93 Journal of Protection, Vol. 55, No. 2, Pages 93-97 (February 1992) Copyright©, International Association of Milk, Food and Environmental Sanitarians

Prevalence of , , Yersinia enterocoiitica, and in Bulk Tank Milk: Risk Factors and Risk of Human Exposure

1 2 2 3

BARTON W. ROHRBACH *, F. ANN DRAUGHON , P. MICHAEL DAVIDSON , and STEPHEN P. OLIVER Downloaded from http://meridian.allenpress.com/jfp/article-pdf/55/2/93/1662951/0362-028x-55_2_93.pdf by guest on 02 October 2021

'Department of Rural Practice, College of Veterinary Medicine; ^Department of and Science; and3Department of Animal Science, Institute of Agriculture, University of Tennessee, Knoxville, Tennessee 37996

(Received for publication August 5, 1991)

ABSTRACT from the bulk tank, and examined the association of se­ lected risk factors for the presence of selected bacterial Milk samples (n=292) from farm bulk tanks were analyzed pathogens in farm bulk tank milk. for selected . Frequency of bacterial isolation was Listeria monocytogenes 12 (4.1%), Campylobacter jejuni 36 (12.3%), METHODS Yersinia enterocolitica 44 (15.1%), and Salmonella 26 (8.9%). The presence of one or more selected bacteria was not associated Study sample with grade classification of dairy, barn type, milking hygiene, The population from which the study sample was drawn reported incidence of clinical mastitis among cows, or the number included all dairy producers (n=355) shipping milk to an East of cows on the farm. Consumption of raw bulk milk was reported Tennessee processing plant which manufactures condensed milk. by 68/195 (34.9%) dairy producers, and of bulk tanks from which was consumed, 17/68 (25%) were contaminated with Questionnaire one or more species of . A questionnaire was used to obtain information on selected demographic, management, husbandry, and health characteristics of the farm and herd, as well as raw milk consumption by dairy Sampling bulk tank milk has been recognized as a producers or others. Questionnaires were distributed prior to practical means for identifying milkborne pathogens at the sampling and collected when bulk tank milk samples were ob­ tained. farm level. Prior surveys identified Salmonella species, Campylobacter jejuni, Listeria monocytogenes, and Yersinia Collection of samples enterocolitica in farm bulk tank milk at the farm level and Independent milk haulers were provided instructions on the in composite samples from tank trucks transporting milk to collection of milk samples and administration of questionnaires. processing plants (4,9,10,12). Prevalence of these bacteria Milk was collected from individual farm bulk tanks at l-to-3 d in bulk tank milk varies among surveys and may be related intervals. Samples and questionnaires were collected from August to geographic (environmental) differences, season, farm 13-15, 1990, in order that all producers shipping to the plant could size, density of animal populations, and regional differ­ be sampled. A 10.82 ml (4 oz) sample of bulk tank milk was ences in management and husbandry of dairy cattle. placed in a sterile Whirl-Pak® bag and refrigerated until arrival at the processing plant. Samples were refrigerated continuously from Numerous milkborne outbreaks of disease, documented the time of collection until analyzed within 24 h. to be the result of by the above pathogens, have been reported in the United States and internationally (5- Laboratory procedure 7,13,16). The true incidence of milkborne disease in the Milk samples were inverted 25 times before dividing into United States, especially where one or a few cases occur, is four aliquots. One aliquot was analyzed for the presence of unknown. Consumption of raw (unpasteurized) milk by Salmonella using the American Public Health Association con­ dairy producers, their employees, family members, and ventional method (12). Milk (25 ml) was inoculated into 225 ml others is thought to be common practice; however, quanti­ of selenite cystine (SC) selective enrichment broth. Selective tative information regarding this practice is unavailable. enrichment broth was incubated at 37°C for 24 h (19). The SC broth was streaked onto brilliant green agar plates and onto XLD Currently, much emphasis for research is placed on agar supplemented with and incubated at 37°C for 24 reduction of contamination of animal agricultural products h. Typical colonies were picked onto slants of triple iron at the farm level (18). Through a bacterial survey of bulk agar and lysine agar. Salmonella were confirmed biochemically tank milk and questionnaires, this study determined the (12). prevalence of selected pathogenic bacteria, estimated the To detect C. jejuni, milk (25 ml) was inoculated into 225 ml risk of exposure due to human consumption of raw milk of brain heart infusion broth supplemented with Blaser Wang

JOURNAL OF FOOD PROTECTION, VOL. 55, FEBRUARY 1992 94 ROHRBACH, DRAUGHON, DAVIDSON AND OLIVER selective supplement and incubated at 42°C for 24 h (_/). Broth TABLE 1. Characteristics of dairies from which questionnaires was streaked onto blood free Campylobacter agar (Oxoid, Inc., 2nd!or milk samples were obtained for a bulk milk survey, East Basinstoke, England) and incubated at 42°C. Plates were exam­ Tennessee and Southwest Virginia, 1990. ined at 24, 48, and 72 h. Typical colonies were picked and examined for , motility, , hippurate hydrolysis, No./total % and biochemical characteristics (12). For detection of Y. enterocolitica, milk (50 ml) was inocu­ Response to questionnaire 200/355* 56.3 lated into primary enrichment broth consisting of 200 ml of Grade B 246/292** 84.3 phosphate buffered physiological saline (M/15, pH 7.6, 0.85% Samples tested 292/355 82.3 Barn type NaCl) containing 0.01% bile and incubated at 4°C. After 7 + d, 1 ml of primary enrichment broth was transferred to bile Stanchion 39/197 19.8 oxylate sorbose broth and incubated at 2I°C for 5 d (15). Second­ Parlor 148/197 75.1 ary enrichment broth was plated onto Yersinia selective agar and Other 10/197 5.1 incubated at 32°C for 24 h. Primary enrichment broth was also Teat dipping 143/196 73.0 streaked onto Yersinia selective agar after 7 and 14 d of incuba­ Dry cow treatment 104/195 53.3 tion at 4°C. Typical colonies were examined for Gram stain, Membership in DHIA++ 15/193 7.8 motility, and biochemical characteristics (12). Environmental Consume raw milk 68/195 34.9 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/55/2/93/1662951/0362-028x-55_2_93.pdf by guest on 02 October 2021 isolates were distinguished from pathogenic isolates using the * Denominator = total samples collected. crystal violet assay (14). **Denominator = total samples analyzed. Milk from farm bulk tanks was analyzed for the presence of + Denominator less than 200 due to incomplete response. L. monocytogenes using a method described previously (8). Milk ++ Dairy Herd Improvement Association. (25 ml) was inoculated into 225 ml of enrichment broth (trypticase soy broth, 30 g; yeast extract, 6 g; acriflavin HC1, 15 mg; nalidixic acid, 40 mg; cycloheximide, 50 mg; distilled water, 1 L) and TABLE 2. Isolation of L. monocytogenes, C. jejuni, Y. entero­ incubated for 48 h at 30°C. After 48 h, the culture from enrich­ colitica, and Salmonella from bulk tank milk samples, East Ten­ ment broth was streaked onto both lithium chloride-phenylethanol- nessee and Southwest Virginia, 1990. moxalactam agar and Listeria Selective Medium (Oxford Formu­ lation) and incubated for 48 h at 30°C. Suspect colonies from No. of both media were picked and streaked for isolation onto trypticase isolates (n=292) % soy agar with 0.6% yeast extract. L. monocytogenes was con­ firmed as follows: Gram stain, catalase production, urea utiliza­ L. monocytogenes 12 4.1 tion, motility, , reactions in triple sugar iron agar and C. jejuni 36 12.3 MR-VP medium, CAMP test and utilization of glucose, maltose, Y. enterocolitica* 44 15.1 esculin, xylose, and rhamnose. Salmonella 26 8.9 One or more isolates 95 32.5 RESULTS Pathogenicity confirmed by crystal violet assay. During a 3 d period, August 13-15, 1990, 355 bulk tank milk samples were collected. All samples collected on days 1 and 2 were analyzed, together with, only those TABLE 3. Combinations of selected zoonotic bacteria* isolated samples collected on day 3 for which completed question­ from individual bulk tank samples positive for at least one bacte­ naires were received (n=292) (Table 1). Grade B dairies rium. comprised 246 of 292 (84.3%) of bulk milk samples; the remainder were classified as Grade A. Questionnaires were No of isolates received from 200 of 355 (56.3%) dairies shipping milk to Pathogen (n=95) % the processing plant. Of 200 questionnaires received, seven L. monocytogenes + C. jejuni 1 1.1 (3.5%) could not be matched to a milk sample but were L. monocytogenes + Y. enterocolitica 5 5.3 included in the analysis. Information on selected character­ L. monocytogenes + Salmonella 1 1.1 istics of the dairy herd and farm management practices C. jejuni + Y. enterocolitica 9 9.5 were summarized from questionnaires (Table 1). In addi­ C. jejuni + Salmonella 1 1.1 tion, information was obtained on whether raw milk from C. jejuni + Salmonella + 1 1.1 the bulk tank was used for human consumption. Y. enterocolitica Y. enterocolitica + Salmonella 4 4.2 Of the 292 bulk tank samples tested, L. monocytogenes L. monocytogenes only 5 5.3 was isolated from 12 (4.1%), C. jejuni from 36 (12.3%), Y. C. jejuni only 24 25.3 enterocolitica from 44 (15.1%), and Salmonella from 26 Y. enterocolitica only 25 26.3 (8.9%) (Table 2). One of the four pathogens was isolated Salmonella only 19 20.0 from 73 of the 95 positive samples, whereas 22 samples contained two or more pathogens (Table 3). The proportion * L. monocytogenes, C. jejuni, Y. enterocolitica, Salmonella. of each selected bacterial pathogen found in combination with one or more of the other pathogens was L. monocyto­ The proportion of dairy producers who returned ques­ genes 7/12 (58%), C. jejuni 11/36 (31%), Y. enterocolitica tionnaires and who had one or more pathogens isolated 19/44 (43%), and Salmonella, 7/26 (27%). The difference from their bulk tank, 60 of 193 (31%), was not different in proportion of each pathogen found singly or in combina­ (P>0.05) from the proportion of contaminated bulk tanks of tion with other organism(s) was not significant (P=0.19) dairy producers not returning the questionnaire, 35 of 99 among genera.

JOURNAL OF FOOD PROTECTION, VOL. 55, FEBRUARY 1992 PATHOGENIC BACTERIA IN BULK TANK MILK 95

(35%) (Table 4). None of the risk factors including grade TABLE 5. Exposure to bacterial pathogens* through consump­ classification of the dairy, facilities for milking, use of teat tion of contaminated bulk tank milk, East Tennessee and South­ dip and/or dry cow therapy, or membership in the Dairy west Virginia, 1990. Herd Improvement Association was associated significantly with bulk tank isolates of specific bacterial pathogens or Not Contaminated** isolation of any pathogen from the bulk tank. Of 84 produc­ contaminated (n=138) (n=57) ers who used teat dipping and dry cow therapy and classi­ fied as having good milking hygiene, 29 (35%) had con­ no. % no. % Total taminated bulk tanks compared to 12 of 39 (31%) with poor milking hygiene and contaminated bulk tanks (OR 1.2, Consumed raw milk P=0.7). Other characteristics of dairies with contaminated Yes 51 75 17 25 68 bulk tanks that were compared to those with no pathogenic No 87 69 40 32 127 bacteria included mean number of cows, producer reported * L. monocytogenes, C. jejuni, Y. enterocolitica, and Salmonella. incidence of clinical mastitis during the preceding month, ** One or more selected bacterial pathogens were isolated from

and percent replacement stock raised on the farm. The Downloaded from http://meridian.allenpress.com/jfp/article-pdf/55/2/93/1662951/0362-028x-55_2_93.pdf by guest on 02 October 2021 the bulk tank sample. frequency of these characteristics was not different (P>0.05) among the groups compared. DISCUSSION

TABLE 4. Selected risk factors for presence of bacterial con­ Sampling in this study was not random so generalizing tamination* of bulk tank milk (n=292) East Tennessee and from these data is difficult. However, the processing plant Southwest Virginia, 1990. from which samples were obtained received milk from most producers in East Tennessee and Southwest Virginia; Not contaminated Contaminated therefore, the sample may be a valid representation of dairies in this geographic area. Bacteria may have gained Factor no./total % no./total % access to milk via environmental contamination during the milking process, along the route to the tank, at the tank, or Questionnaire returned during the process of sampling. Study samples were col­ Yes 133/193 68.9 60/193 31.1 No 64/99 64.7 35/99 35.4 lected according to a standard procedure, and a greater Grade proportion of contaminated samples was not associated A 37/46 80.4 9/46 19.6 with any particular hauler (data not shown). The proportion B 179/246 72.8 67/246 27.2 of producers who answered the questionnaire was similar Barn type among Grade A and Grade B dairies (data not shown). The Stanchion 27/38 71.1 11/38 29.0 proportion of contaminated bulk tanks was similar among Parlor 111/142 78.2 31/142 21.9 dairy producers who responded to the questionnaire when Other 6/10 60 4/10 40 compared to nonrespondents. Thus, results of the present Teat dipping study should not be biased by the strong representation of Yes 105/138 76.1 33/138 23.9 No 38/51 74.5 13/51 25.5 Grade B dairies or the relatively low (56%) response rate to Dry cow therapy the questionnaire. Yes 74/98 75.5 24/98 24.5 The proportion of samples from which L. monocyto­ No 68/90 75.6 22/90 24.4 genes was isolated is similar to that reported in other Membership in DHIA surveys. However, C. jejuni, Y. enterocolitica, and Salmo­ Yes 9/14 64.3 5/14 35.7 nella were isolated from a greater proportion of bulk milk No 132/172 76.7 40/172 23.3 tanks than reported previously (2,4,9-11). If this sample is Clinical Mastitis** 4.5/100 4.5 3.4/100 3.4 representative of the East Tennessee and Southwest Vir­ No. of cows 23.0 ± 1.3+ 22.6 + 2.14 ginia region, this represents a substantial exposure to dairy % Replacement stock 664 + 3.3 68.3 + 6.1 grown on farm producers and others consuming raw bulk tank milk, as well as a potential source of continuous contamination of * Samples analyzed for L. monocytogenes, C. jejuni, Y. entero- milk processing plants. Within individual farms in the colitica, and Salmonella. Southeastern United States, particularly East Tennessee, a ** Producer reported incidence during the preceding 30 d. variety of animal products for both commercial and home + + 1 standard error of the mean. use is produced. The higher prevalence of gram-negative bacteria may reflect cross-contamination between animal species. Increased prevalence of C. jejuni and Salmonella may indicate cross-contamination between poultry and dairy animals. Likewise, the higher prevalence of Y. enteroco­ litica may reflect cross-contamination between swine and Of the 68 (34.9%) dairy producers who consumed raw dairy animals. Future epidemiologic studies should target milk, 17 (25%) had concurrent contamination of the bulk the source of contamination. tank with one or more zoonotic pathogens (Table 5). The prevalence of consumption of raw milk from contaminated The grading system for commercial dairies is based on bulk tanks in the study sample was 17/195 (8.7%). milk quality, observations of farm sanitation, and milking

JOURNAL OF FOOD PROTECTION, VOL. 55, FEBRUARY 1992 96 ROHRBACH, DRAUGHON, DAVIDSON AND OLIVER hygiene practices. Milk quality is based on the number of in farm bulk tanks from East Tennessee and Southwest bacteria in milk and would be expected to be influenced by Virginia is contaminated frequently with pathogenic bacte­ milking and environmental hygiene. Therefore, differences ria, and substantial numbers of persons associated with the in requirements for Grade A versus Grade B milk should dairy industry are exposed to these pathogens. Additional have a public health impact. However, there was no differ­ studies are needed to verify findings of the present study, ence observed among grades in the proportion of samples identify risk factors for the presence of selected pathogens contaminated by one or more of the selected pathogens. If in bulk milk, and to assess the impact of bulk tank contami­ differences in milk quality, farm sanitation, and milking nation on public health. These studies should include mul­ hygiene between grades exist in practice and results of the tiple samples taken at various seasons of the year from present study are reproducible in other dairies from differ­ randomly selected dairies representing broad regions of the ent geographic locations, criteria for grading milk should United States. Serotyping and sensitivity patterns be reviewed. for all bacterial pathogens should be performed so that Pathogens isolated from bulk tank milk in the present comparisons can be made with isolates from other sources. study are infrequently isolated from the mammary gland of The prevalence of raw milk consumption from contami­

the cow (5). The primary source of these pathogens is nated bulk milk tanks emphasizes the need to continue Downloaded from http://meridian.allenpress.com/jfp/article-pdf/55/2/93/1662951/0362-028x-55_2_93.pdf by guest on 02 October 2021 presumed to be the digestive tract of the cow or the efforts toward education of producers about health risks environment. Evaluation of factors associated with udder associated with the consumption of raw milk and empha­ infection in dairy cattle, such as use of teat dipping and dry sizes the importance of legislation prohibiting the sale of cow therapy, the incidence of clinical mastitis, and the unpasteurized milk. proportion of replacement stock raised on the dairy were not associated with isolation of selected pathogenic bacteria ACKNOWLEDGMENTS from bulk tank milk. Bacteria may gain access to bulk tank milk via environmental contamination during the milking Wc are indebted to Mr. Milton Oxford, Mr. Rocky Ottinger, and Mr. Ted Lester of Pet Milk Company, Grceneville. TN, and the independent process, along the route to the bulk tank, at the tank, or milk haulers transporting to Pet Inc. for assistance with distribution of during the process of sampling. A greater number of cows questionnaires and sample collection. Wc are also grateful to Mr. Jimmy on the dairy would be expected to provide a more suitable Hopper, Director, Ellington Agricultural Center, Nashville, TN, for data reservior for pathogenic bacteria, increase environmental on grading and inspection of Tennessee dairies; R. Phebus, C. Hwang, R. Hewitt, B. Compton, V. Juneja, M. Abdalla, D. Yobouct, R. Huff, and J. contamination, and augment the isolation of pathogens W. Wempc for laboratory analysis of milk samples; and Dr. Morris E. from the bulk tank. This was not the case in this study or Potter, Division of Bacterial Disease, Centers for Disease Control, Atlanta, a similar study performed in another geographic area (10). GA, for review of this manuscript. Funding for this project was provided by the University of Tennessee College of Veterinary Medicine Center of Consumption of raw milk from the bulk tank was Excellence Research Program in Livestock Diseases and Human Health, reported by 68 of 195 (35%) dairy producers. This is and the Tennessee Agricultural Experiment Station. probably a minimal estimate, as most dairy producers are aware of potential health risks associated with consumption REFERENCES of raw milk and may be unwilling to admit drinking 1. Blaser, M. J., J. Cravens, B. W. Powers, F. M. LaForce, W. LaForce, unpasteurized milk from the bulk tank. and L. L. Wang. 1979. Campylobacter enteritis associated with The proportion of producers who reportedly did not unpasteurized milk. Am. J. 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JOURNAL OF FOOD PROTECTION, VOL. 55, FEBRUARY 1992