Reduction of Mycobacterium avium ssp. paratuberculosis in colostrum: Development and validation of 2 methods, one based on curdling and one based on centrifugation.

The aim of this study was to develop and validate 2 protocols (for use on-farm and at a central location) for the reduction of Mycobacterium avium ssp. paratuberculosis (MAP) in colostrum while preserving beneficial immunoglobulins (IgG). The on-farm protocol was based on curdling of the colostrum, where the IgG remain in the whey and the MAP bacteria are trapped in the curd. First, the colostrum was diluted with water (2 volumes colostrum to 1 volume water) and 2% rennet was added. After incubation (1 h at 32°C), the curd was cut and incubated again, after which whey and curd were separated using a cheesecloth. The curd was removed and milk powder was added to the whey. Approximately 1 log reduction in MAP counts was achieved. A reduction in total proteins and IgG was observed due to initial dilution of the colostrum. After curd formation, more than 95% of the immunoglobulins remained in the whey fraction. The semi-industrial protocol was based on centrifugation, which causes MAP to precipitate, while the IgG remain in the supernatant. This protocol was first developed in the laboratory. The colostrum was diluted with skimmed colostrum (2 volumes colostrum to 1 volume skimmed colostrum), then skimmed and centrifuged (at 15,600 × g for 30 min at room temperature). We observed on average 1.5 log reduction in the MAP counts and a limited reduction in proteins and IgG in the supernatant. To obtain a semi-industrial protocol, dairy pilot appliances were evaluated and the following changes were applied to the protocol: after 2:1 dilution as above, the colostrum was skimmed and subsequently clarified, after which the cream was heat treated and added to the supernatant. To investigate the effect of the colostrum treatment on the nutritional value and palatability of the colostrum and the IgG transfer, an animal experiment was conducted with 24 calves. Six received the dam's colostrum, 6 were given untreated purchased colostrum (control), and 2 groups of 6 calves received colostrum treated according to both of the above-mentioned methods. No significant differences were found between the test groups and the dam's colostrum group in terms of animal health, IgG uptake in the blood serum, milk, or forage uptake. Two protocols to reduce MAP in colostrum (for use on-farm or at a central location) were developed. Both methods preserve the vital IgG.

Isoepoxydon dehydrogenase (idh) gene expression in relation to patulin production by Penicillium expansum under different temperature and atmosphere.

Penicillium expansum growth and patulin production occur mainly at post-harvest stage during the long-term storage of apples. Low temperature in combination with reduced oxygen concentrations is commonly applied as a control strategy to extend apple shelf life and supply the market throughout the year. Our in vitro study investigated the effect of temperature and atmosphere on expression of the idh gene in relation to the patulin production by P. expansum. The idh gene encodes the isoepoxydon dehydrogenase enzyme, a key enzyme in the patulin biosynthesis pathway. First, a reverse transcription real-time PCR (RT-qPCR) method was optimized to measure accurately the P. expansum idh mRNA levels relative to the mRNA levels of three reference genes (18S, ß-tubulin, calmodulin), taking into account important parameters such as PCR inhibition and multiple reference gene stability. Subsequently, two P. expansum field isolates and one reference strain were grown on apple puree agar medium (APAM) under three conditions of temperature and atmosphere: 20 °C - air, 4 °C - air and 4 °C - controlled atmosphere (CA; 3% O2). When P. expansum strains reached a 0.5 and 2.0 cm colony diameter, idh expression and patulin concentrations were determined by means of the developed RT-qPCR and an HPLC-UV method, respectively. The in vitro study showed a clear reduction in patulin production and down-regulation of the idh gene expression when P. expansum was grown under 4 °C - CA. The results suggest that stress (low temperature and oxygen level) caused a delay of the fungal metabolism rather than a complete inhibition of toxin biosynthesis. A good correlation was found between the idh expression and patulin production, corroborating that temperature and atmosphere affected patulin production by acting at the transcriptional level of the idh gene. Finally, a reliable RT-qPCR can be considered as an alternative tool to investigate the effect of control strategies on the toxin formation in food.

Seafood quality analysis: Molecular identification of dominant microbiota after ice storage on several general growth media.

This study points out the limitations of several general growth media frequently used in seafood research by a systematic identification of the microorganisms on fish samples during ice storage unable to grow on those media. Aerobic psychrotrophic count (APC), replication on various general media and total cultivable microbial community denaturing gradient gel electrophoresis (DGGE) analysis revealed that many potential spoilage microorganisms were overlooked. Those microorganisms overlooked by using only one single growth medium were identified by partial 16S rRNA gene and gyrB gene sequencing. Members of the genera Shewanella, Vibrio, Aliivibrio, Photobacterium, Pseudoalteromonas and Psychrobacter, including Photobacterium phosphoreum, Shewanella baltica and Pseudomonas fluorescens are unable to grow on PCA. APC analysis also confirmed that on plate count agar (PCA) the enumeration of the microbiota was underestimated. Although Long and Hammer agar (LH) and marine agar (MA) obtained the best quantitative (APC analysis) and qualitative (replication and DGGE analyses) results for fish quality analysis, analysts have to keep in mind that some species were also unable to grow on those media, such as Pseudomonas fragi and Acinetobacter sp.

Survival of Mycobacterium avium ssp. paratuberculosis in yoghurt and in commercial fermented milk products containing probiotic cultures.

AIMS: To assess the survival of Mycobacterium avium ssp. paratuberculosis (MAP) in yoghurt and commercial fermented milk products containing probiotic strains. METHODS AND RESULTS: Whole and skimmed UHT milk artificially inoculated with MAP were used to manufacture yoghurt, using two different yoghurt starter cultures. Five commercial fermented milk products were inoculated with MAP. Two different MAP strains were studied. The survival of MAP in all products was monitored by culture over a 6-week storage period at 6°C. In yoghurt, MAP counts did not change appreciably during the storage period. Fat content and type of yoghurt starter culture had no consistent effect on the survival of MAP. In the fermented milk products, survival patterns varied but resulted in a 1·5 to ≥3·8 log reduction for the Niebüll strain and a 1·2-2·2 log reduction for the NIZO strain after 6 weeks, depending on the probiotic starters present in the product. CONCLUSIONS: MAP easily survived in yoghurt but MAP numbers decreased in fermented milk products containing probiotic cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: The results contribute to the lack of knowledge on the behaviour of MAP in yoghurt and fermented milk products containing probiotic cultures. This knowledge is valuable in the context of the risk of MAP transmission to humans via yoghurt and the possible contribution of probiotic fermented milk products to the elimination of MAP.

Localization of Mycobacterium avium subspecies paratuberculosis in artificially inoculated milk and colostrum by fractionation.

The purpose of the study was to determine the distribution of Mycobacterium avium ssp. paratuberculosis (MAP) across the main milk and colostrum fractions (cream, curd, and whey). Raw milk and colostrum were inoculated with 1 of 2 MAP strains, ATCC 19698 or S-23, yielding initial concentrations of 10(6) to 10(7) cfu/mL. After fractionation, for milk as well as for colostrum, 80 to 90% of the recovered MAP cells were found in the curd fraction and 10 to 20% in the cream fraction. Total MAP colony counts in milk whey were 4 to 5 log(10) units lower than colony counts of inoculated milk. In colostrum, colony counts were 2 to 3 log(10) units lower in whey than in inoculated colostrum. Because of the slow growth of MAP and to proceed more smoothly with set-up and optimization of the method, luminescent MAP strains were used. The high correlation coefficient (r=0.960) between colony counts and luminescence measurements showed that the use of luminescent MAP strains during method development was plausible.

Fusarium spp. and Fusarium mycotoxins in maize: a problem for Flanders?

Fusarium species cause not only root, stem and ear rot with severe reductions in crop yield, they produce also toxic secondary metabolites (mycotoxins) such as deoxynivalenol (DON) and zearalenone (ZEA). During several growing seasons the presence of Fusarium spp was followed up. DON and ZEA were determined and related to infection levels. The distribution of DON and ZEA in the different plant parts was studied as well as the influence of the ensiling process on the mycotoxin content. More or less important varietal differences in susceptibility for Fusarium spp. could be detected. DON and ZEA were clearly present in most of the analysed samples. No clear relationship could be detected between visual disease symptoms and mycotoxin content. The accumulation of DON and ZEA was different for the analysed aerial plant parts. The ensiling process gave no reduction of the mycotoxin content.

Differences in susceptibility of winter wheat varieties for Fusarium species under Belgian growing conditions.

Fusarium head blight is an important disease of cereal crops caused by Fusarium species. It causes not only a reduction in yield, but most Fusarium species (F. graminearum. F. culmorum, F. avenaceum. F. poae) produce also a range of toxic metabolites such as deoxynivalenol (DON) and zearalenone (ZEA). The evaluation of Fusarium species was followed up under natural infection conditions during the growing seasons 2001--2002 and 2002--2003 in two varietal winter wheat experiments on the experimental farm of the Hogeschool Gent at Bottelare. Disease pressure, DON and ZEA content, different Fusarium species as well as growth and yield parameters were determined. In both years there were significant differences between the varieties concerning the susceptibility to Fusarium and the DON content. ZEA was not found in the kernels. The mean deoxynivalenol (DON) content was in 2002 (1,126 mg/kg) higher than in 2003 (0.879 mg/kg) although the mean disease severity was bigger in 2003 than in 2002 what means that the DON content was not always correlated with the disease severity. The Fusarium species most frequently identified in our two field trials (Bottelare) were F. graminearum and F. culmorum Varietal differences in susceptibility to Fusarium species and DON contamination could be detected.

Effect of enterocin CCM 4231 on Listeria monocytogenes in Saint-Paulin cheese.

The bacteriocin production by Enterococcus faecium strain in cheese milk and cheese was demonstrated. Purified enterocin CCM 4231 exhibited an anti-listerial effect during Saint-Paulin cheese manufacture. During cheese production the strain grew to a final concentration of 10.1 +/- 0.01 log CFU per mL per g in cheese. Then only a slight decrease of the cell concentration was noticed during ripening and was almost stable for 8 weeks. No significant differences in pH were observed between the experimental and reference cheeses. Bacteriocin production during cheese manufacture was detected only in milk samples and curd, reaching a level of 100 AU/mL. After addition of purified enterocin CCM 4231 (concentration 3200 AU/mL) into the experimental cheese, the initial concentration of 6.7 +/- 0.06 log CFU per mL of Listeria monocytogenes Ohio was reduced up to 1.9 +/- 0.01 log CFU per mL per g. After 6 weeks and at the end of the experiment the difference of surviving cells of L. monocytogenes Ohio in ECH was only one or 0.7 log cycle compared to the control cheese. Although enterocin CCM 4231 partially inhibited L. monocytogenes in Saint-Paulin cheese manufacture, an inhibitory effect of enterocin added was shown in 1-week cheese; however, it was not possible to detect bacteriocin activity by the agar spot test. The traditional fermentation and ripening process was not disturbed, resulting in acceptable end-products, including sensory aspects.

Improvement of the detection of Listeria monocytogenes by the application of ALOA, a diagnostic, chromogenic isolation medium.

A new selective agar medium, ALOA, for the selective and differential isolation of Listeria monocytogenes has been evaluated. All stressed cultures of L. monocytogenes serovars tested grew on the medium as bluish colonies surrounded by a distinctive opaque halo and gave a productivity ratio of at least 0.95. Non-pathogenic Listeria sp. produced bluish colonies without a halo as was also the case for some enterococci and bacilli. Special attention must be paid to some Bacillus cereus strains and L. ivanovii since their colony appearance can be misleading. Only some unidentified listeria-like bacteria gave false-positive results. ALOA detected 4. 3% more positives from naturally contaminated dairy and meat samples compared with the ISO procedure when used with GenprobeTM or VidasTM for confirmation of presumptive colonies; 13.9% false negatives were found compared with 38.9% using PALCAM/Oxford. ALOA was also clearly superior to Oxford and PALCAM when samples containing both L. monocytogenes and L. innocua were examined. The introduction of ALOA in standard isolation procedures as an additional medium would enhance the detection ratio and reduce the time and cost of analysis for L. monocytogenes.

Unidentified Listeria-like bacteria isolated from cheese.

Unidentified Listeria-like bacteria, which lack only one of the phenotypic characteristics used to confirm Listeria spp., were isolated from cheese during routine analysis for Listeria monocytogenes. The VIDAS Listeria assay and the Listeria specific PCR or DNA probe assays used did not identify these strains as Listeria species. This group of bacteria was studied for its homogeneity using rep-PCR and PFGE. Sequence analysis of the 16S rRNA gene showed a homology of 94% to established Listeria spp., implicating a closer relationship than that between Listeria spp. and Brochothrix spp.

Isolation and characterization of two bacteriocins produced by Enterococcus faecium strains inhibitory to Listeria monocytogenes.

A total of 4000 bacterial strains were isolated from milk, cheese and from different samples taken on the farm (silage, faeces) and screened for their antimicrobial activity against Listeria monocytogenes. Only eight of the almost 4000 strains exhibited inhibitory activity in the cell-free supernatant fluid. Two different Enterococcus strains (RZS C5 and RZS C13) were chosen for further study. Plasmid profile analysis of both revealed that RZS C5 contained a 4 kb plasmid while RZS C13 contained a 23 kb plasmid. The inhibitory compounds were proteinaceous and active against all Listeria spp. tested while most of the lactic acid bacteria were insensitive. Weak inhibition was also detected against Clostridium perfringens and some Bacillus spp. Cross-inhibition tests showed different profiles. The bacteriocins were heat-resistant and were very stable during storage, especially at low pH values. Their molecular weight was estimated at +/- 3.0 kDA. Addition of 200 AU/ml of bacteriocin resulted in an initial rapid decrease of the number of viable L. monocytogenes cells followed by slower a logarithmic decrease.

Comparison of Petrifilm™ and Plate Count Methods for Enumerating Molds and Yeasts in Cheese and Yogurt.

The Petrifilm™ method for enumerating yeasts and molds was evaluated on 52 samples of fresh cheese and yogurt. A conventional plate method on glucose yeast extract medium with oxytetracycline was used as reference. Counting the repeatability a value of 0.229 log units was obtained for the Petrifilm™, compared to 0.175 for the conventional plate method. The correlation coefficient was 0.9978 with a residual standard deviation of 0.094 log units. The Petrifilm™ method is practical and fast, and its results may be compared to those of the conventional plate method.