease, 37% are estimated to be exposed to an intake of at least 10(5) ng and 97% to an intake of at least 10(3) ng of fumonisin B1 per person per day. For all people in the Netherlands these percentages would be 1% and 49%, respectively.

Towards internationally acceptable standards for food additives and contaminants based on the use of risk analysis.

Internationally acceptable norms need to incorporate sound science and consistent risk management principles in an open and transparent manner, as set out in the Agreement on the Application of Sanitary and Phytosanitary Measures (the SPS Agreement). The process of risk analysis provides a procedure to reach these goals. The interaction between risk assessors and risk managers is considered vital to this procedure. This paper reports the outcome of a meeting of risk assessors and risk managers on specific aspects of risk analysis and its application to international standard setting for food additives and contaminants. Case studies on aflatoxins and aspartame were used to identify the key steps of the interaction process which ensure scientific justification for risk management decisions. A series of recommendations were proposed in order to enhance the scientific transparency in these critical phases of the standard setting procedure.

Features of the Guillain-Barré syndrome in mice following intraperitoneal injection of patient serum.

Intraperitoneal injection of serum from a patient with the Guillain-Barré syndrome (GBS) produced GBS-like signs in mice: inadequate respiration and weakness in the legs. We studied the clinical, electrophysiological and pathological features of these mice. Three groups of three mice were injected with patient serum from days 6, 10 and 15 after onset of neurological symptoms. GBS-like signs in mice were observed only with serum from day 6 and improved within 48 h. When serum was frozen and thawed more than once no signs were seen. Electrophysiological measurements of the sciatic nerves of injected and control mice were done before and after serum injection. Five days after injection of patient serum of day 6, the mice showed a significant decrease in the ratio between CMAP amplitude from proximal and distal stimulation and increase in H-M interval from proximal stimulation. These electrophysiological changes returned to normal within 12 days. The sciatic nerve showed no morphological abnormalities. Our results indicate that the observed GBS-like signs in mice are caused by peripheral nerve dysfunction.

2-O-methyl-D-mannose residues are immunodominant in extracellular polysaccharides of Mucor racemosus and related molds.

In this study, the structure of the immunodominant carbohydrate epitope of the extracellular polysaccharides from mold species belonging to the order Mucorales reactive with rabbit IgG antibodies was elucidated. An exo-alpha-D-mannanase which was able to abolish the antigenicity of these polysaccharides completely was purified and characterized, and the activity was compared with that of an alpha-D-mannosidase. Analysis of the monomeric reaction products after enzymatic treatment revealed the presence of 2-O-methyl-D-mannose residues. This compound is a constituent of the polysaccharides from the mold genera Mucor, Rhizopus, Rhizomucor, Absidia, Syncephalastrum, and Thamnidium, and its occurrence in fungi has not been reported until now. Two mannan fractions which are highly reactive with rabbit IgG were isolated from the extracellular polysaccharides of Mucor racemosus and characterized with ethylation analysis. The role of the newly found 2-O-methyl-D-mannose residues in the immunoreactivity was assessed by specific degradation of these mannans with the exo-alpha-D-mannanase and subsequent ethylation analysis. It was concluded that the immunodominant carbohydrates reactive with rabbit IgG are chains composed of a single terminal non-reducing 2-O-methyl-D-mannose residue, alpha (1-2)-linked to a short sequence of alpha(1-2)-linked D-mannose residues.

Intervention strategies for Salmonella enteritidis in poultry flocks: a basic approach.

Poultry laying flocks can become infected with Salmonella enteritidis by several routes. In this study a model is presented of the cumulative infection curve of S. enteritidis in laying flocks. Based on this model and practical results the contribution of different routes to the infection can be estimated providing a basis for an effective intervention strategy. For illustration, the cumulative infection curve of S. enteritidis in Dutch laying flocks is analysed. This curve shows a low level of infection at the start of the laying period which indicates that the contribution of the vertical infection route (from infected breeding flocks to progeny) is small. The course of the infection curve indicates that there is a high probability of infection in the first part of the laying period. The result suggests that the laying flocks become infected mainly from the farm environment including not properly cleaned and disinfected poultry houses and infected vermin present on the farm. As a consequence, intervention in The Netherlands should be directed to trace S. enteritidis-contaminated laying farms and eradicate the contamination.

Properties of Bacillus cereus spores in reference materials prepared from artificially contaminated spray dried milk.

A reference material for Bacillus cereus was developed based on spray drying of milk artificially contaminated with B. cereus spores. Various properties of the B. cereus spores in the milk powder were determined. The stability of the materials was good with no detectable decrease in the contamination level during 1 1/2 years storage at -20 degrees C or 4 weeks at 22, 30 or 37 degrees C. The homogeneity of the material was found acceptable for use as a reference material. Heat treatments (10 min at 70 or 80 degrees C) and addition of lysozyme to the enumeration medium did not influence the number of spores counted. The germination of the spores depended on the type of medium in which the milk powder was reconstituted, and on the storage period of the material. The suitability of the material was confirmed in a collaborative study. From the results obtained it was concluded that the material developed meets the general requirements set for reference materials and can therefore be used for, among others, testing laboratory performance.

The production and partial characterization of a monoclonal IgG antibody specific for moulds belonging to the order Mucorales.

A monoclonal antibody (mAb) was raised against extracellular polysaccharides from Mucor racemosus after intrasplenic immunization of mice. An indirect ELISA and a dot-blot assay were developed with this mAb. The IgG antibody was found to be very specific for all mould species tested belonging to the order of Mucorales, except species belonging to the genus Mortierella sensu stricto. No cross-reactions were observed with other moulds or yeasts. The immunoreactivity of the polysaccharides of these moulds with this mAb is based on carbohydrate epitopes, in which fucose residues probably play an important role. The mAb may be suited for specific detection of species of the genera Mucor, Rhizopus, Rhizomucor, Thamnidium, Absidia, Syncephalastrum and species belonging to the Mortierella isabellina group in food, and possibly for diagnosis of mucormycosis in humans.

The identification of Salmonella enteritidis-infected poultry flocks associated with an outbreak of human salmonellosis.

In the summer of 1991 a human outbreak of Salmonella enteritidis infection occurred following a barbecue in which about 100 persons were involved. Eggs, supplied by one or more of 10 different layer farms, were the most probable source of the infection. To identify the S. enteritidis-positive flocks, an immunoassay was used to detect salmonella serogroup D-specific antibodies in the yolk of hens eggs. Antibody titres in the eggs from two layer farms, farm A and B, clearly exceeded the titres found in randomly collected eggs. Further investigation on farm A and B yielded high antibody titres in the eggs from flocks A1, A2 and B2, and low titres in the eggs from flock B1. S. enteritidis was isolated from the faecal samples of flocks A1, A2 and B2, whereas no salmonella was detected in the faecal samples of flock B1. The flocks present on both farms originated from the same breeder flock.

Formation of antigenic extracellular polysaccharides by selected strains of Mucor spp., Rhizopus spp., Rhizomucor spp., Absidia corymbifera and Syncephalastrum racemosum.

In this study, polyclonal IgG antibodies raised against extracellular polysaccharides (EPS) of Mucor racemosus were characterised as almost specific for moulds belonging to the order of Mucorales. Cross-reactivity in the ELISA could be observed only towards the yeast Pichia membranaefaciens. EPS were isolated from various cultures of M. hiemalis growing on six different carbon sources and two nitrogen sources, with ratios varying from 0.13 to 0.44 relative to the amount of biomass. Other strains including Mucor spp., Rhizopus spp., Rhizomucor spp., Absidia corymbifera and Syncephalastrum racemosum also excreted EPS, with ratios varying from 0.05 to 0.23. In all cases, the excreted EPS had similar antigenic properties as determined by ELISA. No enzymatic degradation of the antigenic parts of the polysaccharides could be observed upon prolonged incubation. Considering that all tested strains formed similar amounts of antigenic EPS there might be scope for the specific detection of biomass of Mucoralean moulds using ELISA techniques for example in food.

Detection of Aspergillus and Penicillium extracellular polysaccharides (EPS) by ELISA: using antibodies raised against acid hydrolysed EPS.

Species of the fungal genera Aspergillus and Penicillium produce immunologically active extracellular polysaccharides (EPS) in which galactofuranose residues are immunodominant. The antigenic determinant of the EPS of A. fumigatus, A. niger and P. digitatum could be removed by acid hydrolysis. Due to the hydrolysis of the EPS the immunological reaction between IgG anti-native EPS and hydrolysed EPS disappeared. Antibodies raised in rabbits against the acid hydrolysed EPS revealed new antigenic determinants that were exposed as a result of the acid hydrolysis. Immunological inhibitory experiments showed that the antibodies were no longer directed to galactofuranose residues. Enzyme Linked Immunosorbent Assay, carried out with antibodies raised against the acid hydrolysed EPS showed that the antibodies against the acid hydrolysed EPS were more species specific in comparison with the antibodies against the native EPS.

[Mortality of cattle following feeding of moldy flower bulbs]. / Sterfte van rundvee na het voederen van beschimmelde bloembollen.

Five of eight meat cattle died suddenly without showing prior symptoms of disease. The sudden death occurred in connection with the feeding of mouldy tulip bulbs. A short review is given of the use of flower bulbs as cattle feed, the use of herbicides/fungicides in bulb cultivation, and the relevant legislation. Several toxicological aspects that should be taken into consideration when flower bulbs are used as cattle feed are discussed. Both the Central Veterinary Institute and the State Institute for Public Health and Environmental Hygiene showed, in experiments with mice, the presence of a toxin in extracts of the mouldy tulip bulbs. This toxin is probably produced by moulds present in the tulip bulbs. The death of the animals was probably caused by an as yet unidentified mycotoxin.

Phosphatidylinositol-specific phospholipase C activity as a marker to distinguish between pathogenic and nonpathogenic Listeria species.

In this study, 468 Listeria strains were checked for the presence of phosphatidylinositol-specific phospholipase C (PI-PLC) activity by using a simple assay that consisted of overlaying colonies formed on agar plates with L-alpha-phosphatidylinositol as substrate. In this assay, PI-PLC-active colonies show turbid halos around the colonies as a result of the release of insoluble diacylglycerol from the substrate. This activity was detected only in the pathogenic species Listeria monocytogenes and was not present in any of the 167 strains of Listeria seeligeri, Listeria welshimeri, Listeria innocua, Listeria murrayi, and Listeria grayi tested. Hence, screening for PI-PLC activity permits discrimination between pathogenic and nonpathogenic Listeria species. In particular, the hemolytic but nonpathogenic species L. seeligeri can now be separated from the hemolytic and pathogenic species L. monocytogenes and L. ivanovii. The use of this assay will improve the specific detection and/or isolation of pathogenic Listeria species from clinical samples or food enrichment cultures.

Use of the polymerase chain reaction for direct detection of Listeria monocytogenes in soft cheese.

The polymerase chain reaction (PCR) amplification technique was investigated as a tool for direct detection of Listeria monocytogenes in soft cheeses. Different sets of oligonucleotide primers were used, and parts of the L. monocytogenes Dth 18-gene could be amplified specifically when either a plasmid vector carrying the cloned gene or chromosomal DNA was used a template. The detection limit for L. monocytogenes in dilutions of pure cultures was between 1 and 10 colony-forming units. In extracts from soft cheeses containing L. monocytogenes DNA, the amplification was strongly inhibited. This inhibition could be reduced by an additional purification step. Despite this the detection limit showed a large variation, depending on the brand of cheese used. In some cheeses 10(3) cfu/0.5g could be visualized whereas in others the presence of 10(8) cfu/0.5 g did not yield a detectable quantity of amplified product.

Salmonella contamination of poultry flocks in The Netherlands.

The contamination of poultry in the Netherlands with Salmonella enteritidis was tested. For this, different methods (detection of S. enteritidis in faecal samples of 25 g; detection of S. enteritidis in cloacal swabs; detection of S. enteritidis by serological testing of antibodies in serum) were compared for their efficiency to detect S. enteritidis in flocks of poultry. Testing of faecal samples clearly yielded the best results. This method was used in a transmission study, in which 14 flocks descending from a contaminated primary mother flock were screened for the presence of S. enteritidis. The method was also used for screening 49 flocks of laying hens and 52 flocks of broiler chickens throughout the Netherlands. From the transmission study it became clear that S. enteritidis, phage type 2 (Dutch phage set) was isolated both from the mother flock and from five of the descendent flocks. Screening of poultry flocks for the presence of salmonella revealed that salmonella was present in 47% of the layer flocks and in 94% of the broiler flocks. S. enteritidis was isolated from 15% of the flocks screened.

[Botulism]. / Botulisme.

In view of large-scale outbreaks of botulism among waterfowl and farm animals in the Netherlands, studies were done designed to estimate the risks incurred by man. The presence of C. botulinum in the environment as well as contamination cycles and the potential for multiplication were studied. Raw materials used in the production of food were frequently found to be contaminated with types of C. botulinum pathogenic for man. The growth of C. botulinum in foods and meat preservatives other than nitrite were examined. To reduce the use of laboratory animals in research on botulism, immunological methods to detect botulinum toxins were developed.

[Incidence of Clostridium botulinum on cattle farms]. / Voorkomen van Clostridium botulinum op rundveehouderij bedrijven.

Clostridium botulinum, mainly type B, was constantly found to be present on cattle farms. The organism was isolated both from samples of the soil of pastures and from the faeces of cattle during the winter housing period. The number of C. botulinum type B in samples of soil varied from 10 to 300 organisms per 100 grams. Contamination with C. botulinum was found to be of a similar order of magnitude on farms on which pastures are regularly dressed with sewage sludge. C. botulinum was detected in 13 per cent of the faecal samples (420 samples of one gram each). Particularly grass silage pits prepared with wilted grass were found to provide a possible link between contamination of pastures and cattle.