1580

Journal of Food Protection, Vol. 71, No. 8, 2008, Pages 1580–1589 Copyright ᮊ, International Association for Food Protection

Low Incidence of Foodborne Pathogens of Concern in Raw Milk Utilized for Farmstead Cheese Production

DENNIS J. D’AMICO, ERROL GROVES, AND CATHERINE W. DONNELLY*

Department of Nutrition and Food Sciences, University of Vermont, Burlington, Vermont 05405, USA

MS 08-030: Received 15 January 2008/Accepted 3 March 2008

ABSTRACT Downloaded from http://meridian.allenpress.com/jfp/article-pdf/71/8/1580/1679328/0362-028x-71_8_1580.pdf by guest on 26 September 2021

Overall milk quality and prevalence of four target pathogens in raw milk destined for farmstead cheesemaking was examined. Raw milk samples were collected weekly from June to September 2006 from 11 farmstead cheese operations manufacturing raw milk cheese from cow’s, goat’s, and sheep’s milk. Samples were screened for Listeria monocytogenes, Staphylococcus aureus, Salmonella, and Escherichia coli O157:H7 both quantitatively (direct plating) and qualitatively (PCR). Overall, 96.8% of samples had standard plate counts of Ͻ100,000 CFU/ml, 42.7% of which were Ͻ1,000 CFU/ml. Although no federal standards exist for coliforms in raw milk, 61% of samples tested conformed to pasteurized milk standards under the U.S. Pasteurized Milk Ordinance (PMO) at Ͻ10 CFU/ml. All cow and sheep milk samples and 93.8% of goat milk samples were within the limits dictated by the PMO for somatic cell counts. Of the 11 farms, 8 (73%) produced samples that were positive for S. aureus, which was detected in 34.6% (46 of 133) of milk samples. L. monocytogenes was isolated from three milk samples (2.3%), two of which were from the same farm. E. coli O157:H7 was recovered from one sample of goat’s milk for an overall incidence of 0.75%. Salmonella was not recovered from any of the 133 samples. The findings of this study suggest that most raw milk intended for farmstead cheesemaking is of high microbiological quality with a low incidence of pathogens. These data will help inform risk assessments associated with the microbiological safety of farmstead cheeses, particularly those manufactured from raw milk.

U.S. Standards of Identity for cheese and cheese prod- the Centers for Disease Control (CDC); Campylobacter and ucts (42) permit the manufacture of more than 30 varieties Salmonella infections constituted the majority of cases (16). of cheese from raw milk, most of which must be aged for As a result of these outbreaks, in 1987 a final rule was a minimum of 60 days at a temperature no less than 35ЊF published requiring mandatory pasteurization of all milk (1.67ЊC). U.S. federal regulations (43) governing milk, and milk products intended for human consumption enter- however, do not include microbiological standards, includ- ing interstate commerce (21 CFR §1240.61(a)). Federally, ing the presence of pathogenic bacteria, for raw milk used the Grade ‘‘A’’ Pasteurized Milk Ordinance (PMO) (43) for the manufacture of raw milk products. Because of re- provides guidance to ensure that fluid milk is produced newed interest in specialty cheeses, small-scale artisan and safely. Milk intended for cheesemaking, however, is subject farmstead producers are manufacturing numerous varieties to different regulations as determined by individual states of cheese from raw milk, including cheeses such as surface- and not necessarily governed by the PMO (48); some states ripened soft cheeses that present a higher risk of carrying (e.g., New York) allow the use of grade B (manufacturing foodborne pathogens. grade) milk for the production of cheese. Grade B milk is Unpasteurized milk has been a known vehicle of food- produced under less stringent hygienic guidelines and may borne disease since the inception of the dairy industry. In be at higher risk for the presence of pathogens than is grade the early 1900s, poor sanitation, improper milk handling, A milk (19). and animal health issues resulted in numerous disease out- The presence of pathogenic bacteria in raw milk has breaks associated with milk (37). With the gradual imple- been well documented (21, 24, 29, 30, 36, 44, 46), and the mentation of pasteurization, the risk of milkborne infection dairy farm itself can serve as a reservoir of these bacteria. was greatly reduced (2). Since the late 1970s, changes in Fecal contamination of teats, udder surfaces, and milking agricultural practices and food processing operations and machines can result in subsequent contamination of milk the globalization of the food supply have resulted in the (20). Although the presence of pathogens in the farm en- emergence of newly recognized foodborne pathogens in the vironment does not always lead to the contamination of United States (2), including Listeria monocytogenes, mul- bulk tank milk, artisan cheeses produced in small process- tidrug-resistant Salmonella, and Escherichia coli O157:H7. ing facilities on dairy farms (farmstead) may be at a higher From 1973 to 1992, 46 raw milk–associated foodborne dis- risk for the presence of pathogens because of the potential ease outbreaks resulting in 1,733 illnesses were reported to for environmental contamination of raw materials from the farm environment (32). The excessive workload of on-farm * Author for correspondence. Tel: 802-656-8300; Fax: 802-656-0001; dairies also may deter producers from employing proper E-mail: [email protected]. food hygiene practices (39). For these and other reasons, J. Food Prot., Vol. 71, No. 8 PATHOGEN INCIDENCE IN FARMSTEAD RAW MILK 1581 the use of raw milk for the production of farmstead cheese shifts during refrigeration (23), all samples were processed within may be perceived as a high-risk practice. Typically, a 8 h of collection. cheese classified as ‘‘farmstead’’ (e.g., according to the Standard aerobic bacteria, coliform, and somatic cell American Cheese Society at http://www.cheesesociety.org) counts. Standard counts of aerobic bacteria were determined using is made with milk from the farmer’s own herd or flock on Petrifilm aerobic count plates (3M Microbiology, St. Paul, Minn.) the farm where the animals are raised. The reduction in that were incubated for 48 Ϯ 3hat32Ϯ 1ЊC (official method transport that results from using on-farm milk may help 986.33, AOAC International, Gaithersburg, Md.). Resulting prevent increases in microbial populations that occur from counts were rounded to two significant figures at the time of con- additional surface contact and pumping during gradient version to standard plate counts (SPCs). Coliform counts (CCs) transport (4). The reduced time from milking to cheese- were determined using Petrifilm coliform count plates (3M Mi- Ϯ Ϯ Њ making also limits the outgrowth of undesirable bacteria crobiology) that were incubated for 24 2 h at 32 1 C (official method 986.33). Colonies were counted on all plates containing that may be present in the milk (48). Extended holding of 25 to 250 CFU/ml. Counts outside this range are displayed in Downloaded from http://meridian.allenpress.com/jfp/article-pdf/71/8/1580/1679328/0362-028x-71_8_1580.pdf by guest on 26 September 2021 cooled milk in the bulk tank allows for the selective growth tables as estimates. For the determination of somatic cell counts of psychrotrophic pathogens, including L. monocytogenes (SCCs), vials containing bronopol were filled upon arrival at the and spoilage flora, which are negative contributors to milk laboratory, shaken to ensure even distribution, and shipped to the and cheese quality and safety (23). Vermont Dairy Herd Improvement Association (White River Although the incidence of pathogens in milk from Junction). SCCs were determined with a Somacount 500 (Bentley commercial dairy farms is well documented, there are few Instruments, Inc., Chaska, Minn.) calibrated for cow’s milk. data regarding the microbiological quality and prevalence Quantitative pathogen detection and isolation. Target of pathogenic bacteria in milk used for the manufacture of pathogen population levels were determined through surface plat- artisan and farmstead cheeses (8), which are often made ing of 1 ml of raw milk over six plates (167 ␮l each) of chro- from raw milk. Data concerning changes in bacterial levels mogenic agar (CHROMagar Microbiology, Paris, France) for- in milk from a given farm over time also are scant (8). The mulated for each specific target pathogen (CHROMagar Staph au- objective of this study was to fill data gaps concerning the reus, CHROMagar Salmonella, CHROMagar O157, and threat of pathogens in raw milk intended for the manufac- CHROMagar Listeria). CHROMagar Salmonella was modified by ture of farmstead cheese. We aimed to examine the overall the addition of 5 mg/liter cefsulodin (Sigma-Aldrich, St. Louis, quality and pathogen prevalence in raw milk destined for Mo.). CHROMagar O157 was modified to include 2.5 mg/liter farmstead cheese manufacture throughout the main produc- potassium tellurite (Dynal, Invitrogen, Carlsbad, Calif.), 0.025 mg/liter cefixime (Dynal), and 5 mg/liter cefsulodin (Sigma-Al- tion season. drich). All samples were plated in duplicate. Typical colonies, as defined by the manufacturer, were enumerated after 24 h of in- MATERIALS AND METHODS cubation at 37 Ϯ 0.5ЊC. All presumptive colonies enumerated Milk collection. Study farms meeting the definition of farm- were purified on tryptic soy agar (Becton Dickinson, Sparks, Md.) stead were chosen based on geographic location, willingness to with 0.6% yeast extract (Becton Dickinson) added (TSAYE) and Ϯ Њ participate, and type of milk used for cheese manufacture (raw or incubated for 24 h at 35 0.5 C. A single colony from each pasteurized). Although all study farms produced raw milk cheese, TSAYE plate was then inoculated into 9 ml of tryptic soy broth which was the focus of the study, some also produced additional (Becton Dickinson) with 0.6% yeast extract added and incubated Ϯ Њ products from pasteurized milk. The majority of farms included for 24 h at 35 0.5 C for confirmation by PCR. Samples yielding in this study produced milk and cheese seasonally. A total of 138 at least one confirmed colony of the target pathogen were deemed positive for the presence of that organism. raw milk samples were collected weekly, on the same day each week, from 11 farmstead cheese operations located throughout the Qualitative pathogen detection and detection. The BAX state of Vermont during the main production season between June Q7 automated PCR system (DuPont Qualicon, Wilmington, Del.) and September 2006. Study farms manufactured raw milk cheese was employed for the qualitative detection of target pathogens from the milk of cows (five farms, 67 samples), goats (four farms, according to the manufacturer’s instructions. Each method has 49 samples), or sheep (two farms, 22 samples). The number of been validated by Health Canada (Health Products and Food farms investigated represents approximately one-third of Ver- Branch, Food Directorate, Bureau of Microbial Hazards, Com- mont’s farmstead producers. Information regarding the species as- pendium of Analytical Methods, vol. 3; available at: http://www. sociated with individual study farms is not provided to ensure the hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/volume3). All confidentiality of farm identities. Herd sizes were generally small, PCR-positive enrichments were streaked to respective CHROM- with cow, goat, and sheep dairies ranging from approximately 18 agar media for isolation. to 100, 25 to 75, and 20 to 90 animals, respectively. In general, For the detection of Salmonella, 25 ml of raw milk was add- study farms kept animals on pasture for the duration of this study, ed to 225 ml of lactose broth and incubated for 24 Ϯ 2hat35ЊC and all animals were milked with automated systems. Samples as recommended in the U.S. Food and Drug Administration Bac- were aseptically collected by the farmer-cheesemaker from the teriological Analytical Manual (BAM; chap. 5, Salmonella, avail- farm bulk tank, milk cans, or cheese vat following agitation and able at: http://www.cfsan.fda.gov/ϳebam/bam-5.html). Ten micro- either placed in a refrigerator prior to pickup or immediately liters of the enriched sample was then inoculated into 500 ␮lof stored on ice for transport to the laboratory. Temperatures during brain heart infusion broth (BHI; Becton Dickinson) and incubated transport were monitored with a multipurpose temperature data at 37ЊC for 3 h. Five-microliter aliquots of enriched BHI culture logger (SP125, Dickson, Addison, Ill.) to assure samples were not were removed and processed using the Salmonella assay for the temperature abused. Because milk was collected immediately be- BAX PCR according to the manufacturer’s instructions. fore cheese manufacture and to minimize bacterial population Twenty-five milliliters of raw milk was enriched in 225 ml 1582 D’AMICO ET AL. J. Food Prot., Vol. 71, No. 8

TABLE 1. Raw milk standard plate counts at individual farmstead operations Standard plate counts (CFU/ml) at farma:

Wk ABCDE F GIJK

1 900 41,000 85 820 14,000 16,000 1,200 410 30 –b 2 1,700 76,000 130 1,200 1,600 9,300,000 1,800 79,000 23 15,000 3 3,700 560 160 3,700 1,600 76,000 7,500 10 30 –b 4 7,200 5,500 150 17,000 1,300 150,000 710 100 70 –b 5 1,400 270 130 6,900 1,100 20,000 1,100 1,700 110 –b 6 2,800 530 200 3,100 9,300 20,000 130 600 540 17,000 7 1,100 7,400 120 4,100 3,300 1,300,000 650 100 37 NTc 8 880 NT 93 1,500 4,500 1,500 330 75 20 15,000

9 3,200 140,000 170 1,700 5,700 9,400 1,700 130 120 22,000 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/71/8/1580/1679328/0362-028x-71_8_1580.pdf by guest on 26 September 2021 10 52,000 3,900 73 3,300 NT 7,300 18,000 130 73 9,600 11 NT 600 46 3,700 NT 15,000 230 89 380 13,000 12 7,800 4,000 78 NT 1,500 51,000 950 450 19 NT 13 2,500 1,400 54 1,800 11,000 NT 700 110 25 16,000 GMd 3,000 4,000 100 2,900 3,400 45,000 1,200 260 59 15,000

a Results are expressed as CFU per milliliter for ease of comparison with previous studies. b Data not included because of the presence of starter culture. c NT, not tested. d GM, geometric mean

of complete selective enrichment broth and incubated for 24 Ϯ 2 counts reported in tables as Ͻ1 CFU/ml represent actual observed hat30ЊC for the detection of L. monocytogenes. One hundred average colony counts of 0 or 0.5 CFU/ml. Actual observed av- microliters of the enriched sample was then transferred to 9.9 ml erage colony counts were utilized for statistical analyses. Esti- of 3-morpholinopropanesulphonic acid–buffered Listeria enrich- mated colony counts were not included in analyses. The general ment broth (MOPS-BLEB) for secondary enrichment for 22 Ϯ 2 linear models procedure was used to perform univariate analyses hat35ЊC. Aliquots (5 ␮l) of MOPS-BLEB were removed and of variance on log-transformed SPC and CC data to detect differ- processed with the L. monocytogenes assay for BAX PCR ac- ences in counts within and between species over time. CC data cording to the manufacturer’s instructions. were not log transformed for analyses because of the presence of For the detection of E. coli O157:H7, 25 ml of milk was numerous null values. Spearman’s rank correlation coefficient was enriched in flasks containing 225 ml of enterohemorrhagic E. coli utilized to determine correlations between cell counts and pres- enrichment broth and incubated for 24 h at 37 Ϯ 0.5ЊCinan ence of the pathogen. Results at P Ͻ 0.05 were considered sig- orbital shaker (Lab line Max Q 2508, Barnstead International, nificant. Histograms were constructed with Microsoft Excel (Mi- Dubuque, Iowa) as recommended in the BAM (chap. 4a, Diar- crosoft, Redmond, Wash.). rheagenic Escherichia coli, available at: http://www.cfsan.fda. gov/ϳebam/bam-4a.html). The BAX PCR assay for E. coli O157: RESULTS ␮ H7 was then run on 5- l aliquots of enrichment cultures according SPCs and CCs. A total of 122 and 123 samples from to the manufacturer’s instructions. 11 farms were analyzed for SPCs and CCs, respectively. Staphylococcus aureus was detected solely through direct SPC and CC data from one study farm were removed from plating of raw milk on CHROMagar Staph aureus as previously described. Two samples originally outside our predicted range (Ta- analyses after well water contamination was discovered ble 4) were reanalyzed the following day (Ͻ24hat4ЊC). Isolates through routine state testing as per the PMO; this water obtained were purified on TSAYE incubated for 24 h at 35 Ϯ contaminated the entire dairy. An additional four SPC val- 0.5ЊC. Purified colonies were Gram stained, examined for catalase ues were removed from analysis because of the addition of activity, and regrown in BHI incubated for 24 h at 35 Ϯ 0.5ЊC. starter culture before milk collection (farm K). Changes in To test for coagulase, 10 ␮l of BHI enrichment was added to 0.5 SPCs on individual farms over time are presented in Table ml of reconstituted coagulase plasma with EDTA (BBL coagulase 1. SPCs of the remaining 118 samples ranged from 10 to plasma, Becton Dickinson), mixed thoroughly, and incubated at 9,300,000 CFU/ml with mean, geometric mean (geo-mean), Њ 35 C. Tubes were examined periodically at 6-h intervals for up to and median values of 99,000, 1,400, and 1,450 CFU/ml, 24 h. Any degree of clotting within 24 h was considered a positive respectively (Table 1). The geo-mean is a log transforma- test result. Random coagulase-positive isolates were ribotyped for tion of data obtained over a period of time that tends to S. aureus confirmation. dampen the effects of outlying data. The European Union Ribotyping. Isolates of L. monocytogenes and S. aureus were (EU) employs this method to take into account seasonal subtyped through ribotyping with the automated Riboprinter Mi- variations because regulatory samples are taken multiple crobial Characterization System (DuPont Qualicon) according to times over a 2- or 3-month period. Geo-means may better the manufacturer’s instructions using the restriction endonuclease represent the average value in this study because of the EcoRI (Qualicon). outlying data, which may bias the mean. There was no sig- Statistical analyses. The resulting data were analyzed using nificant effect of sampling week or month on SPCs ob- SPSS for Windows (version 15.0.1, SPSS Inc., Chicago, Ill.). Cell served on individual farms or all farms together. Overall, J. Food Prot., Vol. 71, No. 8 PATHOGEN INCIDENCE IN FARMSTEAD RAW MILK 1583

FIGURE 1. Relative frequency of raw milk standard plate FIGURE 2. Relative frequency of raw milk standard plate counts (SPCs) for all species (n ϭ 118). counts (SPCs) in cow’s (Ⅵ,nϭ 57), sheep’s (Ⅺ,nϭ 12), and Ⅵ ϭ

goat’s ( , n 49) milk. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/71/8/1580/1679328/0362-028x-71_8_1580.pdf by guest on 26 September 2021

96.5% of samples analyzed had SPCs of Ͻ100,000 CFU/ ml; 79.6% had Ͻ10,000 CFU/ml, 43.2% had Ͻ1,000 CFU/ Ͻ ml, and the remaining 16.9% had SPCs of Ͻ100 CFU/ml of 263, 1, and 5.5 CFU/ml, respectively. Changes in CCs (Fig. 1). When analyzed separately by species (Fig. 2), cow on individual farms over time are presented in Table 2. milk SPCs were all Ͻ100,000 CFU/ml, with 82.4% There was no significant effect of sampling week or month Ͻ10,000 CFU/ml, 54.4% Ͻ1,000 CFU/ml, and 24.6% on CCs observed on individual farms or all farms together. Ͻ100 CFU/ml. Mean, geo-mean, and median SPCs were Of 121 samples analyzed for coliforms, 84.3% contained 4,900, 680, and 700 CFU/ml, respectively. Although goats Ͻ100 CFU/ml, with 61.2% Ͻ10 CFU/ml (Fig. 3). Of the were the only species producing milk with SPCs Ͼ100,000 remaining samples, 11.6% had CCs of 101 to 1,000 CFU/ CFU/ml (8.2%; Fig. 2), 73.4% of the SPCs were Ͻ10,000 ml and 4.1% had 1,001 to 10,000 CFU/ml. When separated CFU/ml, 24.5% were Ͻ1,000 CFU/ml, and 12.2% were by species (Fig. 4), 89% of cow milk samples were Ͻ100 Ͻ100 CFU/ml. Mean, geo-mean, and median values for CFU/ml, with 62% Ͻ10 CFU/ml. Mean and median CCs goat milk SPCs were 230,000, 2,543, and 3,100 CFU/ml, for cow’s milk alone were 343 and 6.5 CFU/ml, respec- respectively. Although no samples of sheep’s milk had tively. Seventy-six percent of goat milk samples had CCs SPCs Ͻ100 CFU/ml, all were Ͻ100,000 CFU/ml, with Ͻ100 CFU/ml, with 55% Ͻ10 CFU/ml. For sheep milk mean, geo-mean, and median SPCs of 6,900, 2,758, and samples, 92% were Ͻ100 CFU/ml, with 75% Ͻ10 CFU/ 1,750 CFU/ml, respectively. The overwhelming majority of ml. Mean and median CCs for goat and sheep milk samples sheep milk samples (75%) had Ͻ10,000 CFU/ml, with were 212 and 4.5 CFU/ml and 24.6 and 2.8 CFU/ml, re- 16.7% containing Ͻ1,000 CFU/ml. Observed differences spectively. No significant difference in CCs between spe- in SPCs between species were significant (P ϭ 0.029), with cies (P ϭ 0.723) was observed even with outliers removed lower SPCs observed for cow milk samples. (P ϭ 0.265), probably because of variance within and be- A total of 121 milk samples were analyzed for coliform tween species. Although correlations between SPCs and bacteria. CCs observed in all milks ranged from Ͻ1to CCs were limited, a significant positive correlation was ob- 9,600 CFU/ml, with mean, geo-mean, and median values served on farm E (r ϭ 0.629, P Ͻ 0.05).

TABLE 2. Raw milk coliform counts at individual farmstead operations Coliform counts (CFU/ml) at farma:

Wk ABCDE F GIJK

13Ͼ500b Ͻ1 9.5 15 4.5 4.5 1 Ͻ1 6.5 2 Ͻ1 525 Ͻ1 34.5 52 Ͼ100b 7 9,600 Ͻ1 21.5 312 Ͻ1 Ͻ1 70.5 38.5 265 5.5 3 Ͻ1 17.5 423 Ͻ1 3.5 31 7.5 335 6.5 1.5 Ͻ1 1,300 5 1.5 1 Ͻ1 38 4 1,380 24 2.5 Ͻ113 65Ͻ1 1 8.5 44.5 41 7 25 76 41.5 7 5.5 Ͻ1 Ͻ1 220 420 655 Ͻ14Ͻ14 8 Ͻ1NTc Ͻ1 17 110.5 85.5 5.5 Ͻ1 Ͻ1 58.5 9 2.5 4,250 Ͻ1 23.5 420 450 7 8.5 Ͻ117 10 240 3.5 Ͻ1 19.5 NT 480 3.5 Ͻ1 Ͻ112 11 NT Ͻ1 Ͻ1 3.5 NT 665 15 1 Ͻ1 8,650 12 2 2 Ͻ1 NT 6 320 3 Ͻ1 Ͻ1NT 13 Ͻ1 Ͻ1 3 17.5 425 NT 6.5 Ͻ1 Ͻ162 a Results are expressed as CFU per milliliter for ease of comparison with previous studies. b Estimated value. c NT, not tested. 1584 D’AMICO ET AL. J. Food Prot., Vol. 71, No. 8

FIGURE 3. Relative frequency of raw milk coliform counts (CCs) in milk of all species (n ϭ 121). FIGURE 4. Relative frequency of raw milk coliform counts Ⅵ ϭ Ⅺ ϭ

(CCs) in cow’s ( ,n 63), sheep’s ( ,n 12), and goat’s Downloaded from http://meridian.allenpress.com/jfp/article-pdf/71/8/1580/1679328/0362-028x-71_8_1580.pdf by guest on 26 September 2021 Ⅵ ϭ SSCs. SCCs (Table 3) differed significantly between ( , n 47) milk. species (P Ͻ 0.001), with goat’s milk containing the highest levels followed by sheep’s and then cow’s milk. Because the course of the study, including samples from all species. SCC standards vary by species, statistical analyses were Coagulase-positive isolates formed grade 4ϩ clots within 4 conducted separately. Figure 5 shows the relative frequency to6hofincubation in all but one sample, which formed a distribution of SCCs for cow’s, goat’s, and sheep’s milk. similar clot after 18 h of incubation. All selected isolates All cow milk samples (n ϭ 61) contained Ͻ750,000 so- of coagulase-positive staphylococci were identified as S. matic cells per ml, with 83% under 400,000 cells per ml. aureus by ribotyping. As the most common pathogen de- The remaining 17% fell between 400,000 and 700,000 cells tected, S. aureus was isolated by direct plating in 33.3% of per ml with mean, geo-mean, and median values of samples (44 of 132) at an average level of 250 CFU/ml 179,147, 127,270, and 123,000 cells per ml, respectively. (median, Ͻ1 CFU/ml) (Table 4). Seventeen (27.4%) of 62 Three goat milk samples exceeded 1,000,000 cells per ml, cow milk samples, 9 (18.4%) of 49 goat milk samples, and with 81% Ͻ700,000 cells per ml and almost half (46%) 18 (85.7%) of 21 sheep milk samples were positive for S. Ͻ400,000 cells per ml. Goat milk SCCs were 512,292 cells aureus. per ml on average, with a median of 400,000 cells per ml. L. monocytogenes was isolated from 2.3% of all milk All sheep milk samples contained fewer than 700,000 cells samples (3 of 133 samples) or 4.8% of cow milk samples per ml. Mean SCC for raw sheep’s milk was 383,955 cells (3 of 62 samples). The three isolates of L. monocytogenes per ml, with a median value of 460,000 cells per ml. Sig- were obtained from two of the five cow farms. Two of the nificant correlations between SCCs and SPCs on farm G (r three isolates were found at the same farm 10 weeks apart ϭ 0.767, P Ͻ 0.01) and CCs on farm F (r ϭ 0.615, P Ͻ and were identified by direct plating at Ͻ1 CFU/ml. Sub- 0.05) were observed. SCCs were not correlated with the typing revealed that the two isolates were of different ri- presence of pathogens, including S. aureus. botypes (DUP-1030B and DUP-1045B). The first isolate Pathogen incidence. Of the 11 farms, 8 (73%) had at obtained in sampling week 1 was of the same ribotype least one milk sample that tested positive for S. aureus over (DUP-1030B) as the isolate recovered through enrichment

TABLE 3. Somatic cell counts at individual farmstead operations Somatic cell counts (cells/ml) at farm:

Wk ABC D E FGH IJK

1 400,000 348,000 264,000 1,213,000 81,000 264,000 246,000 566,000 81,000 62,000 606,000 2 606,000 696,000 230,000 650,000 50,000 200,000 200,000 27,000 66,000 50,000 460,000 3 264,000 857,000 303,000 NTa 29,000 373,000 230,000 NT 76,000 71,000 348,000 4 606,000 857,000 283,000 81,000 33,000 400,000 187,000 162,000 123,000 47,000 325,000 5 650,000 919,000 303,000 303,000 71,000 650,000 162,000 93,000 87,000 62,000 373,000 6 47,000 303,000 152,000 1,493,000 38,000 123,000 123,000 650,000 283,000 41,000 373,000 7 460,000 696,000 246,000 606,000 54,000 460,000 141,000 528,000 200,000 50,000 325,000 8 460,000 NT 264,000 492,000 54,000 492,000 200,000 214,000 100,000 29,000 400,000 9 492,000 650,000 325,000 303,000 115,000 400,000 230,000 303,000 141,000 50,000 460,000 10 460,000 606,000 460,000 460,000 NT 985,000 528,000 107,000 230,000 71,000 606,000 11 NT 528,000 348,000 528,000 NT 528,000 187,000 174,000 162,000 66,000 400,000 12 650,000 919,000 303,000 NT 71,000 1,393,000 214,000 NT 132,000 71,000 NT 13 528,000 800,000 230,000 303,000 66,000 NT 152,000 NT NT 66,000 373,000 GMb 404,995 645,895 276,977 459,340 55,832 431,190 201,075 198,587 126,671 54,972 411,707 a NT, not tested. b GM, geometric mean. J. Food Prot., Vol. 71, No. 8 PATHOGEN INCIDENCE IN FARMSTEAD RAW MILK 1585

ically consisted of single samples of single-species milk collected from a large number of farms. Thus, direct com- parisons are not intended and associations should be inter- preted with caution. Results from samples collected early in the study re- vealed widespread contamination on one study farm; the contamination was later traced to well water. These findings highlight the benefits of routine raw milk and water screen- ing for microbiological quality at farmstead cheese opera- tions. Although there are no standards for SPCs in raw milk used for the manufacture of raw milk products in the United FIGURE 5. Relative frequency of raw milk somatic cell counts States, the SPC limit for prepasteurized raw milk is 100,000 Ⅵ ϭ Ⅺ ϭ (SCCs) for cow’s ( ,n 61), sheep’s ( ,n 22), and goat’s CFU/ml (43); only 3.5% of all samples in this study ex- Downloaded from http://meridian.allenpress.com/jfp/article-pdf/71/8/1580/1679328/0362-028x-71_8_1580.pdf by guest on 26 September 2021 Ⅵ ϭ ( , n 48) milk. ceeded this limit, and 79.6% of milk samples contained Ͻ10,000 CFU/ml, the target recommended as the best prac- from a milk sample collected at the other positive farm in tice for raw milk intended for the manufacture of raw milk week 6. Detection following enrichment also suggests con- cheese. Overall, cow milk SPCs were significantly lower tamination levels of Ͻ1 CFU/ml. E. coli O157:H7 was re- than those for both sheep’s and goat’s milk. When analyzed covered from one sample (0.75%) of goat’s milk following separately by species, 100, 91.8, and 100% of cow, goat, enrichment. Study farms on which pathogens were found and sheep milk samples, respectively, contained SPCs of Ͻ in raw milk samples are not identified to ensure the confi- 100,000 CFU/ml (Fig. 2). All dairies processing cow’s dentiality of participants. Salmonella was not recovered milk also had geo-mean SPCs well below 100,000 CFU/ml from any of the 133 samples analyzed. Raw milk cheeses (Table 1) as per EU regulations for raw cow’s milk (10). produced from contaminated milks were examined after Cow, goat, and sheep milk SPCs in this study were similar manufacture and at time of sale using the BAX PCR system to those obtained as part of the National Animal Health as previously described, and no viable pathogens were de- Monitoring System (NAHMS) Dairy 2002 survey (44). In tected or recovered (data not shown). In another instance, the NAHMS survey, 92.1% of cow milk samples were the contaminated raw milk was pasteurized shortly after within the PMO limits compared with 100% compliance sample collection. (96.5% for all species combined) reported here. The dif- ferences lie in overall distribution; 54.4% of cow milk sam- DISCUSSION ples (43.2% of samples for all species combined) in our In this study, trends in bacterial and somatic cell loads study had SPCs Ͻ1,000 CFU/ml compared with only 12% in raw milk used for the production of farmstead cheese of samples in the NAHMS survey, where the majority of were evaluated through repeated collection of samples from SPCs were between 1,000 and 4,999 (48%) compared with farms over time. Although laboratory procedures were per- only 21% (27.1% for all species) in our study. Of 855 raw formed in a central laboratory and remained consistent milk samples from New York State taken over 2 years, only throughout the study, the methodology and experimental 26.8% of these samples had SPCs of Ͻ5,000 and the re- design differ from those of many other studies, which typ- maining 73.2% had Ͼ5,000 CFU/ml (1), almost twice the

TABLE 4. Staphylococcus aureus counts on farms where this pathogen was found S. aureus counts (CFU/ml) at farm:

Wk ABDF G H IJ

153Ͻ1 Ͻ11 Ͻ1 12,500a Ͻ1 Ͻ1 218Ͻ17Ͻ1 28.5 15 Ͻ117 3 Ͻ1 Ͻ1 Ͻ1 Ͻ1 Ͻ1NTb 1.5 Ͻ1 48Ͻ144Ͻ1 39 72 7.5 Ͻ1 53Ͻ14Ͻ1 18.5 4.5 Ͻ1 Ͻ1 6 13 47.5 Ͻ1 Ͻ1 12.5 4 Ͻ1 25.5 7 55.5 51 Ͻ1 Ͻ1 19.5 8.5 Ͻ1 Ͻ1 85NTϽ1 Ͻ1 24 85.5 Ͻ1 Ͻ1 9 Ͻ156 Ͻ1 Ͻ12659Ͻ1 Ͻ1 10 9 11 Ͻ1 Ͻ1 20,600a Ͻ1 2.5 Ͻ1 11 NT Ͻ1 Ͻ1 Ͻ1 30.5 8.5 3.5 Ͻ1 12 NT Ͻ1NTϽ1 26.5 NT Ͻ1NT 13 4 Ͻ1 Ͻ1 NT 24.5 NT Ͻ1 Ͻ1 a Result obtained after 24 h of incubation at 4ЊC. b NT, not tested. 1586 D’AMICO ET AL. J. Food Prot., Vol. 71, No. 8 percentage reported in the NAHMS survey (40%) (44) and Belgium (2.45 log CFU/ml) (6), yet 10-fold lower than the almost three times that observed in our study (25%). The United States (3.4 log CFU/ml) (20). geo-mean of cow milk SPCs reported here (680 CFU/ml) All cow milk samples (n ϭ 65) were within the limits is also substantially lower than that observed in New York of the PMO for SCC at Ͻ750,000 cells per ml. The overall State (11,400 CFU/ml) (1). Mean SPCs for cow’s milk in geometric mean SCCs for all cow milk samples of 127,270 the present study (4,900 CFU/ml) were also far lower than cells per ml was within the EU limit of 400,000 cells per the 26,600 CFU/ml calculated using data from approxi- ml. On an individual basis, only one farm exceeded this mately 90% of the commodity fluid milk produced in Ver- limit. Average SCC in farmstead cow’s milk reported here mont during the same sampling period (45). Although all (179,147 cells per ml) was nearly half that of producers sheep milk samples were within the standards of the PMO shipping milk in the state of Vermont during the same sam- for raw milk to be pasteurized (100,000 CFU/ml), 8.2% of pling time frame (341,608 cells per ml) (45). Compared goat milk samples exceeded the standards. All sheep and with average SCCs from throughout the year, our results

goat milk farms maintained geo-mean SPCs (Table 1) with- are still below the national average of 288,000 cells per ml Downloaded from http://meridian.allenpress.com/jfp/article-pdf/71/8/1580/1679328/0362-028x-71_8_1580.pdf by guest on 26 September 2021 in the EU limits for raw milk intended for the manufacture and the state of Vermont average (250,000 cells per ml) for of raw milk products by a process that does not involve milk from Dairy Herd Improvement herds during 2006 (26) any heat treatment (Ͻ500,000 CFU/ml) (10). In addition, and lower than the recommended SCCs in raw milk used 73 and 75% of goat and sheep milk samples, respectively, to make cheese (Ͻ250,000 cells per ml). All sheep milk were below the target of 10,000 CFU/ml set for best prac- samples (n ϭ 22) were within the limits of the PMO at tice when making cheese from raw cow’s milk, and only Ͻ750,000 cells per ml. Although the average SCC in raw two goat milk samples exceeded the Ͻ250,000 CFU/ml tar- goat’s milk was the highest of the three milk types in the get recommended for small ruminants. Because SPCs do present study (512,000 cells per ml), it was slightly lower not differ significantly between sheep’s and goat’s milk, than that reported for raw goat’s milk used for cheese pro- similar microbiological limits have been used for both spe- duction in Italy (13). Three samples (6.2%) exceeded the cies (27). In our study geo-mean and median values were SCC limits for goat’s milk under the PMO, and 93.8% of similar for sheep’s and goat’s milk but arithmetic means samples were in compliance. Because the instrument uti- were not because of the presence of outlying data. The me- lized to determine SCCs for small ruminant milk samples dian SPCs for goat and sheep bulk tank milk in our study was calibrated to cow’s milk, the resulting counts may have of 3.5 (goat) and 3.44 (sheep) log CFU/ml are more than been affected. 1-log lower than levels typically reported in the literature, The prevalence of four target pathogens in raw milk including reports from Switzerland of 4.69 and 4.78 log for farmstead cheesemaking was evaluated in this study. CFU/ml for goat’s and sheep’s milk, respectively (27). The Numerous researchers have examined the prevalence of goat milk mean SPC in the present study (5.36 log CFU/ foodborne pathogens in bulk tank milk (19, 24, 29, 30, 36, ml) was higher than that reported for raw goat’s milk des- 44, 46). However, the data range widely because of differ- tined for the manufacture of raw milk cheese in Italy (mean, ences in geographic location, season, hygiene, herd size 4.7 log CFU/ml) (13) and for a grade A research herd in (36), and detection methodology. Oklahoma (4.16 CFU/ml) (48), likely the result of outlying The repeated sampling employed in our study resulted data. Although data on coliform levels in goat’s milk are in pathogen incidence rates probably higher than those that limited, our results (2.33 log CFU/ml) are in agreement would result from single point-in-time surveys (18). In our with those of others (13, 48). study, 50 (36.5%) of 137 samples contained one or more Although no U.S. federal standards exist for coliform pathogens. Overall pathogen prevalence rates reported in levels in raw milk, 61.2% of all samples tested (n ϭ 121) the United States range from 10.5 to 32% (18, 19, 36), conformed to PMO standards for grade ‘‘A’’ pasteurized although target pathogens differ. Many surveys have not (not raw) milk at Ͻ10 CFU/ml (Fig. 3), and 84.3% of sam- included S. aureus as a pathogen of interest, although S. ples were within the recommended standards for raw milk aureus constituted 92% of the samples testing positive for for cheese at Ͻ100 CFU/ml. Levels of coliform bacteria pathogens in our study. When only Salmonella, E. coli reported in the literature vary widely. Ranges observed here O157:H7, and L. monocytogenes are considered, the inci- for all milks (Ͻ1 CFU/ml to 4.89 log CFU/ml) are similar dence of pathogenic bacteria drops drastically to 3%. to those reported by Desmasures and others (7) for cow’s As a major causative agent of mastitis, S. aureus is one milk from the Camembert region of Normandy (Ͻ1 to 4.34 of the most common contagious pathogens infecting dairy log CFU/ml) and Jayarao and Wang (20) in the U.S. (0 to cows. Commonly isolated from raw milk, S. aureus has 4.7 log CFU/ml). When analyzed separately, the maximum been frequently associated with foodborne outbreaks related coliform count in cow’s milk was 1 log less than all milks to cheese made from raw or unspecified milk in France (5). combined with a range from Ͻ1 CFU/ml to 3.98 CFU/ml. S. aureus was detected in 17 (27.4%) of 62 cow milk sam- In a study by Desmasures and others (7), 84% of cow milk ples in our study, similar to the 25.1% observed by De Reu samples contained coliform levels Ͻ2 log CFU/ml, similar et al. (6) in Belgium. Similar contamination levels of 240 to the 90.3% in our study yet slightly higher than found for CFU/ml also were found in a study on S. aureus prevalence dairy herds in the United States at 73% (20) and 76.5% (1) at a Norwegian dairy that produced raw cow’s milk cheese as well as in Belgium at 67.1% (6). Mean coliform counts (21). Small ruminant milk also can contain S. aureus, which (2.29 log CFU/ml) were also similar to those reported in has been detected in 30 to 40% of goat (27, 41) and sheep J. Food Prot., Vol. 71, No. 8 PATHOGEN INCIDENCE IN FARMSTEAD RAW MILK 1587

(27) milk samples, including those destined for raw milk dairy cows, which can serve as reservoirs for human food- cheese manufacture (41). Although the incidence in goat’s borne salmonellosis via fecal contamination, and unpas- milk in the present study falls below this range, sheep’s teurized milk and milk products have been implicated in milk had a higher incidence because of the small number numerous outbreaks of salmonellosis (9). Although low in- of samples, most of which were taken from a single farm cidence rates of 0.1 to 2.9% have been reported in Europe (n ϭ 12). The overall variation in S. aureus levels and (7, 34) and of 0.17% in Canada (40), higher isolation rates isolation across and within species is likely due to the ten- of 1.5 to 8.9% from cow’s milk have been reported in the dency for intermittent shedding of the organism. The lack United States (18, 19, 29, 36, 44), depending on geographic of a positive correlation between SCCs and S. aureus could location. In the present study, Salmonella was not detected be explained by the presence of other mastitis pathogens in any samples analyzed, similar to the result obtained for not included in the survey. Mean levels of S. aureus ob- raw cow’s milk and raw cow’s milk cheese in Belgium (6). served in this study (250 CFU/ml) are below the target for Although incidence data for small ruminant milk are lim-

raw milk cheese manufacture (500 CFU/ml) and do not ited, Salmonella has been detected in goat’s milk (13). Tra- Downloaded from http://meridian.allenpress.com/jfp/article-pdf/71/8/1580/1679328/0362-028x-71_8_1580.pdf by guest on 26 September 2021 necessarily present a food safety risk because virulence is ditional culture methods for the isolation of Salmonella are associated with the production of heat-stable enterotoxins, time-consuming and labor intensive, and the use of PCR which generally occurs when populations are Ͼ5 log CFU/ can improve detection in bulk tank milk (22) through en- ml (Ͼ100,000 CFU/ml). Thus, cheeses made from raw milk hanced sensitivity in a much shorter time. The failure to under EU regulation (11) must contain Ͻ5 log CFU/ml at detect Salmonella in the present study is most likely due to the time of manufacture, when the number of staphylococci low levels of the organism in milk, an issue possibly con- is expected to be highest. If values Ͼ5 log CFU/ml are founded by the enrichment protocol employed before the detected, the cheese batch must be tested for the presence PCR assay. In raw milk, a high load of background flora, of enterotoxins. However, S. aureus is generally regarded notably coliforms, may outcompete Salmonella present at as a poor competitor, resulting in limited growth during low levels during primary enrichment in lactose broth. Op- cheesemaking in the presence of active starter culture. To timization of enrichment protocols for PCR assays requires better understand the risk of foodborne illness attributed to further work. the presence of S. aureus on study farms, an examination Although the ruminant farm environment may serve as of the genetic diversity and antibiotic resistance within our a significant reservoir for L. monocytogenes, including sub- isolates and their propensity for toxin production warrants types linked to human listeriosis (31), L. monocytogenes further investigation. was detected in only three samples of cow’s milk at Ͻ1 Dairy cattle are known reservoirs of Shiga toxin–pro- CFU/ml, with corresponding prevalence rates of 4.8% (3 of ducing E. coli, including serotype O157:H7 (47). Incidence 62 samples) for cow’s milk and 2.3% (3 of 133 samples) appears to be low (0.75%) in the bulk tank milk of cull for all milks combined. Two isolates were recovered from cows (30) but may be higher (4.3%) in raw milk obtained the milk of a single farm, and although L. monocytogenes from case farms (47). Although potentially pathogenic Shi- subtypes can persist in a herd’s bulk milk (12, 28), our ga toxin–producing E. coli strains have been isolated from subtyping data suggest recontamination of milk from a sep- 2.4 to 3.8% of milk samples in South Dakota, western Min- arate source rather than persistent contamination. One of nesota (19), and Pennsylvania (18), none of the isolates these two subtypes shared a ribotype with a third isolate were of serotype O157:H7. Similarly, no E. coli O157:H7 obtained from a separate farm 6 weeks later. Although ge- isolates were detected in surveys conducted in southeast netically indistinguishable isolates were shared between Scotland (3), Italy (25), or The Netherlands (17) in 500, processors, transmission is improbable because of the dis- 100, and 1,011 samples, respectively. Although our sam- tance and the lack of an epidemiological link between fa- pling was conducted during late spring and summer when cilities. In accordance with our data, detection through di- isolation rates generally are higher (30), E. coli O157:H7 rect plating and enrichment has revealed that contamination was not detected in any cow milk samples. Results are not levels are typically low, ranging from Ͻ1 to 37 CFU/ml likely to have been affected by intermittent shedding as in (12, 14, 24, 44), including milk of high microbiological other studies because of the repeated sampling of individual quality used for cheesemaking (8). Although in some sur- farms. Our data and those of others suggest that contami- veys, no L. monocytogenes was detected in bulk tank milk nation of milk may be uncommon (3). Non-O157:H7 Shiga (24), the incidence rate for cow’s milk reported here is on toxin–producing E. coli also has been detected in both the lower end of the range reported in surveys conducted goat’s and sheep’s milk at 16.3 and 12.7%, respectively throughout the United States of 2.8 to 7% (18, 19, 24, 28, (27). In our study, E. coli O157:H7 was isolated from one 36, 44). Our results also are in accordance with detection sample of goat’s milk (0.75%) through enrichment, sug- rates from outside the United States, ranging from 1 to gesting a contamination level of Ͻ1 CFU/ml. Contamina- 5.3% throughout Europe (14, 15, 34, 38, 46) and in Ontar- tion of goat’s milk may not be uncommon; raw goat’s milk io, Canada (40). Higher incidence rates of up to 33.3% (15) was implicated in an outbreak of E. coli O157:H7 infection have been reported for dairy silo milk, probably because of in British Columbia in 2001 (33) and was isolated at 1.5 the longer storage time and the commingling of milk from CFU/ml from 1.7% of raw goat’s milk samples in an Italian numerous producers (46). Although both goats and sheep survey (13). are known carriers of L. monocytogenes (35), no samples Salmonella also can be found in the intestinal tracts of of raw milk from small ruminant farms were positive for 1588 D’AMICO ET AL. J. Food Prot., Vol. 71, No. 8 this pathogen. Other investigators have reported prevalence the Dairy Section of the Vermont Agency of Agriculture for providing rates of 2.56% in raw goat’s milk (14) and 2.19% in sheep’s producer milk quality data from calendar year 2006. We also thank Tyler Curran for her help in sample collection and processing and Alan Howard milk (35) in Spain. In the latter study, failure to isolate from for his assistance with statistical analyses. direct plating indicates that contamination levels were Ͻ5 CFU/ml (35). L. monocytogenes prevalence reported here REFERENCES may, however, underestimate the yearly incidence; Listeria spp. contamination seems to increase in the winter months 1. Boor, K. J., D. P. Brown, S. C. Murphy, S. M Kozlowski, and D. K. Bandler. 1998. Microbiological and chemical quality of raw milk in while animals are indoors, grouped together, and feeding New York State. J. Dairy Sci. 81:1743–1748. on silage (38). 2. Centers for Disease Control and Prevention. 1999. Achievements in The findings of this study indicate that most raw milk public health 1900–1999: safer and healthier foods. Morb. Mortal. intended for farmstead cheesemaking was of high micro- Wkly. Rep. 48:905–913. biological quality according to current U.S. and EU stan- 3. Coia, J., Y. Johnston, N. Steers, and M. Hanson. 2001. A survey of the prevalence of Escherichia coli O157:H7 in raw cow’s milk and dards and in comparison with quality reports from the state raw milk cheeses in southeast Scotland. Int. J. Food Microbiol. 66: Downloaded from http://meridian.allenpress.com/jfp/article-pdf/71/8/1580/1679328/0362-028x-71_8_1580.pdf by guest on 26 September 2021 of Vermont, the United States as a whole, and other coun- 63–69. tries. Pathogen incidence and milk quality data also are sim- 4. Costello, M., M.-S. Rhee, M. P. Bates, S. Clark, L. O. Luedecke, ilar to those reported in surveys examining the quality of and D.-H. Kang. 2003. Eleven-year trends of microbiological quality small scale and farmstead milk destined for the manufacture in bulk tank milk. Food Prot. Trends 23:393–400. 5. De Buyser, M.-L., B. Dufour, M. Maire, and V. Lafarge. 2001. Im- of raw milk products, including cheese, in European coun- plication of milk and milk products in food-borne diseases in France tries. Thus, factors inherent to small-scale operations such and in different industrialised countries. Int. J. Food Microbiol. 67: as the lack of milk pooling and holding, pasture feeding, 1–17. and very small herd sizes may play a role in the overall 6. De Reu, K., K. Grijspeerdt, and L. Herman. 2004. A Belgian survey quality of raw milk. However, milk quality may not be cor- of hygiene indicator bacteria and pathogenic bacteria in raw milk and direct marketing of raw milk farm products. J. Food Saf. 24: (4, 40, related with milk safety. In accordance with others 17–36. 44), our results suggest that one microbiological measure- 7. Desmasures, N., F. Bazin, and M. Gueguen. 1997. Microbiological ment, such as the SCC or SPC (44), may not serve as a composition of raw milk from selected farms in the Camembert re- predictor of other bacterial values, including pathogen pres- gion of Normandy. J. Appl. Microbiol. 83:53–58. ence, and that compliance with bacteriological limits does 8. Desmasures, N., and M. Gueguen. 1997. Monitoring the microbiol- ogy of high quality milk by monthly sampling over 2 years. J. Dairy not guarantee the absence of pathogenic bacteria. Res. 64:271–280. The variation from farm to farm, regardless of species, 9. El-Gazzar, F. E., and E. H. Marth. 1992. Salmonellae, salmonellosis, indicates that some operations practice strict hygienic con- and dairy foods: a review. J. Dairy Sci. 75:2327–2343. trols but that additional effort is needed at other operations. 10. European Union. 2004. Commission regulation (EC) no. 853/2004 of Although repeated sampling may provide results suggesting the European Parliament and of the Council of 29 April 2004 laying Off. J. Eur. a higher incidence of a pathogen at the farm level (20), the down specific hygiene rules for food of animal origin. Union L226:22–82. Available at: http://europa.eu.int/eur-lex/lex/ incidence reported here is low in comparison to that found LexUriServ/site/en/oj/2004/l࿞226/l࿞22620040625en00220082.pdf. Ac- in other surveys and therefore may be conservative. Our cessed 1 November 2007. data suggest that if present, these pathogens are present at 11. European Union. 2005. Commission regulation (EC) no. 2073/2005 of extremely low population levels. However, improper stor- 15 November 2005 on microbiological criteria for foodstuffs. Off. J. age of raw milk and raw milk products facilitates pathogen Eur. Union L338:1–29. Available at: http://europa.eu.int/eur-lex/lex/ LexUriServ/site/en/oj/2005/l࿞338/l࿞33820051222en00010026.pdf. Ac- growth to levels of public health concern. The physico- cessed 1 November 2007. chemical parameters of some cheeses produced from raw 12. Fenlon, D. R., T. Stewart, and W. Donachie. 1995. The incidence, milk permit the survival and possible growth of certain numbers and types of Listeria monocytogenes isolated from farm pathogens. Because this study was limited to a small num- bulk tank milks. Lett. Appl. Microbiol. 20:57–60. ber of farms sampled over time, future investigations 13. Foschino, R., R. Barucco, and K. Stradiotto. 2002. Microbial com- position, including the incidence of pathogens, of goat milk from should include the sampling of farmstead operations rep- the Bergamo region of Italy during a lactation year. J. Dairy Res. resenting multiple regions over each season. Further inves- 69:213–225. tigation is also warranted to determine sources of contam- 14. Gaya, P., C. Saralegui, M. Medina, and M. Nunez. 1996. Occurrence ination in an effort to establish control programs. The data of Listeria monocytogenes and other Listeria spp. in raw caprine presented here form part of an exposure assessment and risk milk. J. Dairy Sci. 79:1936–1941. reduction plan to promote continuous production of micro- 15. Harvey, J., and A. Gilmour. 1992. Occurrence of Listeria species in raw milk and dairy products produced in Northern Ireland. J. Appl. biologically safe artisan and farmstead cheese. These data Bacteriol. 72:119–125. also will help inform risk assessments of the microbiolog- 16. Headrick, M. L., S. Korangy, N. H. Bean, F. J. Angulo, S. F. Altek- ical safety of farmstead cheeses, particularly those manu- ruse, M. E. Potter, and K. C. Klontz. 1998. The epidemiology of raw factured from raw milk. milk–associated foodborne disease outbreaks reported in the United States, 1973 through 1992. Am. J. Public Health 88:1219–1221. ACKNOWLEDGMENTS 17. Heuvelink, A., B. Bleumink, F. Biggelaar, M. Te Giffel, R. Beumer, and E. De Boer. 1998. Occurrence and survival of verocytotoxin- This study was supported by a U.S. Department of Agriculture, An- producing Escherichia coli O157 in raw cow’s milk in The Neth- imal and Plant Health Inspection Service grant (06-9650-0196GR) award- erlands. J. Food Prot. 61:1597–1601. ed to Dr. Catherine Donnelly. The authors gratefully acknowledge the 18. Jayarao, B. M., S. C. Donaldson, B. A. Straley, A. A. Sawant, N. cheese companies for their participation and assistance with this study and V. Hegde, and J. L. Brown. 2006. A survey of foodborne pathogens J. Food Prot., Vol. 71, No. 8 PATHOGEN INCIDENCE IN FARMSTEAD RAW MILK 1589

in bulk tank milk and raw milk consumption among farm families Available at: http://www.phac-aspc.gc.ca/publicat/ccdr-rmtc/02vol28/ in Pennsylvania. J. Dairy Sci. 89:2451–2458. dr2801eb.html. Accessed 13 December 2007. 19. Jayarao, B. M., and D. R. Henning. 2001. Prevalence of foodborne 34. Rea, M. C., T. M. Cogan, and S. Tobin. 1992. Incidence of patho- pathogens in bulk tank milk. J. Dairy Sci. 84:2157–2162. genic bacteria in raw milk in Ireland. J. Appl. Bacteriol. 73:331– 20. Jayarao, B. M., and L. Wang. 1999. A study on the prevalence of 336. gram-negative bacteria in bulk tank milk. J. Dairy Sci. 82:2620– 35. Rodriguez, J. L., P. M. Gaya, M. Medina, and M. Nunez. 1994. 2624. Incidence of Listeria monocytogenes and other Listeria spp. in ewes’ 21. Jorgensen, H., J. T. Mork, and L. M. Rorvik. 2005. The occurrence raw milk. J. Food Prot. 57:571–575. of Staphylococcus aureus on a farm with small-scale production of 36. Rohrbach, B. W., F. A. Draughon, P. M. Davidson, and S. P. Oliver. raw milk cheese. J. Dairy Sci. 88:3810–3817. 1992. Prevalence of Listeria monocytogenes, Campylobacter jejuni, 22. Karns, J. S., J. S. Van Kessel, B. J. McCluskey, and M. L. Perdue. Yersinia enterocolitica, and Salmonella in bulk tank milk: risk fac- 2005. Prevalence of Salmonella enterica in bulk tank milk from U.S. tors and risk of human exposure. J. Food Prot. 55:93–97. dairies as determined by polymerase chain reaction. J. Dairy Sci. 88: 37. Ryser, E. T. 2001. Public health concerns, p. 397–546. In E. H. Marth 3475–3479. and J. L. Steele (ed.), Applied dairy microbiology, 2nd ed. Marcel 23. Lafarge, V., J. C. Ogier, V. Girard, V. Maladen, J. Y. Leveau, A. Dekker, New York. Gruss, and A. Delacroix-Buchet. 2004. Raw cow milk bacterial pop- 38. Sanaa, M., B. Poutrel, J. L. Menard, and F. Serieys. 1993. Risk fac- Downloaded from http://meridian.allenpress.com/jfp/article-pdf/71/8/1580/1679328/0362-028x-71_8_1580.pdf by guest on 26 September 2021 ulation shifts attributable to refrigeration. Appl. Environ. Microbiol. tors associated with contamination of raw milk by Listeria mono- 70:5644–5650. cytogenes in dairy farms. J. Dairy Sci. 76:2891–2898. 24. Lovett, J., D. W. Francis, and J. M. Hunt. 1987. Listeria monocy- 39. Schoder, D. P. W., A. Kareem, W. Baumgartner, and M. Wagner. togenes in raw milk: detection, incidence, and pathogenicity. J. Food 2003. A case of sporadic ovine mastitis caused by Listeria mono- Prot. 50:188–192. cytogenes and its effect on contamination of raw milk and raw-milk 25. Massa, S., E. Goffredo, C. Altieri, and K. Natola. 1999. Fate of cheeses produced in the on-farm dairy. J. Dairy Res. 70:395–401. Escherichia coli O157:H7 in unpasteurized milk stored at 8ЊC. Lett. 40. Steele, M. L., W. B. McNab, C. Poppe, M. W. Griffiths, S. Chen, S. Appl. Microbiol. 28:89–92. A. Degrandis, L. C. Fruhner, C. A. Larkin, J. A. Lynch, and J. A. 26. Miller, R. H., H. D. Norman, and L. L. M. Thornton. 2007. Somatic Odermeru. 1997. Survey of Ontario bulk tank milk for foodborne cell counts of milk from dairy herd improvement herds during 2006. pathogens. J. Food Prot. 60:1341–1346. Animal Improvement Programs Laboratory research report SCC8. 41. Tham, W., L. J. Hajdu, and M. L. Danielsson-Tham. 1990. Bacte- Animal Improvement Programs Laboratory, Agricultural Research riological quality of on-farm manufactured goat cheese. Epidemiol. Service, U.S. Department of Agriculture. Available at: http://aipl. Infect. 104:87–100. arsusda.gov/publish/dhi/dhi07/sccrpt.htm. Accessed 10 November 42. U.S. Department of Health and Human Services, Food and Drug 2007. Administration. 2003. Cheeses and cheese products. Definitions and 27. Muehlherr, J. E., C. Zweifel, S. Corti, J. E. Blanco, and R. Stephan. standards under the Federal Food, Drug and Cosmetic Act, part 133, 2003. Microbiological quality of raw goat’s and ewe’s bulk-tank milk title 21, Code of Federal Regulations. Available at: http://www. in Switzerland. J. Dairy Sci. 86:3849–3856. cfsan.fda.gov/ϳlrd/FCF133.html. Accessed 14 January 2008. 28. Muraoka, W., C. Gay, D. Knowles, and M. Borucki. 2003. Preva- 43. U.S. Department of Health and Human Services, Food and Drug lence of Listeria monocytogenes subtypes in bulk milk of the Pacific Administration, Center for Food Safety and Applied Nutrition. 2004. Northwest. J. Food Prot. 66:1413–1419. Grade ‘‘A’’ Pasteurized Milk Ordinance 2003 revision. Available at: 29. Murinda, S. E., L. T. Nguyen, S. J. Ivey, B. E. Gillespie, R. A. http://www.cfsan.fda.gov/ϳear/pmo03toc.html. Accessed 10 No- Almeida, F. A. Draughon, and S. P. Oliver. 2002. Molecular char- vember 2007. acterization of Salmonella spp. isolated from bulk tank milk and cull 44. Van Kessel, J. A., J. S. Karns, L. Gorski, B. J. McCluskey, and M. dairy cow fecal samples. J. Food Prot. 65:1100–1105. L. Perdue. 2004. Prevalence of salmonellae, Listeria monocytogenes 30. Murinda, S. E., L. T. Nguyen, S. J. Ivey, B. E. Gillespie, R. A. and fecal coliforms in bulk tank milk on US dairies. J. Dairy Sci. Almeida, F. A. Draughon, and S. P. Oliver. 2002. Prevalence and 87:2822–2830. molecular characterization of Escherichia coli O157:H7 in bulk tank 45. Vermont Agency of Agriculture. Unpublished data. milk and fecal samples from cull cows: a 12-month survey of dairy 46. Waak, E., W. Tham, and M. L. Danielsson-Tham. 2002. Prevalence farms in east Tennessee. J. Food Prot. 65:752–759. and fingerprinting of Listeria monocytogenes strains isolated from 31. Nightingale, K. K., Y. H. Schukken, C. R. Nightingale, E. D. Fortes, raw whole milk in farm bulk tanks and in dairy plant receiving tanks. A. J. Ho, Z. Her, Y. T. Grohn, P. L. McDonough, and M. Wiedmann. Appl. Environ. Microbiol. 68:3366–3370. 2004. Ecology and transmission of Listeria monocytogenes infecting 47. Wells, J. G., L. D. Shipman, K. D. Greene, E. G. Sowers, J. H. ruminants and in the farm environment. Appl. Environ. Microbiol. Green, D. N. Cameron, F. P. Downes, M. L. Martin, P. M. Griffin, 70:4458–4467. and S. M. Ostroff. 1991. Isolation of Escherichia coli serotype O157: 32. Pritchard, T. J., C. M. Beliveau, K. J. Flanders, and C. W. Donnelly. H7 and other Shiga-like-toxin–producing E. coli from dairy cattle. 1994. Increased incidence of Listeria species in dairy processing J. Clin. Microbiol. 29:985–989. plants having adjacent farm facilities. J. Food Prot. 57:770–775. 48. Zeng, S. S., S. S. Chen, B. Bah, and K. Tesfai. 2007. Effect of 33. Public Health Agency of Canada. 2002. Escherichia coli O157:H7 extended storage on microbiological quality, somatic sell count, and outbreak associated with the ingestion of unpasteurized goat’s milk composition of raw goat milk on a farm. J. Food Prot. 70:1281– in British Columbia, 2001. Canada Communicable Disease Report. 1285.