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* Department of Health and Safety, and
Department of Processing, NIZO Food Research, PO Box 20, 6710 BA Ede, the Netherlands
Chair of Dairy Science, Wageningen University and Research Centre, PO Box 8129, 6700 EV Wageningen, the Netherlands
1 Corresponding author: marc.vissers{at}nizo.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Key Words: butyric acid bacteria • bulk tank milk • silage • late-blowing
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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To prevent late-blowing, cheese manufacturers have 3 main options: 1) minimizing BAB spore concentrations in raw milk; 2) removing BAB spores from raw milk via bactofugation; and 3) preventing the growth of BAB in cheese by adding inhibitory agents, such as nitrate and lysozyme, to cheese milk (Stadhouders 1990; Waes et al., 1990). In the Netherlands, farmers are encouraged to produce FTM with low concentrations of BAB spores via the milk quality payment systems. Two 0.1-mL tubes of FTM are tested monthly for the presence of BAB spores. If both tubes test positive, the farmer receives a deduction from the milk price. The Dutch dairy industry wants farmers to produce FTM with a maximum spore limit (MSL) of 3 log10 spores/L to obtain (after bactofugation) fewer than 1 log10 spores/L in pasteurized cheese milk.
The BAB spores in FTM originate from the farm environment. Grass and corn silage are the most important sources of BAB spores. Grass and corn silage are fed to cows together (referred to as mixed silage). The BAB spores in the mixed silage accumulate in feces (Te Giffel, 2002). Via the exterior of the cow’s teats, feces and BAB spores are transmitted to milk during milking (Bergère et al., 1968).
Using model simulations, we defined a strategy to achieve a BAB spore concentration in FTM below the MSL. Control of the spore concentration in the mixed silage offered to the cows was shown to be the most important control strategy (Vissers et al., 2006). The model showed that the BAB spore concentration in mixed silage should ideally be less than 3 log10 spores/g and should not exceed 5 log10 spores/g. If the mixed silage contains more than 5 log10 spores/g, it is not possible to ensure an FTM concentration of less than 3 log10 spores/L.
The aim of this study of Dutch farms was to determine the effect of different aspects of farm management on BAB spore concentrations in FTM using a year-long survey. In addition, the results of the survey were used to validate the control strategy derived from model simulations.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Sample Collection
Samples were collected between January and December 2005. Each farm was visited 6 times at intervals of 8 wk. At each visit, in addition to sample collection, a questionnaire was answered to collect actual farm management data. The questionnaire contained questions related to the feed ration fed to the cows, housing conditions, and milking practices. The questionnaire was completed by the researcher visiting the farm.
The following sample types were collected: FTM (every visit), feces from the barn (when cows were housed half a day or all day), feces from the pasture (when cows were grazing all day), bedding material from the barn (when cows were housed half a day or all day), soil (when cows were at pasture half a day or all day), and mixed silage in the barn (every visit when appropriate). No samples of individual grass and corn silage were taken because earlier studies had shown that a sample of the mixed silage offered to the cows is a better indicator of the microbial quality of the silage fed (Vissers et al., 2007). All but the FTM sample were composite samples. Each composite sample consisted of 10 randomly collected subsamples that were mixed on location. Samples were stored at 4 to 6°C until microbial analysis within 24 h after sampling.
Microbial Analyses
The concentrations of BAB spores in FTM, feces, bedding material, mixed silage, and soil were determined by a most probable number method according to the Dutch Standard (NEN-ISO-6877, 1994). Feces, bedding, soil, silage, and feed extracts were prepared by adding 90 mL of peptone physiological salt solution (PPS; 1 g of bacteriological peptone and 8.5 g of sodium chloride/L) to 10 g of sample and homogenizing for 2 min in a laboratory blender (Stomacher, Seward Medical, London, UK). Decimal dilution series of extracts were prepared in PPS. A 0.1-mL volume of diluted extract was added to tubes containing 10 mL of sterilized milk supplemented with glucose (0.5%) and lactic acid (0.18%). The tubes were heated for 5 min at 80°C to inactivate vegetative cells and to trigger the germination of spores, then sealed with paraffin and incubated for 4 d at 37°C. A tube scored positive if, after incubation, gas formation was visible. The detection limit was 30 spores/L for FTM and 300 spores/g for feces, bedding, mixed silage, and soil.
Statistical Analyses
Statistical calculations were performed using Statistica (StatSoft Inc., Tulsa, OK) and Microsoft Excel (Microsoft, Redmond, WA). To perform statistical analyses, samples containing BAB spore concentrations below the detection limit were assigned a concentration equaling half the detection limit. All statistical analyses were performed on log10 transformed data.
Student’s t-tests were used to determine significant differences between farms and spore concentrations in different carriers (i.e., feces, bedding material, mixed silage, and soil) and to detect possible effects of farm management practices on BAB spore concentrations in FTM. The Fisher exact test was used to determine whether high concentrations occurred more frequently in a specific carrier or group.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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gives an overview of BAB spore concentrations measured in FTM and carriers of BAB spores in the farm environment (environmental carriers) on all 24 farms during the survey. The average BAB spore concentration in FTM was close to the MSL (3 log10 spores/L). In 33% of the FTM, the MSL was exceeded. Also in feces, bedding material, and mixed silage, high concentrations (above 5 log10 spores/g) were frequently detected. Spore concentrations in feces and bedding material did not statistically differ (P > 0.2). Spore concentrations in feces were higher than those in the mixed silage (P < 0.01), most likely because of a concentration effect (Te Giffel, 2002). The BAB spore concentrations in soil varied much less than the concentrations in the other environmental carriers and never exceeded 5 log10 spores/g. No relationship between soil type (e.g., clay, sand, and peat) and BAB spore concentration was found (P > 0.2).
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). Second, farms were grouped based on the average BAB spore concentration detected in FTM per farm in the survey (survey grouping, Table 2). Based on the historical grouping, group A farms had lower spore concentrations in FTM than group B farms, and group B farms had lower spore concentrations than group C farms. In the survey grouping (Table 2) the low group represents the 6 farms with the lowest average BAB spore concentrations in FTM, the high group represents the 6 farms with the highest average BAB spore concentration in FTM, and the medium group represents the remaining 12 farms. Average BAB spore concentrations in FTM per farm ranged from 1.8 to 3.4 log10 spores/L.
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Clear relationships were found after grouping based on the survey data (Table 2
). The 6 farms that fed mixed silage with the lowest average spore concentration (3.4 log10 spores/g) also produced FTM with the lowest average concentrations (2.1 log10 spores/g). Conversely, the 6 farms with the highest FTM concentration (3.2 log10 spores/g) also fed silage with the highest average concentration (4.7 log10 spores/g). These concentrations were around the critical levels defined for FTM and silage by Vissers et al. (2006). Survey data in Table 2 also indicated that the relationship between average concentrations in mixed silage, feces, and FTM was almost linear on a log10 scale because the absolute differences observed at the low, medium, and high farms were similar for these 3 carriers (i.e., 
1.1 log10 difference between low and high and 0.7 log10 difference between low and medium). Historical grouping (Table 2) gave less clear relations than grouping based on the survey data. However, the farms that performed best based on the historical data (group A) also had the lowest average BAB spore concentrations in mixed silage and FTM.
On all farms in the survey, substantial fluctuations were observed in BAB spore concentrations in FTM and environmental carriers. On only 4 farms (3 from the low group and 1 from the medium group) was the BAB spore concentration in FTM consistently below the MSL. Also in feces, bedding material, and mixed silage, critically high concentrations (>5 log10 spores/g) were occasionally found on farms with the lowest spore concentration in FTM. For example, the highest BAB spore concentration in feces (7 log10 spores/g) was observed on the farm with the third lowest average BAB spore concentration in FTM. At the visit to this farm, the feces sample contained 7 log10 spores/g and the BAB spore concentration in FTM was 4.1 log10 spores/L. However, critically high BAB spore concentrations in feces, bedding material, and mixed silage were more frequently observed on the farms with the highest average concentrations in FTM. Nevertheless, all farms except one were able to meet the MLS on at least 2 of the visits. On all farms, the concentration was above the MSL at least once.
Effects of Farm Management
In addition to the determination of BAB spore concentrations, data about farm management practices at the participating farms were collected by using a questionnaire. Table 3 gives an overview of farm management practices related to the housing of cows, milking hygiene, and type of silage fed. Statistically significant relationships among the BAB spore concentrations in FTM were found only for the location of the cows during the summer period and for the presence in the herd of cows preferring to lie down on dirty patches. Therefore, these aspects were analyzed in more detail.
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shows the BAB spore concentration measured in the winter period and for the different housing situations observed during the summer period. During the winter period, cows were housed 24 h of the day at 96% of the farm visits. Table 4 shows that keeping cows 24 h a day at pasture leads to a reduction of the level of FTM with BAB spores. The observed lower BAB spore concentration in FTM for this situation seems to be related to the lower concentration in feces. The BAB spore concentration in mixed silage did not differ (P > 0.2). Further analysis suggested, however, that it was probably not the location of the cows that was important but whether the cows were fed silage or not. The questionnaire revealed that in 66% of the cases in which cows were grazing all day, the diet of fresh grass was supplemented with silage. In these cases, cows were offered silage in the barn before milking. When the diet of grazing cows was not supplemented with silage, the BAB spore concentration in FTM never exceeded 1.6 log10 spores/L (5 samples), whereas when silage was fed to cows grazing all day, the BAB spore concentration in FTM ranged from <1.5 to 3.7 log10 spores/L (9 samples).
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Critical Concentrations in Feces, Bedding, and Mixed Silage
Figures 1 and 2 show the fraction of FTM samples with a BAB spore concentration below and above the MSL in relation to BAB spore concentrations in feces and mixed silage. Figure 1 shows that BAB spore concentrations in feces above 5 log10 spores/g were critical with respect to the production of FTM with fewer spores than the MSL. When feces contained more than 5 log10 spores/g, almost 60% of the FTM exceed the MSL. On the other hand, when feces contained less than 4 log10 spores/g, none of the FTM exceeded the criterion. These results suggest that farmers should try to take measures that result in a BAB spore concentration in feces of less than 4 log10 spores/g.
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shows that also in mixed silage a critical concentration can be distinguished. A BAB spore concentration of greater than 5 log10 spores/g in mixed silage should be prevented. When mixed silage contained more than 5 log10 spores, almost 70% of the FTM samples contained more spores than the MSL. Conversely, when the silage contained less than 3 log10 spores/g, only about 13% of the FTM contained more spores than the MSL of 3 log10 spores/L. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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The survey in this study confirmed that control of the BAB spore concentration in silage was by far the most important option to control the BAB spore concentration in FTM. Other measures that were presumed important, such as teat cleaning (Stadhouders and Jörgenson, 1990), were much less important. In line with the model of Vissers et al. (2006), a more or less linear relation was found among BAB spore concentrations in mixed silage offered to the cows, feces, and FTM (Table 2). On the rare occasions (5 of the 144 visits) when no grass silage or corn silage was fed to the cows, the spore concentration in FTM never exceeded 1.6 log10 spores/L.
The most efficient way to control the BAB spore concentration in FTM was shown to be by controlling the BAB spore concentration in mixed silage. Farmers that produced milk with the lowest average BAB spore concentrations in FTM (2.1 log10 spores/L) also fed mixed silage to the cows with the lowest average spore concentrations (3.4 log10 spores/g). Conversely, farmers with the highest average BAB spore concentrations in FTM (3.2 log10 spores/L) fed mixed silage with the highest average concentrations (4.7 log10 spores/g). Apart from silage feed and silage quality, no relevant relations with other aspects of farm management were detected.
In the modeling study by Vissers et al. (2006), it was postulated that to consistently produce FTM meeting the MSL, farmers should aim for a concentration of BAB spores in the silage fed of below 3 log10 spores/g (in combination with a moderate level of teat hygiene) and should prevent concentrations of more than 5 log10 spores/g (Vissers et al., 2006). Figures 1 and 2 confirm that these concentrations are critical for control. When the mixed silage offered to the cows contained more than 5 log10 spores/g, the majority of the FTM samples exceeded the MSL. On the other hand, when the silage fed contained fewer than 3 log10 spores/g of FTM, only a minority of the samples (13%) exceeded the MSL. At concentrations in mixed silage between 3 and 5 log10, the number of FTM samples with a BAB spore concentration above and below the MSL was similar. At these concentrations in mixed silage, presumably the level of fecal contamination of the teat exterior plays a role, as also stated in the modeling study. The agreement between the survey results and results obtained in a modeling study emphasizes the usefulness of the modeling approach for identifying measures to control the microbial contamination of FTM.
Incident Management
In achieving and maintaining BAB spore concentrations of less than 3 log10 spores/g in silage fed to cows, the heterogeneous distribution of BAB spores in grass and corn silage is a difficulty. A number of studies have shown that BAB spore concentrations in grass and corn silage can be highly elevated locally (up to >7 log10 spores/g), whereas the concentration in the bulk of the silage is low (less than 3 log10 spores/g; Jonsson, 1991; Driehuis and Te Giffel, 2005; Vissers et al., 2007). When such local spots with high BAB spore concentrations are mixed with the bulk of the silage and other silage, the average BAB spore concentration in the mixed silage fed to cows increases significantly, as was confirmed in a recent study (Vissers et al., 2007). The heterogeneous distribution of BAB spores in silage may explain why even farms with a low average BAB spore concentration in FTM occasionally produce FTM with a BAB spore concentration above the MSL (Table 2). As such, efficient farm management with respect to control of the contamination of FTM with BAB spores probably depends on the ability of farmers to minimize the incidence of elevated BAB spore concentrations (>5 log10 spores/g) in the mixed silage fed to cows in the barn.
Control of BAB Spores in Mixed Silage
To ensure that the mixed silage fed contains fewer than 3 log10 spores/g, it is important to limit the initial contamination of silage with BAB spores and to prevent the growth of BAB during ensilage, storage, and feed-out. Silage is initially contaminated with BAB spores as soil is enclosed during harvesting (Pahlow et al., 2003). The BAB spore concentrations measured in soil (Table 1) indicate that an initial contamination level of less than 3 spores/g of silage can be secured when less than 1% of soil is enclosed. To prevent the growth of BAB, it is first important to produce an anaerobically stable silage by ensuring a fast and sufficiently deep decline of the pH by lactic acid fermentation (Weissbach, 1996; Pahlow et al., 2003). Second, penetration of oxygen into the silage during storage and feed-out should be prevented. Penetration of oxygen is the most likely cause of locally increased BAB spore concentrations in silage (Jonsson, 1991; Vissers et al., 2007). The presence of oxygen in silage enables yeasts to grow and consume the organic acids. As a consequence, pH rises locally and BAB may start to proliferate, resulting in small pockets with a very high BAB spore concentration. Adequate sealing of silage and a high silage density limit the penetration of oxygen (Parsons, 1991; McGechan and Williams, 1994).
Late-Blowing in Summer
Traditionally, late-blowing of cheeses was exclusively associated with milk produced in the winter period. In the Dutch milk quality payment system, FTM was therefore initially tested for BAB spores during the winter period only. In recent years, however, incidents of late-blowing in cheese have occurred more often during the summer, suggesting that also during the summer period the FTM has contained significant concentrations of BAB spores. As a reaction, since 2004 FTM has been tested for BAB spores all year round. The results shown in Table 4 concerning the BAB spore concentrations in FTM in summer and winter confirm that this change in the milk payment system was justified. The survey presented in this paper clearly demonstrates that the increased frequency of late-blowing during summer is most likely due to changed feeding and housing patterns in the summer. In the past in the Netherlands, cows were fed silage and housed all day only during the winter period. In summertime, cows were grazing all day and no silage was fed. Nowadays in the summer period, cows are generally in the barn for at least half of the day (Table 3), but more importantly, silage is fed all year round, even when cows graze all day.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Received for publication November 30, 2006. Accepted for publication March 12, 2007.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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This article has been cited by other articles:
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  M. M. M. Vissers, F. Driehuis, M. C. Te Giffel, P. De Jong, and J. M. G. Lankveld Short Communication: Quantification of the Transmission of Microorganisms to Milk via Dirt Attached to the Exterior of Teats J Dairy Sci, August 1, 2007; 90(8): 3579 - 3582. [Abstract] [Full Text] [PDF]
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M. M. M. Vissers, M. C. Te Giffel, F. Driehuis, P. De Jong, and J. M. G. Lankveld Minimizing the Level of Bacillus cereus Spores in Farm Tank Milk J Dairy Sci, July 1, 2007; 90(7): 3286 - 3293. [Abstract] [Full Text] [PDF]
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