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Prevalence of Foodborne Pathogens in Bulk Tank Milk1
J. Dairy Sci. 84:2157–2162 American Dairy Science Association, 2001.
Prevalence of Foodborne Pathogens in Bulk Tank Milk1 B. M. Jayarao2 and D. R. Henning Minnesota-South Dakota Dairy Food Research Center Department of Dairy Science South Dakota State University Brookings, 57007-0647
ABSTRACT
INTRODUCTION
Bulk tank milk from 131 dairy herds in eastern South Dakota and western Minnesota was examined for the presence of for foodborne pathogens. Campylobacter jejuni, shiga-toxin producing Escherichia coli, Listeria monocytogenes, Salmonella spp., and Yersinia enterocolitica were detected in 9.2, 3.8, 4.6, 6.1, and 6.1% of bulk tank milk samples, respectively. Thirty-five of 131 (26.7%) bulk tank milk samples contained one or more species of pathogenic bacteria. Isolates of Salmonella belonged to group D (n = 4), B (n = 2), C (n = 1), and E (n = 1) “O” serogroups. All six isolates of Listeria monocytogenes were identified as O antigen type 1. Four of five isolates of E. coli encoded for the shiga-toxin 2 gene, while one strain encoded for the shiga-toxin 1 gene. Escherichia coli O157:H7 was not isolated from bulk tank milk samples. Based on autoagglutination testing, it was inferred that all eight isolates of Yersinia enterocolitica were likely to be virulent. Non A-grade (manufacturing grade) raw milk producers were at a higher risk (odd’s ratio, 4.98; confidence interval, 1.96 to 12.22) of having one or more pathogens in their bulk tank milk than were Grade A producers. It was observed that 21 of 79 (26.6%) dairy producers who consumed raw milk had one or more pathogenic bacteria in their bulk tank milk. The findings of the study warrant the need for educational programs for dairy producers about the risks associated with consumption of raw milk. (Key words: bulk tank milk, foodborne pathogens)
Several studies have identified milkborne pathogens including Campylobacter jejuni, Shiga-toxin producing Escherichia coli (STEC), Listeria monocytogenes, Salmonella spp., and Yersinia enterocolitica in farm bulk tank milk (BTM) (Lovett et al., 1987; McManus and Lanier, 1987; Moustafa et al., 1983; O’Donnell, 1995; Rahn et al., 1997; Rohrbach et al., 1992; Steele et al., 1997). The prevalence rates of these pathogens in BTM vary considerably among surveys, and could be influenced by several factors such as geographical area, season, farm size, number of animals on farm, hygiene, and farm management practices (Rohrbach et al., 1992). Consumption of raw or inadequately pasteurized milk has been associated with several outbreaks of enteric infections associated with pathogens such as STEC (Borczyk et al., 1987; Keene et al., 1997; Martin et al., 1986), Salmonella spp. (D’Aoust, et al., 1985; Ryan et al., 1987; Spake et al., 1997), L. monocytogenes (Fleming et al., 1985; Linnan et al., 1988) and C. jejuni (Evans et al., 1996; Jones, and Willis, 1981; Potter et al., 1983). Rohrbach et al. (1992) reported that 34.9% of dairy producers in eastern Tennessee and southwest Virginia consumed raw milk. In California, where it is legal to sell certified milk, about 3.2% of respondents to a survey on raw milk consumption practices consumed raw milk (Headrick et al., 1997). A questionnaire-based survey conducted by Jayarao and Cassel (1999) showed that nearly 60% of dairy producers in eastern South Dakota and western Minnesota consumed raw milk. Limited epidemiological data exist on foodborne illnesses caused by the consumption of raw milk from the bulk tank. To complicate the issue further, the long-term effects of continued exposure to milkborne pathogens on human health are virtually unknown. The emergence of multiple antibiotic-resistant strains of Salmonella ser. Typhimurium DT104 should be of great concern to the public, especially dairy producers, their families, and employees, because this organism is resistant to antibiotics that are commonly used in medical and veterinary practices. An outbreak following the handling of sick calves and consumption of
Abbreviation key: BTM = bulk tank milk, STEC = shiga-toxin producing Escherichia coli.
Received January 29, 2001. Accepted May 21, 2001. Corresponding author: B. M. Jayarao; e-mail: [email protected]. 1 Published with the approval of the director of the South Dakota Agricultural Experiment Station as Publication Number 3231 of the Journal Series. 2 Current address: Department of Veterinary Science, Pennsylvania State University, PA 16802.
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raw milk that contained Salmonella ser. Typhimurium DT104 received national attention (Spake et al., 1997). Clinical and laboratory-based findings have shown that following an episode of severe foodborne illness some individuals tend to develop painful, often debilitating, reactive arthritis caused by sensitization to some gramnegative foodborne pathogens such as Campylobacter jejuni, Yersinia enterocolitica, and Salmonella spp. (Sieper et al., 1996). The purposes of this study were to ascertain the prevalence of foodborne pathogens in BTM and determine the percentage of dairy producers who consume raw milk. MATERIALS AND METHODS Bulk Tank Milk BTM from dairy herds (n = 131) was examined for the presence of milkborne pathogens including C. jejuni, shiga-toxin producing E. coli, L. monocytogenes, Salmonella spp., and Y. enterocolitica. A single BTM sample (∼120 ml) was collected in sterile snap cap milk collection vials (Capital Vials, Fultonville, NY) from each of the 131 dairy producers. Milk samples were collected between March 1 and 15, 1997, by milk truck haulers of a milk cooperative. The samples were shipped on ice to the laboratory and examined within 36 h of collection. A survey of the same 131 dairy producers indicated that 79 of 131 (60%) of producers consumed raw milk from their bulk tank. Isolation, Identification, and Characterization of Foodborne Pathogens Campylobacter jejuni from raw milk was isolated as described by Hunt (1992) with a modification that included Oxyrase enrichment as described by Tran (1995). Briefly, 10 ml of milk was centrifuged at 14,000 rpm for 20 min at 4°C, and the resulting pellet was resuspended in 45 ml of Campylobacter enrichment broth (Hunt, 1992) supplemented with vancomycin (15 mg/ml), trimethoprim (7.5 mg/ml), and polymixin B (500 U/l), in 38 × 200 mm glass tubes. One milliliter of Oxyrase enzyme (30 enzyme units) was added to each tube, followed by incubation at 42°C for 48 h in a shaker water bath. Ten microliters of enriched sample was plated on CCDA-Preston agar (Oxoid Ltd., Basingstoke, England) and incubated in a microaerophilic atmosphere that was created by using a gas generating kit for Campylobacter (Oxoid) in an anaerobic jar (Oxoid). Plates were incubated at 42°C for 48 h. Campylobacter was identified to genus level as described by Hunt (1992) and species identified using API-CAMPY identification kit (BioMe´rieux, Hazelwood, MO). Journal of Dairy Science Vol. 84, No. 10, 2001
Escherichia coli from raw milk was isolated as described by Ansay and Kaspar (1997) with modifications. Briefly, 10 ml of raw milk was enriched in an equal volume of 2 × modified EC broth (Difco Laboratories, Detroit, MI) supplemented with 20 µg/ml of novobiocin (Sigma Chemical Co., St. Louis, MO) and incubated overnight at 37°C. Escherichia coli was isolated by subculturing 50 µl of enriched sample onto MacConkey agar supplemented with 4-methylumbelliferyl-beta glucuronide (Difco). The plates were incubated for 48 h at 42°C. At least 5 to 10 fluorescent colonies from MacConkey-4-methylumbelliferyl-beta glucuronide plates were selected for further characterization. For detection of E. coli O157:H7, 50 µl of enriched sample was spread on sorbitol MacConkey agar (Difco) supplemented with cefixime (0.05 mg/L) and potassium tellurite (2.5 mg/L) (Dynal Inc., Lake Success, NY) and incubated for 48 h at 42°C. At least 5 to 10 sorbitol-negative colonies from sorbitol MacConkey agar were tested with O157 antigen by latex agglutination (Unipath Co., Ogdensburg, NY). Isolates identified as belonging to the genus Escherichia were tested for shiga-toxins by the specific PCR assay (Pollard et al., 1990). Isolates that produced shiga-toxins were identified and speciated with API 20E (BioMe´rieux). Isolation of L. monocytogenes was done as described by Hitchins (1992). Briefly, 25 ml of milk sample was added to 225 ml of Listeria enrichment broth (Difco) and incubated for 24 and 48 h at 30°C. A loopful of enriched broth was streaked on Oxford medium (Unipath) and Lithium chloride-phenylethanol moxalactam agar (Unipath) at 24 and 48 h, respectively. At least five colonies that were presumptive positive on Oxford and Lithium chloride-phenylethanol moxalactam agar were transferred to trypticase soy agar (Difco) with 0.6% yeast extract. The plates were incubated for 48 h at 30°C. All isolates were examined for Gram’s reaction, hemolysis, and cAMP reaction on 5% sheep blood agar, catalase production, nitrate reduction, and motility on SIM medium (Becton Dickinson, Cockeysville, MD). All isolates were confirmed to species by use of API-Listeria (BioMe´rieux) identification kit. Isolates identified as L. monocytogenes were serotyped using O antisera (Difco). Isolation of Salmonella from raw milk was done as described by Andrews et al. (1992). Briefly, 25 ml of milk was added to 225 ml of lactose broth (Difco) and incubated for 24 h at 37°C. About 1 ml of preenriched sample was transferred to 10 ml each of selenite cystine broth (Unipath) and tetrathionate broth (Unipath), respectively. Enrichment was carried out for 24 h at 37°C. The enriched broths were subcultured on Hektoen enteric agar (Difco) and xylose lysine desoxycholate agar (Unipath) and incubated for 24 h at 35°C. Colonies presumptive for Salmonella were inoculated on triple
PATHOGENS IN BULK TANK MILK
sugar agar (Difco) and urease agar (Difco) and then tested with Salmonella polyvalent O antiserum (Difco). Organisms that gave typical reactions for Salmonella were then confirmed by using API-20E identification kit (BioMe´rieux). Isolates were then tested by seroagglutination using Salmonella O group (A-I) antisera (Difco). Yersinia enterocolitica was isolated from raw milk as described by Weagant et al. (1992). Briefly, 10 ml of milk was added to 90 ml of peptone sorbitol bile broth and incubated at 10°C for 10 d. On d 10, the enriched broth was treated with 0.5% KOH and plated on MacConkey’s agar and Celfsulodin-irgasan-novobiocin agar (Difco). The plates were incubated for 48 h at 22°C. Colonies with characteristics for Yersinia were examined on lysine arginine iron agar (Difco), Christensen’s urea agar (Difco) and bile esculin agar (Difco). Organisms identified as Yersinia were speciated with API20E identification kit (BioMe´rieux). Isolates of Y. enterocolitica were presumed to be pathogenic based on a positive reaction to the autogglutination test described by Laird and Cavanaugh (1980). Data Analyses Epi-info version 6.0, a database and statistics system for epidemiology on microcomputers was used for data analysis (Centers for Disease Control and Prevention, Atlanta, GA). The occurrence of one or more pathogenic bacteria in raw milk was compared to the grade of milk and milk consumption practices using chi-square tests. RESULTS AND DISCUSSION Farm BTM from 131 dairy herds in eastern South Dakota and western Minnesota was examined for the presence of foodborne pathogens. Isolation rates of C. jejuni, STEC, Salmonella spp., and Y. enterocolitica have been reported from several regions of the United States and Canada; however, this is the first time that the isolation rate of foodborne pathogens has been reported from the eastern part of South Dakota and western Minnesota (Tables 1 and 2). It was observed that 35 of 131 (26.7%) BTM samples had one or more pathogens. An isolation rate of 25% was reported by Rohrbach et al. (1992) from BTM milk in eastern Tennessee and southwestern Virginia, which contained one or more pathogenic species of bacteria. BTM from Grade A and non-Grade A producers met the requirements for the states of South Dakota and Minnesota in effect at the time of the study. All Grade A milk had a SCC of <750,000 cells/ml, a standard plate count of <100,000 cfu/ml, and was free of drug residues. All non-Grade A milk or manufacturing grade was free
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of drug residues, had SCC <750,000 cells/ml for South Dakota producers and <1,000,000 cells/ml for Minnesota producers. In this study, 18 of 34 (53%) non-Grade A raw milk samples had one or more pathogenic bacteria. The percentage was significantly higher than the isolation rate from Grade A milk, which was 17.5%. The odds ratio analysis indicated that the likelihood of isolating one or more pathogenic bacteria from nonGrade A raw milk was 4.98 fold higher than Grade A raw milk (χ2 = 14.98, P < 0.0001) (Table 1). In this study, C. jejuni was isolated from 12 of 131 (9.2%) of BTM samples (Table 1). Campylobacter jejuni was also isolated concurrently with E. coli (n = 1 bulk tank), L. monocytogenes (n = 1), and Y. enterocolitica (n = 1). The isolation rate observed is higher than most of the reported isolation rates, with the exception of that of Rohrbach et al. (1992) (Table 2). Campylobacter jejuni frequently has been isolated from raw BTM (Beumer et al., 1988, Rohrbach et al., 1992; Steele et al., 1997) and from feces of cows (Beumer et al., 1988; Humphrey and Beckett, 1987). Contamination of milk with feces containing Campylobacter spp. is the primary cause. A study conducted by Lander and Gill (1980) showed that following intramammary inoculation with C. jejuni, C. jejuni was shed in milk for 3 to 73 d postinfection in variable numbers. Outbreaks of Campylobacteriosis caused by consumption of raw milk and certified raw milk have been reported in California, Pennsylvania, Michigan, Minnesota, and Vermont (Potter et al., 1983). STEC belong to several serotypes including O157:H7 and have been associated with human disease. Foods of animal origin including raw milk have been implicated as important vehicles for STEC infections in humans. It is now widely documented that STEC, including O157:H7, can be isolated from feces of asymptomatic cattle and raw milk (Wells et al., 1991). Seldom has raw milk been implicated in outbreaks of disease caused by STEC; from 1982 to 1990 only 14 confirmed cases of STEC-associated illness following consumption of raw milk occurred in the United States (Erskine, et al., 1991). In this study, STEC strains were isolated from 5 of 131 (3.8%) BTM samples. Klie et al. (1997) reported a similar STEC incidence rate of 3.9% for raw milk and 2.1% for certified raw milk in Germany. A recent study conducted by Steele et al. (1997) reported that only 0.87% of the BTM samples in Ontario contained STEC. Four of the five isolates of E. coli encoded for shiga-toxin 2 gene, while one strain encoded for shiga-toxin 1 gene. On the contrary, Montenegro et al. (1990) reported that most of the STEC isolates of bovine origin encoded for shiga-toxin 1 gene. Listeria monocytogenes was isolated from 6 of 131 (4.6%) of BTM samples (Table 1). The isolation rate Journal of Dairy Science Vol. 84, No. 10, 2001
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Table 1. Prevalence of selected pathogens in bulk tank milk from eastern South Dakota and western Minnesota. No. (%) of isolates from bulk tank milk (n = 131)
Organism
Milk grade1 (n = 131)
Combination of pathogens Bulk tanks (n = 131)
Organism(s)
A (n = 97)
Non A (n = 34)
Consume raw milkb (n = 131) Yes (n = 79)
No (n = 52)
Salmonella spp.
8 (6.1)
Salmonella + E. coli
7 1
3 ...
4 1
5 1
2 ...
Listeria monocytogenes
6 (4.6)
L. monocytogenes + C. jejuni + C. jejuni + Y. enterocolitica C. jejuni + E. coli Y. enterocolitica
4 1 1
1 ... ...
3 1 1
2 ... ...
2 1 1
9 1 7
7 1 3
2 ... 4
5 1 4
4 ... 3
Campylobacter jejuni
12 (9.2)
Yersinia enterocolitica
8 (6.1)
STEC Escherichia coli Total
5 (3.8) 39 ...
E. coli ... ...
4 35 26.7%
2 17 17.5%
2 18 52.9%
3 21 26.6%
1 14 26.9%
Milk grade: test of significance, χ2 (P) = 14.98 (0.0001); Odds ratio (confidence interval) = 4.98 (1.96 to 12.22). Consumption of raw milk: test of significance, χ2 (P) = 0.26 (0.6113); Odds ratio (confidence interval) = 0.82 (0.37 to 5.39).
1 b
observed in this study was similar to isolation rates reported in the literature (Table 2). All isolates of L. monocytogenes belonged to O antigen type 1. The O antigen includes serotypes 1/2a, 1/2b, and 1/2c. In this study, the serotype analysis was limited to the O antigen type, the six isolates could have been any of the 3
serotypes(1/2a, 1/2b, or 1/2c). Listeria monocytogenes serotypes 1, 1/2a, and 4b have been reported to occur in raw milk (Fenlon et al., 1995; Liewen and Plautz, 1988; Yoshida et al., 1998). As observed with gramnegative bacteria, fecal contamination could be the source of L. monocytogenes contamination in raw BTM.
Table 2. Isolation rates for foodborne pathogens from raw milk reported from Canada and United States. Percent isolation rate
Year
Country
Reference
Campylobacter jejuni
0.9 1.5 0.4 1.2 12.3 9.2 0.47
1982 1983 1987 1989 1992 1997 1998
US USA USA Canada USA USA Canada
Doyle & Roman (1982) Lovett et al. (1983) McManus and Lanier (1987) Davaidson et al. (1989) Rohrbach et al. (1992) This study Steele et al. (1997)
Shiga-toxin producing Escherichia coli
3.8 0.87
1997 1998
USA Canada
This study Steele et al. (1997)
Listeria monocytogenes
4.2 1.3 5.4 4.0 1.6 1.9 4.1 4.6 2.7 4.7 2.9 8.9 6.1 0.17
1987 1988 1988 1988 1989 1990 1992 1997 1998 1987 1988 1992 1997 1998
USA Canada Canada USA Canada Canada USA USA Canada USA Canada USA USA Canada
Lovett et al. (1987) Farber et al. (1988) Slade et al. (1988) Liewen and Plautz (1988) Davidson et al. (1989) Fedio and Jackson (1990) Rohrbach et al. (1992) This study Steele et al. (1997) McManus and Lanier (1987) McEwen et al. (1988) Rohrbach et al. (1992) This study Steele et al. (1997)
1983 1987 1989 1992 1997
USA USA Canada USA USA
Moustafa et al. (1983) McManus and Lanier (1987) Davidson et al. (1989) Rohrbach et al. (1992) This study
Pathogen
Salmonella spp.
Yersinia enterocolitica
Journal of Dairy Science Vol. 84, No. 10, 2001
12 48.1 2.7 15.1 6.1
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The low incidence of L. monocytogenes in raw milk does not pose any practical problem for pasteurized milk and processed dairy products but may be of significance for raw milk consumption (O’Donnell, 1995). Salmonella was isolated from 8 of 131 (6.1%) BTM samples, which is within the range of isolation rates reported in the literature (Table 2). Isolates of Salmonella belonged to group D (n = 4), B (n = 2), C (n = 1), and E (n = 1) “O” serogroups. These serogroups have been identified as important causes of diseases in calves, dairy cows, and humans (Carter et al., 1995). The isolation rate of Y. enterocolitica (6.1%; Table 1) observed in this study is considerably lower than that reported previously (Table 2). Based on an autoagglutination test, it was inferred that all eight isolates of Y. enterocolitica were pathogenic. To date there have been two reported outbreaks of foodborne infection caused by Y. enterocolitica, in which contaminated milk was incriminated (Black et al., 1978; Tackett et al., 1984). Although Y. enterocolitica has been isolated from the intestines of dairy cattle, generally milk cows are not considered as reservoirs of pathogenic strains of Y. enterocolitica. However, a major concern is that Y. enterocolitica can grow to large numbers at refrigeration temperatures so that contaminated raw milk could become significant health risk when consumed. The isolation of foodborne disease-causing bacteria in raw milk has been reported extensively from Canada and United States (Table 2). The isolation rates reported over the last two decades vary considerably. This can be attributed partly to the isolation identification, true prevalence, season, geographic area, farm size, number of animals on farm, hygiene, and farm management practices. These reported findings clearly suggest pathogens do occur in BTM and may pose a health hazard if raw milk is consumed. In this study, 79 of 131 (60%) dairy producers who participated in the BTM pathogen survey reported they consumed raw milk. Of the 79 dairy producers who consumed raw milk, 21 (26.6%) had one or more pathogenic bacteria in their BTM. There was no significant difference in the incidence of pathogenic bacteria in raw milk of dairy producers who did and did not consume raw milk (χ2 = 0.26; P = 0.6113); odds ratio = 0.82 (confidence interval, 0.37 to 5.39) (Table 1). An epidemiological study conducted by Headrick et al. (1998) on raw-milk associated foodborne disease outbreaks in the United States revealed that 46 raw milk associated outbreaks occurred between 1973 and 1992; of which 40 (87%) of the outbreaks occurred in states where the intrastate sale of raw milk was legal. They concluded that consumption of raw milk remains a preventable cause of foodborne disease outbreaks.
CONCLUSIONS The findings of this study suggest that pathogenic bacteria of human health significance are in BTM. This observation and with previously reported data on consumption of raw milk from the same geographical area indicate that dairy producers and farm families are at risk of ingesting foodborne pathogens when consuming raw milk. This risk can be further compounded when it is legally permitted to sell raw milk. Based on the findings of this study, it is recommended that further studies be conducted to understand the behaviorial risk factors associated with raw milk consumption and that educational programs be developed to address issues related to consumption of raw milk. Based on the findings C. jejuni, STEC, L. monocytogenes, Salmonella spp., and Y. enterocolitica were isolated from BTM samples collected from western South Dakota and eastern Minnesota. The prevalence rates for most foodborne pathogens in BTM were similar to that previously reported from Canada and United States. Foodborne pathogens were more likely to be present in non-Grade A milk than Grade A milk. REFERENCES Andrews W. K., V. R. Bruce, G. June, F. Satchell, and P. Sherrod. 1992. Salmonella. Pages 51–70 in FDA Bacteriological Analytical Manual. 7th ed. Association of Official Analytical Chemists, Arlington, VA. Ansay, S. E., and C. W. Kaspar. 1997. Survey of retail cheeses, dairy processing environments and raw milk for Escherichia coli O157:H7. Lett. Appl. Microbiol. 25:131–134. Beumer, R. R., J. J. M. Cruysen, and J. R. K. Birtantie. 1988. The occurrence of Campylobacter jejuni in raw cow’s milk. J. Appl. Bacteriol. 65:93–96. Black, R. E., R. J. Jackson, T. Tsai, M. Medvesky, M. Shayegani, J. C. Feeley, K. I. E. MaCleod, and A. M. Wakeler. 1978. Epidemic Yersinia enterocolitica infection due to contaminated chocolate milk. N. Engl. J. Med. 298:70–76. Borczyk, A. A., M. A. Karmali, H. Loir, and L. M. C. Duncan. 1987. Bovine reservoir for verotoxin-producing Escherichia coli O157:H7. Lancet (8524):98. Carter, G. R., M. M. Chengappa, and R. A Wayne. 1995. Essentials of Veterinary Microbiology. 5th ed. Williams and Wilkins, Baltimore, MD. pp 151–165. Davidson, R. J., D. W. Sprung, C. E. Park, and M. K. Raymond. 1989. Occurrence of Listeria monocytogenes, Campylobacter spp., and Yersinia enterocolitica in Manitoba raw milk. Can. Inst. Food Sci. Technol. J. 22:70–74. D’Aoust, J. Y., D. W. Warburton, and A. M. Sewell. 1985. Salmonella typhimurium phage-type 10 from cheddar cheese implicated in a major Canadian foodborne outbreak. J. Food Prot. 48:1062–1066. Doyle, M. P., and D. J. Roman. 1982. Prevalence and survival of Campylobacter jejuni in unpasteurized milk. Appl. Environ. Microbiol. 44:1154–1158. Erskine, R. J., J. W. Tyler, M. G. Riddell, Jr., and R. C. Wilson. 1991. Theory, use, and realities of efficacy and food safety of antimicrobial treatment of acute coliform mastitis. J. Am. Vet. Med. Assoc. 198:980–984. Evans, M. R, R. J. Roberts, C. D. Ribeiro, D. Gardner, and D. Kembrey. 1996. A milk-borne Campylobacter outbreak following an educational farm visit. Epidemiol. Infect. 117:457–462. Journal of Dairy Science Vol. 84, No. 10, 2001
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Farber, J. M., G. W. Sander, and S. A. Malcolm. 1988. The presence of Listeria spp. in raw milk in Ontario. Can. J. Microbiol. 34:95–100. Fedio, W. M., and H. Jackson. 1990. Incidence of Listeria monocytogenes in raw milk in Alberta. Can. Inst. Food Sci. Technol. J. 23:236–238. Fenlon, D. R., and J. Wilson. 1989. The incidence of Listeria monocytogenes in raw milk from farm bulk tanks in North-East Scotland. J. Appl. Bacteriol. 66:191–196. Fleming, D. W., S. L. Cochi, K. L. MacDonald, J. Brondum, P. S. Hayes, B. D. Plikaytis, M. B. Holmes, A. Audurier, C. V. Broome, and A. L. Reingold. 1985. Pasteurized milk as a vehicle of infection in an outbreak of listeriosis. New Engl. J. Med. 312:404–407. Headrick, M. L., B. Timbo, K. C. Klontz, and S. B. Werner, 1997. Profile of raw milk consumers in California. Public Health Rep 112:418–422. Headrick, M. L., S. Korangy, N. H. Bean, F. J. Angulo, S. F. Altekruze, M. E. Potter, and K. C. Klontz. 1998. The epidemiology of raw milk-associated foodborne disease outbreaks reported in the United States, 1973 through 1992. Am. J. Public Health 88:1219–1221. Hitchins, A. D. 1992. Listeria monocytogenes. Pages 141–151 in FDA Bacteriological Analytical Manual. 7th ed. Association of Official Analytical Chemists, Arlington, VA. Humphrey, T. J., and P. Beckett. 1987. Campylobacter jejuni in dairy cows and raw milk. Epidemiol. Infect. 98:263–269. Hunt, J. M. 1992. Campylobacter. Pages 77–94 in FDA Bacteriological Analytical Manual. 7th ed. Association of Official Analytical Chemists, Arlington, VA. Jayarao, B. M., and E. K. Cassel. 1999. Mastitis prevention and milk hygiene practices adopted by dairy producers. Large Anim. Pract. 20:6–14. Jones, P. H., and A. T. Willis. 1981. Campylobacter enteritis associated with the consumption of free school milk. J. Hyg. Camb. 87:155–170. Keene, W. E., K. Hedberg, D. E. Herriott, D. D. Hancock, R. W. McKay, T. J. Barrett, and D. W. Fleming. 1997. A prolonged outbreak of Escherichia coli O157:H7 infections caused by commercially distributed raw milk. J. Infect. Dis. 176:815–818. Klie, H., M. Timm, H. Richter, P. Gallien, K. W. Perlberg, and H. Steinruck. 1997. Detection and occurrence of vertoxin-forming and/or shigatoxin producing Escherichia coli (VTEC and/or STEC in milk). Berl. Munch. Tierarztl. Wochenschr. 110:337–341. Laird, W. J., and D. C. Cavanaugh. 1980. Correlation of autoagglutination and virulence of Yersinia. J. Clin. Microbiol. 27:1667–1670. Lander, K. P., and K. P. W. Gill. 1980. Experimental infection of the bovine udder with Campylobacter coli/jejuni. J. Hyg. 84:421–428. Liewen, M. B., and M. W. Plautz. 1988. Occurrence of Listeria monocytogenes in raw milk in Nebraska. J. Food Prot. 51:840–841. Linnan, M. J., L. Mascola, X. Dong Lou, V. Goulet, S. May, C. Salinem, D. W. Hird, M. L. Yonekura, P. Hayes, R. Weaver, A. Audurier, B. D. Plikaytis, S. L. Fannin, A. Kleks, and C. V. Broome. 1988. Epidemic listeriosis associated with Mexican-style cheese. New Engl. J. Med. 319:823–828. Lovett, J. D., W. Francis, and J. M. Hunt. 1983. Isolation of Campylobacter jejuni from raw milk. Appl. Environ. Microbiol. 46:459–462. Lovett, J. D., W. Francis, and J. M. Hunt. 1987. Listeria monocytogenes in raw milk: Detection, incidence, and pathogenicity. J. Food. Prot. 50:188–192. Martin, M. L., L. D. Shipman, J. G. Wells, M. E. Potter, K. Hedberg, I. K. Wachsmuth, R. V. Tauxe, J. P. Davis, J. Arnolai, and J. Tilleli. 1986. Isolation of Escherichia coli O157:H7 from dairy cattle associated with two cases of hemolytic uremic syndrome. Lancet. 1;2(85414):1043. McEwen, S. A., L. H. McClure, and S. W. Martin. 1988. Farm inspection scores and milk quality criteria as indices of Salmonella in bulk tank milk. J. Food Prot. 51:958–962. McManus, C., and J. M. Lanier. 1987. Salmonella, Campylobacter jejuni, and Yersinia enterocolitica in raw milk. J. Food Prot. 50:51–55.
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Montenegro, M. A., M. Bu¨lte, T. Trumpf, S. Aleksic˙, G. Reuter, E. Bulling, and R. Helmuth. 1990. Detection and characterization of fecal verotoxin-producing Escherichia coli from healthy cattle. J. Clin. Microbiol. 28:1417–1421. Moustafa, M. K., A. A-H. Ahmed, and E. H. Marth. 1983. Occurrence of Yersinia enterocolitica in raw and pasteurized milk. J. Food Prot. 46:276–278. O’Donnell, E. T. 1995. The incidence of Salmonella and Listeria in raw milk from farm bulk tanks in England and Wales. J. Soc. Dairy Technol. 48:25–29. Pollard, D. R., W. M. Johnson, H. Lior, S. D. Tyler, and K. R. Rozee. 1990. Rapid and specific detection of verotoxin genes in Escherichia coli by polymerase chain reaction. J. Clin. Microbiol. 28:540–545. Potter, M. E., M. J. Blaser, R. K. Sikes, A. F. Kaufmann, and J. G. Wells. 1983. Human Campylobacter infection associated with certified raw milk. Am. J. Epidemiol. 117:475–483. Rahn, K., S. A. Renwick, R. P. Johnson, J. B. Wilson, R. C. Clarke, D. Alves, S. McEwen, H. Loir, and J. Spika. 1997. Persistence of Escherichia coli O157:H7 in dairy cattle and the dairy farm environment. Epidem. Infect. 119:251–259. Rohrbach, R. W., F. A. Draughon, P. M. Davidson, and S. P. Oliver. 1992. Prevalence of Listeria monocytogenes, Campylobacter jejuni, Yersinia enterocolitica and Salmonella in bulk tank milk: risk factors and risk of human exposure. J. Food Prot. 55:93–97. Ryan, C. A., M. K. Nickels, N. T. Hargrett-Bean, M. E. Potter, T. Endo, L. Mayer, C. W. Langkop, C. Gibson, R. C. McDonald, R. T. Kenny, N. D. Puhr, S. J. McDonnell, R. J. Martin, M. L. Cohen, and P. A. Blake. 1987. Massive outbreak of antimicrobial-resistant salmonellosis traced to pasteurized milk. J. Am. Med. Assoc. 258:3269–3274. Sieper, J., G. H. Kingsley, and E. Marker-Herman. 1996. Aetiological agents and immune mechanisms in enterogenic reactive arthritis. Baillieres. Clin. Rheumatol. 10:105–121. Slade, P. J., D. L. Collins-Thompson, and F. Fletcher. 1988. Incidence of Listeria species in Ontario raw milk. Can. Inst. Food Sci. Technol. J. 21:425–429. Spake, A., M. B. Marcus, and D. McGraw. 1997. O is for outbreak: When a drug-resistant Salmonella struck a Vermont farm, health officials knew it might be just the beginning. US News World Rep. 23:70–79. Steele, M. L., W. B. McNab, C. Poppe, M. W. Griffiths, S. Chen, S. A. Degrandis, L. C. Fruhner, C. A. Larkin, J. A. Lynch, and J. A. Odermeru. 1997. Survey of Ontario bulk tank milk for foodborne pathogens. J. Food Prot. 60:1341–1346. Tackett, C. O., J. P. Narain, R. Sattin, J. P. Lofgren, C. Konigsberg, Jr., R. C. Rendtorff, A. Rausa, B. R. Davis, and M. L. Cohen. 1984. A multistate outbreak of infections caused by Yersinia enterocolitica transmitted by pasteurized milk. J. Am. Med. Assoc. 251:483–486. Tran, T. T., 1995. Evaluation of Oxyrase enrichment method for isolation of Campylobacter jejuni from inoculated foods. Lett. Appl. Microbiol. 21:345–347. Weagant, S. D., P. Feng, and J. T. Stanfield. 1992. Yersinia enterocolitica and Yersinia pseudotuberculosis. Pages 95–109 in FDA Bacteriological Analytical Manual. 7th ed. Association of Official Analytical Chemists, Arlington, VA. Wells, J. G., L. D. Shipman, K. D. Greene, E. G. Sowers, J. H. Green, D. N. Cameron, M. E. Potter, R. V. Tauxe, and I. K. Wachsmuth. 1991. Isolation of Escherichia coli serotype O157:H7 and other shiga-like-toxin producing E. coli from dairy cattle. J. Clin. Microbiol. 29:985–989. Wray, C., Q. C. Wadsworth, and D. W. Richards, and J. H. Morgan. 1989. A three-year study of Salmonella dublin infection in a closed dairy herd. Vet. Rec. 124:532–535. Yoshida, T., M. Sato, and K. Hirai. 1998. Prevalence of Listeria species in raw milk from farm bulk tanks in Nagano prefecture. J. Vet. Med. Sci. 60:311–314.
Prevalence of Foodborne Pathogens in Bulk Tank Milk1 B. M. Jayarao2 and D. R. Henning Minnesota-South Dakota Dairy Food Research Center Department of Dairy Science South Dakota State University Brookings, 57007-0647
ABSTRACT
INTRODUCTION
Bulk tank milk from 131 dairy herds in eastern South Dakota and western Minnesota was examined for the presence of for foodborne pathogens. Campylobacter jejuni, shiga-toxin producing Escherichia coli, Listeria monocytogenes, Salmonella spp., and Yersinia enterocolitica were detected in 9.2, 3.8, 4.6, 6.1, and 6.1% of bulk tank milk samples, respectively. Thirty-five of 131 (26.7%) bulk tank milk samples contained one or more species of pathogenic bacteria. Isolates of Salmonella belonged to group D (n = 4), B (n = 2), C (n = 1), and E (n = 1) “O” serogroups. All six isolates of Listeria monocytogenes were identified as O antigen type 1. Four of five isolates of E. coli encoded for the shiga-toxin 2 gene, while one strain encoded for the shiga-toxin 1 gene. Escherichia coli O157:H7 was not isolated from bulk tank milk samples. Based on autoagglutination testing, it was inferred that all eight isolates of Yersinia enterocolitica were likely to be virulent. Non A-grade (manufacturing grade) raw milk producers were at a higher risk (odd’s ratio, 4.98; confidence interval, 1.96 to 12.22) of having one or more pathogens in their bulk tank milk than were Grade A producers. It was observed that 21 of 79 (26.6%) dairy producers who consumed raw milk had one or more pathogenic bacteria in their bulk tank milk. The findings of the study warrant the need for educational programs for dairy producers about the risks associated with consumption of raw milk. (Key words: bulk tank milk, foodborne pathogens)
Several studies have identified milkborne pathogens including Campylobacter jejuni, Shiga-toxin producing Escherichia coli (STEC), Listeria monocytogenes, Salmonella spp., and Yersinia enterocolitica in farm bulk tank milk (BTM) (Lovett et al., 1987; McManus and Lanier, 1987; Moustafa et al., 1983; O’Donnell, 1995; Rahn et al., 1997; Rohrbach et al., 1992; Steele et al., 1997). The prevalence rates of these pathogens in BTM vary considerably among surveys, and could be influenced by several factors such as geographical area, season, farm size, number of animals on farm, hygiene, and farm management practices (Rohrbach et al., 1992). Consumption of raw or inadequately pasteurized milk has been associated with several outbreaks of enteric infections associated with pathogens such as STEC (Borczyk et al., 1987; Keene et al., 1997; Martin et al., 1986), Salmonella spp. (D’Aoust, et al., 1985; Ryan et al., 1987; Spake et al., 1997), L. monocytogenes (Fleming et al., 1985; Linnan et al., 1988) and C. jejuni (Evans et al., 1996; Jones, and Willis, 1981; Potter et al., 1983). Rohrbach et al. (1992) reported that 34.9% of dairy producers in eastern Tennessee and southwest Virginia consumed raw milk. In California, where it is legal to sell certified milk, about 3.2% of respondents to a survey on raw milk consumption practices consumed raw milk (Headrick et al., 1997). A questionnaire-based survey conducted by Jayarao and Cassel (1999) showed that nearly 60% of dairy producers in eastern South Dakota and western Minnesota consumed raw milk. Limited epidemiological data exist on foodborne illnesses caused by the consumption of raw milk from the bulk tank. To complicate the issue further, the long-term effects of continued exposure to milkborne pathogens on human health are virtually unknown. The emergence of multiple antibiotic-resistant strains of Salmonella ser. Typhimurium DT104 should be of great concern to the public, especially dairy producers, their families, and employees, because this organism is resistant to antibiotics that are commonly used in medical and veterinary practices. An outbreak following the handling of sick calves and consumption of
Abbreviation key: BTM = bulk tank milk, STEC = shiga-toxin producing Escherichia coli.
Received January 29, 2001. Accepted May 21, 2001. Corresponding author: B. M. Jayarao; e-mail: [email protected]. 1 Published with the approval of the director of the South Dakota Agricultural Experiment Station as Publication Number 3231 of the Journal Series. 2 Current address: Department of Veterinary Science, Pennsylvania State University, PA 16802.
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raw milk that contained Salmonella ser. Typhimurium DT104 received national attention (Spake et al., 1997). Clinical and laboratory-based findings have shown that following an episode of severe foodborne illness some individuals tend to develop painful, often debilitating, reactive arthritis caused by sensitization to some gramnegative foodborne pathogens such as Campylobacter jejuni, Yersinia enterocolitica, and Salmonella spp. (Sieper et al., 1996). The purposes of this study were to ascertain the prevalence of foodborne pathogens in BTM and determine the percentage of dairy producers who consume raw milk. MATERIALS AND METHODS Bulk Tank Milk BTM from dairy herds (n = 131) was examined for the presence of milkborne pathogens including C. jejuni, shiga-toxin producing E. coli, L. monocytogenes, Salmonella spp., and Y. enterocolitica. A single BTM sample (∼120 ml) was collected in sterile snap cap milk collection vials (Capital Vials, Fultonville, NY) from each of the 131 dairy producers. Milk samples were collected between March 1 and 15, 1997, by milk truck haulers of a milk cooperative. The samples were shipped on ice to the laboratory and examined within 36 h of collection. A survey of the same 131 dairy producers indicated that 79 of 131 (60%) of producers consumed raw milk from their bulk tank. Isolation, Identification, and Characterization of Foodborne Pathogens Campylobacter jejuni from raw milk was isolated as described by Hunt (1992) with a modification that included Oxyrase enrichment as described by Tran (1995). Briefly, 10 ml of milk was centrifuged at 14,000 rpm for 20 min at 4°C, and the resulting pellet was resuspended in 45 ml of Campylobacter enrichment broth (Hunt, 1992) supplemented with vancomycin (15 mg/ml), trimethoprim (7.5 mg/ml), and polymixin B (500 U/l), in 38 × 200 mm glass tubes. One milliliter of Oxyrase enzyme (30 enzyme units) was added to each tube, followed by incubation at 42°C for 48 h in a shaker water bath. Ten microliters of enriched sample was plated on CCDA-Preston agar (Oxoid Ltd., Basingstoke, England) and incubated in a microaerophilic atmosphere that was created by using a gas generating kit for Campylobacter (Oxoid) in an anaerobic jar (Oxoid). Plates were incubated at 42°C for 48 h. Campylobacter was identified to genus level as described by Hunt (1992) and species identified using API-CAMPY identification kit (BioMe´rieux, Hazelwood, MO). Journal of Dairy Science Vol. 84, No. 10, 2001
Escherichia coli from raw milk was isolated as described by Ansay and Kaspar (1997) with modifications. Briefly, 10 ml of raw milk was enriched in an equal volume of 2 × modified EC broth (Difco Laboratories, Detroit, MI) supplemented with 20 µg/ml of novobiocin (Sigma Chemical Co., St. Louis, MO) and incubated overnight at 37°C. Escherichia coli was isolated by subculturing 50 µl of enriched sample onto MacConkey agar supplemented with 4-methylumbelliferyl-beta glucuronide (Difco). The plates were incubated for 48 h at 42°C. At least 5 to 10 fluorescent colonies from MacConkey-4-methylumbelliferyl-beta glucuronide plates were selected for further characterization. For detection of E. coli O157:H7, 50 µl of enriched sample was spread on sorbitol MacConkey agar (Difco) supplemented with cefixime (0.05 mg/L) and potassium tellurite (2.5 mg/L) (Dynal Inc., Lake Success, NY) and incubated for 48 h at 42°C. At least 5 to 10 sorbitol-negative colonies from sorbitol MacConkey agar were tested with O157 antigen by latex agglutination (Unipath Co., Ogdensburg, NY). Isolates identified as belonging to the genus Escherichia were tested for shiga-toxins by the specific PCR assay (Pollard et al., 1990). Isolates that produced shiga-toxins were identified and speciated with API 20E (BioMe´rieux). Isolation of L. monocytogenes was done as described by Hitchins (1992). Briefly, 25 ml of milk sample was added to 225 ml of Listeria enrichment broth (Difco) and incubated for 24 and 48 h at 30°C. A loopful of enriched broth was streaked on Oxford medium (Unipath) and Lithium chloride-phenylethanol moxalactam agar (Unipath) at 24 and 48 h, respectively. At least five colonies that were presumptive positive on Oxford and Lithium chloride-phenylethanol moxalactam agar were transferred to trypticase soy agar (Difco) with 0.6% yeast extract. The plates were incubated for 48 h at 30°C. All isolates were examined for Gram’s reaction, hemolysis, and cAMP reaction on 5% sheep blood agar, catalase production, nitrate reduction, and motility on SIM medium (Becton Dickinson, Cockeysville, MD). All isolates were confirmed to species by use of API-Listeria (BioMe´rieux) identification kit. Isolates identified as L. monocytogenes were serotyped using O antisera (Difco). Isolation of Salmonella from raw milk was done as described by Andrews et al. (1992). Briefly, 25 ml of milk was added to 225 ml of lactose broth (Difco) and incubated for 24 h at 37°C. About 1 ml of preenriched sample was transferred to 10 ml each of selenite cystine broth (Unipath) and tetrathionate broth (Unipath), respectively. Enrichment was carried out for 24 h at 37°C. The enriched broths were subcultured on Hektoen enteric agar (Difco) and xylose lysine desoxycholate agar (Unipath) and incubated for 24 h at 35°C. Colonies presumptive for Salmonella were inoculated on triple
PATHOGENS IN BULK TANK MILK
sugar agar (Difco) and urease agar (Difco) and then tested with Salmonella polyvalent O antiserum (Difco). Organisms that gave typical reactions for Salmonella were then confirmed by using API-20E identification kit (BioMe´rieux). Isolates were then tested by seroagglutination using Salmonella O group (A-I) antisera (Difco). Yersinia enterocolitica was isolated from raw milk as described by Weagant et al. (1992). Briefly, 10 ml of milk was added to 90 ml of peptone sorbitol bile broth and incubated at 10°C for 10 d. On d 10, the enriched broth was treated with 0.5% KOH and plated on MacConkey’s agar and Celfsulodin-irgasan-novobiocin agar (Difco). The plates were incubated for 48 h at 22°C. Colonies with characteristics for Yersinia were examined on lysine arginine iron agar (Difco), Christensen’s urea agar (Difco) and bile esculin agar (Difco). Organisms identified as Yersinia were speciated with API20E identification kit (BioMe´rieux). Isolates of Y. enterocolitica were presumed to be pathogenic based on a positive reaction to the autogglutination test described by Laird and Cavanaugh (1980). Data Analyses Epi-info version 6.0, a database and statistics system for epidemiology on microcomputers was used for data analysis (Centers for Disease Control and Prevention, Atlanta, GA). The occurrence of one or more pathogenic bacteria in raw milk was compared to the grade of milk and milk consumption practices using chi-square tests. RESULTS AND DISCUSSION Farm BTM from 131 dairy herds in eastern South Dakota and western Minnesota was examined for the presence of foodborne pathogens. Isolation rates of C. jejuni, STEC, Salmonella spp., and Y. enterocolitica have been reported from several regions of the United States and Canada; however, this is the first time that the isolation rate of foodborne pathogens has been reported from the eastern part of South Dakota and western Minnesota (Tables 1 and 2). It was observed that 35 of 131 (26.7%) BTM samples had one or more pathogens. An isolation rate of 25% was reported by Rohrbach et al. (1992) from BTM milk in eastern Tennessee and southwestern Virginia, which contained one or more pathogenic species of bacteria. BTM from Grade A and non-Grade A producers met the requirements for the states of South Dakota and Minnesota in effect at the time of the study. All Grade A milk had a SCC of <750,000 cells/ml, a standard plate count of <100,000 cfu/ml, and was free of drug residues. All non-Grade A milk or manufacturing grade was free
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of drug residues, had SCC <750,000 cells/ml for South Dakota producers and <1,000,000 cells/ml for Minnesota producers. In this study, 18 of 34 (53%) non-Grade A raw milk samples had one or more pathogenic bacteria. The percentage was significantly higher than the isolation rate from Grade A milk, which was 17.5%. The odds ratio analysis indicated that the likelihood of isolating one or more pathogenic bacteria from nonGrade A raw milk was 4.98 fold higher than Grade A raw milk (χ2 = 14.98, P < 0.0001) (Table 1). In this study, C. jejuni was isolated from 12 of 131 (9.2%) of BTM samples (Table 1). Campylobacter jejuni was also isolated concurrently with E. coli (n = 1 bulk tank), L. monocytogenes (n = 1), and Y. enterocolitica (n = 1). The isolation rate observed is higher than most of the reported isolation rates, with the exception of that of Rohrbach et al. (1992) (Table 2). Campylobacter jejuni frequently has been isolated from raw BTM (Beumer et al., 1988, Rohrbach et al., 1992; Steele et al., 1997) and from feces of cows (Beumer et al., 1988; Humphrey and Beckett, 1987). Contamination of milk with feces containing Campylobacter spp. is the primary cause. A study conducted by Lander and Gill (1980) showed that following intramammary inoculation with C. jejuni, C. jejuni was shed in milk for 3 to 73 d postinfection in variable numbers. Outbreaks of Campylobacteriosis caused by consumption of raw milk and certified raw milk have been reported in California, Pennsylvania, Michigan, Minnesota, and Vermont (Potter et al., 1983). STEC belong to several serotypes including O157:H7 and have been associated with human disease. Foods of animal origin including raw milk have been implicated as important vehicles for STEC infections in humans. It is now widely documented that STEC, including O157:H7, can be isolated from feces of asymptomatic cattle and raw milk (Wells et al., 1991). Seldom has raw milk been implicated in outbreaks of disease caused by STEC; from 1982 to 1990 only 14 confirmed cases of STEC-associated illness following consumption of raw milk occurred in the United States (Erskine, et al., 1991). In this study, STEC strains were isolated from 5 of 131 (3.8%) BTM samples. Klie et al. (1997) reported a similar STEC incidence rate of 3.9% for raw milk and 2.1% for certified raw milk in Germany. A recent study conducted by Steele et al. (1997) reported that only 0.87% of the BTM samples in Ontario contained STEC. Four of the five isolates of E. coli encoded for shiga-toxin 2 gene, while one strain encoded for shiga-toxin 1 gene. On the contrary, Montenegro et al. (1990) reported that most of the STEC isolates of bovine origin encoded for shiga-toxin 1 gene. Listeria monocytogenes was isolated from 6 of 131 (4.6%) of BTM samples (Table 1). The isolation rate Journal of Dairy Science Vol. 84, No. 10, 2001
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Table 1. Prevalence of selected pathogens in bulk tank milk from eastern South Dakota and western Minnesota. No. (%) of isolates from bulk tank milk (n = 131)
Organism
Milk grade1 (n = 131)
Combination of pathogens Bulk tanks (n = 131)
Organism(s)
A (n = 97)
Non A (n = 34)
Consume raw milkb (n = 131) Yes (n = 79)
No (n = 52)
Salmonella spp.
8 (6.1)
Salmonella + E. coli
7 1
3 ...
4 1
5 1
2 ...
Listeria monocytogenes
6 (4.6)
L. monocytogenes + C. jejuni + C. jejuni + Y. enterocolitica C. jejuni + E. coli Y. enterocolitica
4 1 1
1 ... ...
3 1 1
2 ... ...
2 1 1
9 1 7
7 1 3
2 ... 4
5 1 4
4 ... 3
Campylobacter jejuni
12 (9.2)
Yersinia enterocolitica
8 (6.1)
STEC Escherichia coli Total
5 (3.8) 39 ...
E. coli ... ...
4 35 26.7%
2 17 17.5%
2 18 52.9%
3 21 26.6%
1 14 26.9%
Milk grade: test of significance, χ2 (P) = 14.98 (0.0001); Odds ratio (confidence interval) = 4.98 (1.96 to 12.22). Consumption of raw milk: test of significance, χ2 (P) = 0.26 (0.6113); Odds ratio (confidence interval) = 0.82 (0.37 to 5.39).
1 b
observed in this study was similar to isolation rates reported in the literature (Table 2). All isolates of L. monocytogenes belonged to O antigen type 1. The O antigen includes serotypes 1/2a, 1/2b, and 1/2c. In this study, the serotype analysis was limited to the O antigen type, the six isolates could have been any of the 3
serotypes(1/2a, 1/2b, or 1/2c). Listeria monocytogenes serotypes 1, 1/2a, and 4b have been reported to occur in raw milk (Fenlon et al., 1995; Liewen and Plautz, 1988; Yoshida et al., 1998). As observed with gramnegative bacteria, fecal contamination could be the source of L. monocytogenes contamination in raw BTM.
Table 2. Isolation rates for foodborne pathogens from raw milk reported from Canada and United States. Percent isolation rate
Year
Country
Reference
Campylobacter jejuni
0.9 1.5 0.4 1.2 12.3 9.2 0.47
1982 1983 1987 1989 1992 1997 1998
US USA USA Canada USA USA Canada
Doyle & Roman (1982) Lovett et al. (1983) McManus and Lanier (1987) Davaidson et al. (1989) Rohrbach et al. (1992) This study Steele et al. (1997)
Shiga-toxin producing Escherichia coli
3.8 0.87
1997 1998
USA Canada
This study Steele et al. (1997)
Listeria monocytogenes
4.2 1.3 5.4 4.0 1.6 1.9 4.1 4.6 2.7 4.7 2.9 8.9 6.1 0.17
1987 1988 1988 1988 1989 1990 1992 1997 1998 1987 1988 1992 1997 1998
USA Canada Canada USA Canada Canada USA USA Canada USA Canada USA USA Canada
Lovett et al. (1987) Farber et al. (1988) Slade et al. (1988) Liewen and Plautz (1988) Davidson et al. (1989) Fedio and Jackson (1990) Rohrbach et al. (1992) This study Steele et al. (1997) McManus and Lanier (1987) McEwen et al. (1988) Rohrbach et al. (1992) This study Steele et al. (1997)
1983 1987 1989 1992 1997
USA USA Canada USA USA
Moustafa et al. (1983) McManus and Lanier (1987) Davidson et al. (1989) Rohrbach et al. (1992) This study
Pathogen
Salmonella spp.
Yersinia enterocolitica
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12 48.1 2.7 15.1 6.1
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PATHOGENS IN BULK TANK MILK
The low incidence of L. monocytogenes in raw milk does not pose any practical problem for pasteurized milk and processed dairy products but may be of significance for raw milk consumption (O’Donnell, 1995). Salmonella was isolated from 8 of 131 (6.1%) BTM samples, which is within the range of isolation rates reported in the literature (Table 2). Isolates of Salmonella belonged to group D (n = 4), B (n = 2), C (n = 1), and E (n = 1) “O” serogroups. These serogroups have been identified as important causes of diseases in calves, dairy cows, and humans (Carter et al., 1995). The isolation rate of Y. enterocolitica (6.1%; Table 1) observed in this study is considerably lower than that reported previously (Table 2). Based on an autoagglutination test, it was inferred that all eight isolates of Y. enterocolitica were pathogenic. To date there have been two reported outbreaks of foodborne infection caused by Y. enterocolitica, in which contaminated milk was incriminated (Black et al., 1978; Tackett et al., 1984). Although Y. enterocolitica has been isolated from the intestines of dairy cattle, generally milk cows are not considered as reservoirs of pathogenic strains of Y. enterocolitica. However, a major concern is that Y. enterocolitica can grow to large numbers at refrigeration temperatures so that contaminated raw milk could become significant health risk when consumed. The isolation of foodborne disease-causing bacteria in raw milk has been reported extensively from Canada and United States (Table 2). The isolation rates reported over the last two decades vary considerably. This can be attributed partly to the isolation identification, true prevalence, season, geographic area, farm size, number of animals on farm, hygiene, and farm management practices. These reported findings clearly suggest pathogens do occur in BTM and may pose a health hazard if raw milk is consumed. In this study, 79 of 131 (60%) dairy producers who participated in the BTM pathogen survey reported they consumed raw milk. Of the 79 dairy producers who consumed raw milk, 21 (26.6%) had one or more pathogenic bacteria in their BTM. There was no significant difference in the incidence of pathogenic bacteria in raw milk of dairy producers who did and did not consume raw milk (χ2 = 0.26; P = 0.6113); odds ratio = 0.82 (confidence interval, 0.37 to 5.39) (Table 1). An epidemiological study conducted by Headrick et al. (1998) on raw-milk associated foodborne disease outbreaks in the United States revealed that 46 raw milk associated outbreaks occurred between 1973 and 1992; of which 40 (87%) of the outbreaks occurred in states where the intrastate sale of raw milk was legal. They concluded that consumption of raw milk remains a preventable cause of foodborne disease outbreaks.
CONCLUSIONS The findings of this study suggest that pathogenic bacteria of human health significance are in BTM. This observation and with previously reported data on consumption of raw milk from the same geographical area indicate that dairy producers and farm families are at risk of ingesting foodborne pathogens when consuming raw milk. This risk can be further compounded when it is legally permitted to sell raw milk. Based on the findings of this study, it is recommended that further studies be conducted to understand the behaviorial risk factors associated with raw milk consumption and that educational programs be developed to address issues related to consumption of raw milk. Based on the findings C. jejuni, STEC, L. monocytogenes, Salmonella spp., and Y. enterocolitica were isolated from BTM samples collected from western South Dakota and eastern Minnesota. The prevalence rates for most foodborne pathogens in BTM were similar to that previously reported from Canada and United States. Foodborne pathogens were more likely to be present in non-Grade A milk than Grade A milk. REFERENCES Andrews W. K., V. R. Bruce, G. June, F. Satchell, and P. Sherrod. 1992. Salmonella. Pages 51–70 in FDA Bacteriological Analytical Manual. 7th ed. Association of Official Analytical Chemists, Arlington, VA. Ansay, S. E., and C. W. Kaspar. 1997. Survey of retail cheeses, dairy processing environments and raw milk for Escherichia coli O157:H7. Lett. Appl. Microbiol. 25:131–134. Beumer, R. R., J. J. M. Cruysen, and J. R. K. Birtantie. 1988. The occurrence of Campylobacter jejuni in raw cow’s milk. J. Appl. Bacteriol. 65:93–96. Black, R. E., R. J. Jackson, T. Tsai, M. Medvesky, M. Shayegani, J. C. Feeley, K. I. E. MaCleod, and A. M. Wakeler. 1978. Epidemic Yersinia enterocolitica infection due to contaminated chocolate milk. N. Engl. J. Med. 298:70–76. Borczyk, A. A., M. A. Karmali, H. Loir, and L. M. C. Duncan. 1987. Bovine reservoir for verotoxin-producing Escherichia coli O157:H7. Lancet (8524):98. Carter, G. R., M. M. Chengappa, and R. A Wayne. 1995. Essentials of Veterinary Microbiology. 5th ed. Williams and Wilkins, Baltimore, MD. pp 151–165. Davidson, R. J., D. W. Sprung, C. E. Park, and M. K. Raymond. 1989. Occurrence of Listeria monocytogenes, Campylobacter spp., and Yersinia enterocolitica in Manitoba raw milk. Can. Inst. Food Sci. Technol. J. 22:70–74. D’Aoust, J. Y., D. W. Warburton, and A. M. Sewell. 1985. Salmonella typhimurium phage-type 10 from cheddar cheese implicated in a major Canadian foodborne outbreak. J. Food Prot. 48:1062–1066. Doyle, M. P., and D. J. Roman. 1982. Prevalence and survival of Campylobacter jejuni in unpasteurized milk. Appl. Environ. Microbiol. 44:1154–1158. Erskine, R. J., J. W. Tyler, M. G. Riddell, Jr., and R. C. Wilson. 1991. Theory, use, and realities of efficacy and food safety of antimicrobial treatment of acute coliform mastitis. J. Am. Vet. Med. Assoc. 198:980–984. Evans, M. R, R. J. Roberts, C. D. Ribeiro, D. Gardner, and D. Kembrey. 1996. A milk-borne Campylobacter outbreak following an educational farm visit. Epidemiol. Infect. 117:457–462. Journal of Dairy Science Vol. 84, No. 10, 2001
2162
JAYARAO AND HENNING
Farber, J. M., G. W. Sander, and S. A. Malcolm. 1988. The presence of Listeria spp. in raw milk in Ontario. Can. J. Microbiol. 34:95–100. Fedio, W. M., and H. Jackson. 1990. Incidence of Listeria monocytogenes in raw milk in Alberta. Can. Inst. Food Sci. Technol. J. 23:236–238. Fenlon, D. R., and J. Wilson. 1989. The incidence of Listeria monocytogenes in raw milk from farm bulk tanks in North-East Scotland. J. Appl. Bacteriol. 66:191–196. Fleming, D. W., S. L. Cochi, K. L. MacDonald, J. Brondum, P. S. Hayes, B. D. Plikaytis, M. B. Holmes, A. Audurier, C. V. Broome, and A. L. Reingold. 1985. Pasteurized milk as a vehicle of infection in an outbreak of listeriosis. New Engl. J. Med. 312:404–407. Headrick, M. L., B. Timbo, K. C. Klontz, and S. B. Werner, 1997. Profile of raw milk consumers in California. Public Health Rep 112:418–422. Headrick, M. L., S. Korangy, N. H. Bean, F. J. Angulo, S. F. Altekruze, M. E. Potter, and K. C. Klontz. 1998. The epidemiology of raw milk-associated foodborne disease outbreaks reported in the United States, 1973 through 1992. Am. J. Public Health 88:1219–1221. Hitchins, A. D. 1992. Listeria monocytogenes. Pages 141–151 in FDA Bacteriological Analytical Manual. 7th ed. Association of Official Analytical Chemists, Arlington, VA. Humphrey, T. J., and P. Beckett. 1987. Campylobacter jejuni in dairy cows and raw milk. Epidemiol. Infect. 98:263–269. Hunt, J. M. 1992. Campylobacter. Pages 77–94 in FDA Bacteriological Analytical Manual. 7th ed. Association of Official Analytical Chemists, Arlington, VA. Jayarao, B. M., and E. K. Cassel. 1999. Mastitis prevention and milk hygiene practices adopted by dairy producers. Large Anim. Pract. 20:6–14. Jones, P. H., and A. T. Willis. 1981. Campylobacter enteritis associated with the consumption of free school milk. J. Hyg. Camb. 87:155–170. Keene, W. E., K. Hedberg, D. E. Herriott, D. D. Hancock, R. W. McKay, T. J. Barrett, and D. W. Fleming. 1997. A prolonged outbreak of Escherichia coli O157:H7 infections caused by commercially distributed raw milk. J. Infect. Dis. 176:815–818. Klie, H., M. Timm, H. Richter, P. Gallien, K. W. Perlberg, and H. Steinruck. 1997. Detection and occurrence of vertoxin-forming and/or shigatoxin producing Escherichia coli (VTEC and/or STEC in milk). Berl. Munch. Tierarztl. Wochenschr. 110:337–341. Laird, W. J., and D. C. Cavanaugh. 1980. Correlation of autoagglutination and virulence of Yersinia. J. Clin. Microbiol. 27:1667–1670. Lander, K. P., and K. P. W. Gill. 1980. Experimental infection of the bovine udder with Campylobacter coli/jejuni. J. Hyg. 84:421–428. Liewen, M. B., and M. W. Plautz. 1988. Occurrence of Listeria monocytogenes in raw milk in Nebraska. J. Food Prot. 51:840–841. Linnan, M. J., L. Mascola, X. Dong Lou, V. Goulet, S. May, C. Salinem, D. W. Hird, M. L. Yonekura, P. Hayes, R. Weaver, A. Audurier, B. D. Plikaytis, S. L. Fannin, A. Kleks, and C. V. Broome. 1988. Epidemic listeriosis associated with Mexican-style cheese. New Engl. J. Med. 319:823–828. Lovett, J. D., W. Francis, and J. M. Hunt. 1983. Isolation of Campylobacter jejuni from raw milk. Appl. Environ. Microbiol. 46:459–462. Lovett, J. D., W. Francis, and J. M. Hunt. 1987. Listeria monocytogenes in raw milk: Detection, incidence, and pathogenicity. J. Food. Prot. 50:188–192. Martin, M. L., L. D. Shipman, J. G. Wells, M. E. Potter, K. Hedberg, I. K. Wachsmuth, R. V. Tauxe, J. P. Davis, J. Arnolai, and J. Tilleli. 1986. Isolation of Escherichia coli O157:H7 from dairy cattle associated with two cases of hemolytic uremic syndrome. Lancet. 1;2(85414):1043. McEwen, S. A., L. H. McClure, and S. W. Martin. 1988. Farm inspection scores and milk quality criteria as indices of Salmonella in bulk tank milk. J. Food Prot. 51:958–962. McManus, C., and J. M. Lanier. 1987. Salmonella, Campylobacter jejuni, and Yersinia enterocolitica in raw milk. J. Food Prot. 50:51–55.
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Montenegro, M. A., M. Bu¨lte, T. Trumpf, S. Aleksic˙, G. Reuter, E. Bulling, and R. Helmuth. 1990. Detection and characterization of fecal verotoxin-producing Escherichia coli from healthy cattle. J. Clin. Microbiol. 28:1417–1421. Moustafa, M. K., A. A-H. Ahmed, and E. H. Marth. 1983. Occurrence of Yersinia enterocolitica in raw and pasteurized milk. J. Food Prot. 46:276–278. O’Donnell, E. T. 1995. The incidence of Salmonella and Listeria in raw milk from farm bulk tanks in England and Wales. J. Soc. Dairy Technol. 48:25–29. Pollard, D. R., W. M. Johnson, H. Lior, S. D. Tyler, and K. R. Rozee. 1990. Rapid and specific detection of verotoxin genes in Escherichia coli by polymerase chain reaction. J. Clin. Microbiol. 28:540–545. Potter, M. E., M. J. Blaser, R. K. Sikes, A. F. Kaufmann, and J. G. Wells. 1983. Human Campylobacter infection associated with certified raw milk. Am. J. Epidemiol. 117:475–483. Rahn, K., S. A. Renwick, R. P. Johnson, J. B. Wilson, R. C. Clarke, D. Alves, S. McEwen, H. Loir, and J. Spika. 1997. Persistence of Escherichia coli O157:H7 in dairy cattle and the dairy farm environment. Epidem. Infect. 119:251–259. Rohrbach, R. W., F. A. Draughon, P. M. Davidson, and S. P. Oliver. 1992. Prevalence of Listeria monocytogenes, Campylobacter jejuni, Yersinia enterocolitica and Salmonella in bulk tank milk: risk factors and risk of human exposure. J. Food Prot. 55:93–97. Ryan, C. A., M. K. Nickels, N. T. Hargrett-Bean, M. E. Potter, T. Endo, L. Mayer, C. W. Langkop, C. Gibson, R. C. McDonald, R. T. Kenny, N. D. Puhr, S. J. McDonnell, R. J. Martin, M. L. Cohen, and P. A. Blake. 1987. Massive outbreak of antimicrobial-resistant salmonellosis traced to pasteurized milk. J. Am. Med. Assoc. 258:3269–3274. Sieper, J., G. H. Kingsley, and E. Marker-Herman. 1996. Aetiological agents and immune mechanisms in enterogenic reactive arthritis. Baillieres. Clin. Rheumatol. 10:105–121. Slade, P. J., D. L. Collins-Thompson, and F. Fletcher. 1988. Incidence of Listeria species in Ontario raw milk. Can. Inst. Food Sci. Technol. J. 21:425–429. Spake, A., M. B. Marcus, and D. McGraw. 1997. O is for outbreak: When a drug-resistant Salmonella struck a Vermont farm, health officials knew it might be just the beginning. US News World Rep. 23:70–79. Steele, M. L., W. B. McNab, C. Poppe, M. W. Griffiths, S. Chen, S. A. Degrandis, L. C. Fruhner, C. A. Larkin, J. A. Lynch, and J. A. Odermeru. 1997. Survey of Ontario bulk tank milk for foodborne pathogens. J. Food Prot. 60:1341–1346. Tackett, C. O., J. P. Narain, R. Sattin, J. P. Lofgren, C. Konigsberg, Jr., R. C. Rendtorff, A. Rausa, B. R. Davis, and M. L. Cohen. 1984. A multistate outbreak of infections caused by Yersinia enterocolitica transmitted by pasteurized milk. J. Am. Med. Assoc. 251:483–486. Tran, T. T., 1995. Evaluation of Oxyrase enrichment method for isolation of Campylobacter jejuni from inoculated foods. Lett. Appl. Microbiol. 21:345–347. Weagant, S. D., P. Feng, and J. T. Stanfield. 1992. Yersinia enterocolitica and Yersinia pseudotuberculosis. Pages 95–109 in FDA Bacteriological Analytical Manual. 7th ed. Association of Official Analytical Chemists, Arlington, VA. Wells, J. G., L. D. Shipman, K. D. Greene, E. G. Sowers, J. H. Green, D. N. Cameron, M. E. Potter, R. V. Tauxe, and I. K. Wachsmuth. 1991. Isolation of Escherichia coli serotype O157:H7 and other shiga-like-toxin producing E. coli from dairy cattle. J. Clin. Microbiol. 29:985–989. Wray, C., Q. C. Wadsworth, and D. W. Richards, and J. H. Morgan. 1989. A three-year study of Salmonella dublin infection in a closed dairy herd. Vet. Rec. 124:532–535. Yoshida, T., M. Sato, and K. Hirai. 1998. Prevalence of Listeria species in raw milk from farm bulk tanks in Nagano prefecture. J. Vet. Med. Sci. 60:311–314.