Short Communication: Quantification of the Transmission of Microorganisms to Milk via Dirt Attached to the Exterior of Teats

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Abstract 

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 ∼3 to 300mg/L, with an average of 59mg/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.

Key words: risk analysis, raw milk, microbial contamination, spores of mesophilic bacteria

The dairy industry assures the microbial quality and safety of dairy products via a chain management approach. Therefore, from “grass to glass”, quality assurance systems such as good farming practices, good hygienic practices, and hazard analysis and critical control points are implemented (Te Giffel, 2003; Morgan, 2004). In addition, risk analyses can be used to quantitatively assess risks throughout the food chain and to identify effective measures to improve food safety and the microbial quality of farm tank milk (FTM; Notermans et al., 1997; Cassin et al., 1998; Vissers et al., 2006).

At the farm level, many microorganisms [e.g., butyric acid bacteria (spores), Bacillus cereus (spores), and Listeria monocytogenes] are transmitted from the environment to FTM via the exterior of teats (Bergère et al., 1968; Sanaa et al., 1993; Slaghuis et al., 1997). Contamination of milk via the exterior of the cows’ teats occurs when teats are contaminated with dirt consisting of feces, bedding material, soil, or a combination of these. Dirt attached to the exterior of teats rinses off during milking, the dirt dilutes in the milk, and subsequently milk is contaminated with microorganisms originating from the dirt. The concentration of microorganisms transmitted via this route (C_transmitted in cfu/L) depends on the amount of rinsed-off dirt (Mass_dirt in g/L) and the microbial concentration in rinsed-off dirt (C_dirt in cfu/g), equation [1].

[1]

Equation [1] implies that for quantitative risk analysis of the microbial quality of FTM, data are required with respect to the amount of dirt transmitted via the exterior of teats and microbial concentrations in transmitted dirt. Data on the microbial concentrations in dirt or constituents of dirt (e.g., feces, bedding material, and soil) are generally available (e.g., Slaghuis et al., 1997; Cooke and Sandeman, 2000; Vissers et al., 2007b). However, only Stadhouders and Jørgensen (1990) give insight into the amount of transmitted dirt. They estimated that FTM contains on average 40mg of dirt/L of milk. The objective of this study was to obtain more and up-to-date data about the amount of dirt transmitted to FTM via the exterior of teats.

Probably due to the heterogeneous composition of dirt and its low concentrations in comparison to the gross components of milk, a reliable method to directly determine the amount of dirt in milk is currently not available. Stadhouders and Jørgensen (1990) applied an indirect method based on the transmission route and equation [1] as an alternative. If a specific dirt component is transmitted from the farm environment to milk exclusively via the dirt attached to the exterior of teats, this specific component can be used as a marker for transmitted dirt: when the marker concentrations are measured in samples of milk and dirt, the amount of transmitted dirt can be calculated using equation [1] (Stadhouders and Jørgensen (1990).

Stadhouders and Jørgensen (1990) used spores of butyric acid bacteria (BAB) as a microbial marker for transmitted dirt. To obtain more robust data in our study, spores of mesophilic aerobic bacteria are used as a microbial marker. As for BAB spores, there is ample evidence that these spores are transmitted from the farm environment to milk via the exterior of teats (Slaghuis et al., 1997; Cooke and Sandeman, 2000). The major advantage of using spores of mesophilic aerobic bacteria instead of spores of BAB as the microbial marker is that the concentration of spores of mesophillic aerobic bacteria is generally above the detection limit (Slaghuis et al., 1997). Stadhouders and Jørgensen (1990) were not able to obtain a value for the concentration of BAB spores in 36% of their milk samples. Consequently, it was not possible for Stadhouders and Jørgensen (1990) to calculate the amount of transmitted dirt for these farms. An additional advantage of our method, using mesophilic aerobic spores as the microbial marker, is that a plate count method could be used for enumeration. Stadhouders and Jørgensen (1990) had to use a most-probable-number method to determine the concentration of BAB spores in dirt and milk.

Milk samples are taken from milk recording jars to minimize the possibility that milk is contaminated with spores of mesophilic aerobic bacteria with surfaces of the milk equipment. Milk of a single cow is collected in these jars before further transportation through the milklines to the farm tank. Dirt samples were collected from the underside of the udder. Dirt attached to the udder was assumed to have the same history and composition as dirt attached to the teats. It is not possible to take dirt samples from the teats because this would affect the amount of transmitted dirt. Using a milk sample from the recording jar and a dirt sample from the udder of the same cow, the amount of transmitted dirt for an individual cow can be calculated.

To obtain accurate data it is important that the dirt sample is representative of dirt rinsed off teats. Therefore, first the representativeness of dirt samples taken from the udder for dirt attached to teats was determined at an experimental farm (Animal Sciences Group, Wageningen UR, Lelystad, the Netherlands). At the experimental farm, cows were housed 24h/d, as is common practice in the Netherlands. Dirt samples were collected from the underside of the udders and teats of 6 randomly selected cows that had visible dirt attached to the teats. Dirt samples mainly consisted of feces with small amounts of bedding. The concentrations of mesophilic aerobic spores in the samples were determined via dilution of the collected dirt in physiological salt solution followed by pasteurization of 5mL of the diluted samples for 10min at 80°C. Pasteurized samples were cooled in melted ice prior to serial dilution in physiological salt solution. Appropriate dilutions were plated on plate count milk agar. Spore counts were determined after 2 d of incubation at 30°C. After log₁₀ transformation, necessary to obtain normally distributed data, the average spore concentrations in dirt attached to the udder and dirt attached to the teats were 6.9 log₁₀ spores/g (SD=0.4) and 6.6 log₁₀ spores/g (SD=0.3) respectively. The observed difference is not significant (t-test; P=0.14). These results imply that a dirt sample from the udder is representative of dirt rinsed off teats during milking.

In the main study the methodology described was used to determine the amount of dirt transmitted to milk at 11 farms, selected at random, in different regions of the Netherlands. The visited farms milked between 40 and 125 cows, and cows were housed 24h a day during the experimental period. Per-farm dirt and milk samples were collected from 10 randomly selected cows. Again, dirt samples were collected from the underside of the udder and mainly consisted of feces and bedding. Per cow the amount of dirt transmitted to milk was calculated. These data were then used to determine the farm average.

Table 1 shows the spore concentration measured in dirt and milk plus the calculated amount of transmitted dirt per farm. Spore concentrations measured in milk and dirt corresponded to the concentration in milk, feces, bedding, and soil previously observed in the Netherlands (Slaghuis et al., 1997). Spore concentration in dirt and milk were highly correlated (correlation coefficient=0.79; P=0.02). This implies that the spores of mesophilic aerobic bacteria in milk most likely originated from dirt attached to the teats and that no relevant contamination of the milk via the milk cups and tubing to the milk recording jar had occurred. The calculated average amount of dirt transmitted to milk per farm ranged from ∼3 to ∼300mg/L, with an average across farms of 59mg/L. Significant differences among farms existed. At some farms a high variation of the calculated amount of dirt in milk of individual cows was observed (e.g., farms 8 to 11). It shows that this variation mainly occurred at the high end of the values observed (i.e., at all farms the lowest value observed was between 1 and 8mg/L, whereas the highest values ranged from 10 to 1,160mg/L). This could imply that the difference between the most and least hygienic farms mainly lies in the ability of farms to prevent the (incidental) transmission of a high amount of dirt to milk of individual cows. Incidental transmission of a high amount could, for example, be due to inconsistent cleaning of boxes and teats. However, the obtained data set was too small to identify specific measures that reduce the average amount of dirt in milk.

Table 1. Concentrations of spores of mesophilic bacteria in milk of individual cows and in dirt attached to the udder and the calculated amount of transferred dirt for the 11 farms visited¹

The minimum (3mg/L) and average (59mg/L) values corresponded with previous estimations of the amount of dirt transmitted to milk during housing of cows; that is, a minimum of 2mg/L and average of 40mg/L (Stadhouders and Jørgensen, 1990). However, the maximum average amount of dirt observed at a farm in our survey (∼300mg/L) was less than the maximum found in tank milk by Stadhouders and Jørgensen (1990; up to 1,000mg/L). Also, values above 100mg/L were observed far less frequently in our survey compared with the study of Stadhouders and Jørgensen (1990). Thus, it seems that implementation and improvement of quality control systems and hygienic measures taken at the farm level have mainly reduced the highest values occurring but have not improved the average. In addition, it should be kept in mind that Stadhouders and Jørgensen (1990) had to estimate the average value because they could not measure the concentration of their marker for transmitted dirt, spores of BAB, in 36% of the milk samples.

The usefulness of the data obtained can be illustrated using the contamination of FTM with spores of BAB as a case study. Minimization of the contamination of FTM with spores of BAB is important for the production of semihard cheeses (Klijn et al., 1995). The FTM is contaminated with spores of BAB when feces are transmitted to milk via the exterior of teats. The BAB spores in feces mainly originate from silage (Bergère et al., 1968). The BAB spore concentrations in feces vary by more than a factor of 10,000 (from 2 to 6 log spores/g; Kalzendorf, 1996; Vissers et al., 2007b). The amount of transmitted dirt varies across farms by a factor of only 100 (Table 1). This implies that BAB spore concentrations in FTM depend more on the spore concentration in feces than on the amount of transmitted dirt. Consequently, it is more important to minimize BAB spore concentration in feces than to minimize the contamination of teats with feces or to maximize the efficiency of teat cleaning, as has been shown by Vissers et al. (2006). The BAB spore concentrations in silage and thus in feces can be reduced via proper ensiling practice and the prevention of air infiltration during storage and feed out of the silage (Pahlow et al., 2003; Vissers et al., 2007a).

The role of teat hygiene should not be neglected because a factor of 100 difference between the amount of dirt transmitted to milk at the best and worst farms is substantial. For example, according to Table 1, the variation of the mesophilic aerobic spore count in feces at one farm (factor 10 to 100) is roughly the same as the variation in the average amount of dirt transmitted to FTM (factor 100). This could imply that to reduce the concentration of spores of mesophilic aerobic bacteria, hygienic measures are preferable over measures that reduce the spore concentration in feces.

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References 

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