Risk Factors Associated with Contamination of Raw Milk by Listeria monocytogenes in Dairy Farms

Risk Factors Associated with Contamination of Raw Milk by Llsterls monocytogenes In Dairy Farms M. SANAA. B. POUTREL,1 J. L MENARD,2 end F. SERIEYS2 Epidemiology and AnImal Health Management Laboratory Ecole Nationale Veterlnaire d'A1fort 7, avenue du General-de-Gaulle 94704 Malsons-Alfort, France ABSTRACT

A case-control study involving 128 selected dairy fanns was conducted to assess the association of several suspected risk factors with the odds of contamination of raw milk by Listeria ~nocytogenes. Using logistic regresSIon, we found that poor quality of silage (pH >4.0), inadequate frequency of cleaning the exercise area., poor cow cleanliness, insufficient lighting of milking bams and parlors, and incorrect disinfection of towels between milkings were significantly associated with milk contamination by L monocytogenes. More attention to preparing silage and good milking and bam hygiene are important for diminishing the risks of exogenous contamination of raw milk by L monocytogenes. (Key words: Listeria monocytogenes, raw milk, bacteriology quality) Abbreviation key: OR

= odds

ratio.

INTRODUCTION

Listeria monocytogenes is regularly implicated in human and animal diseases. Pregnant women, their fetuses or newborn children, and, more generally, any individual whose immune system is weak are particularly at high risk. Some disease incidence occurs in apparently healthy people. Listeriosis is usually sporadic, but large outbreaks related to consumption of contaminated foods have been reported (2).

Received November 24, 1992. Accepted May 17, 1993. Ilnstitut National de Ia Recherche Agrooomique. Laboratoire de Pathologic Infectieuse et Immunologic, Centre de Recherche de TOUB, 37380 Nouzilly, Prance. 21nstitut de I'Elevage. 149, roe de Bercy, 75012 Paris.

France. 1993 J Dairy Sci 76:2891-2898

Listeria monocytogenes is widely distributed in the environment and may be transmitted to humans through contamination of food products. Milk and milk products appear to be particularly susceptible to contamination (10). Listeria ssp. have been isolated from raw milk; prevalence has ranged from 1 to 45% (1, 3, 7, 8, 16), depending on survey design and isolation methods. All of the five recognized Listeria species were found in raw milk. The incidence of Listeria ssp. varies seasonally, and the concentration in raw milk often is < 1 bacterium/mi. Heat treatment significantly reduces the concentration of L. monocytogenes so that pasteurized milk does not present appreciable risk to human health (17). Thus, the presence of L monocytogenes in dairy products is mainly due to indirect contamination from raw milk. Furthermore, raw milk must be considered to be a potential source of plant contamination, which can affect the entire product production chain. On dairy farms, raw milk may be contaminated from feces (12, 13). Two studies (5, 24) have confirmed the relationship between poor quality of silage and the presence of Listeria spp. in feces. Listeria monocytogenes is one of the factors involved in the development of bovine mastitis. However, cases of bovine mastitis linked to L monocytogenes infection have been rare (4, 9, 14, 23). When mastitis occurs, the milk contains 2000 to 20,000 bacteria/mi. Thus, except for bovine mastitis, the milking procedure determines the main potential hazard of raw milk contamination. Milk can also be contaminated from unclean udders, teats, human hands, and equipment. The goal of the present study was to evaluate the association between raw milk contamination by L monocytogenes and herd factors, such as housing, feeding, and hygienic conditions. Our aim was to identify the main hazards related to herd management and to specify the critical control points that must be moni-

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SANAA ET AL.

tored in order to avoid milk contamination and, possibly, subsequent multiplication of L

monocytogenes. MATERIALS AND METHODS Study Design

The study population for the case-control study was selected from a group of 2000 dairy herds from which the milk was delivered raw to cheese factories. During the last 2 yr, these factories have initiated a program to monitor Listeria spp. based on analysis of the herd raw milk. The bulk tank milk samples were analyzed monthly to detect the presence of L monocytogenes using the isolation method developed by Van Netten et al. (25, 26). Milk from farm bulk tanks (30 ml) was inoculated into 225 ml of enrichment broth (trypticase soy broth, 30 g; yeast extract, 6 g; acriflavine·Hel, 15 mg; nalidixic acid, 40 mg; cycloheximide, 50 mg; distilled water, 1 L) and incubated at 37·C. The culture from enrichment broth was streaked onto Palcam medium (prepared in our laboratory) after 48 h and 7 d of incubation. All plates were incubated 48 h at 37·C. Suspect colonies from Palcam medium were selected and streaked for isolation onto trypticase soy agar with .6% yeast extract. Listeria monocytogenes was confirmed as follows: Gram stain, catalase production, urea utilization. motility, hemolysis, reactions in triple sugar iron agar and MR-VP (methyl redVoges-Proskauer) medium, CAMP test, and utilization of glucose, maltose, esculin, xylose, and rhamnose. Calculation of sample Size

We have made the following assumptions to calculate sample size: 1) the proportion of herds with no milk contamination by Listeria spp. (herd controls) exposed to the hypothetical risk factors was 30%; 2) relative risk >2 [estimated by odds ratio; (OR)] of the occurrence of L monocytogenes in raw milk was biologically important; 3) the threshold of significance for this estimated risk (a) was .05; and 4) the threshold of significance for the second type error (/1) was .20. The formula given by Schwartz et al. (22) indicated that at Journal of Dairy Science Vol. 76, No. 10, 1993

least 50 herds with contaminated bulk milk (herd cases) and an equal number of controls were needed in the study population. selection of Cases

The monitoring program initiated in the study area showed that the incidence of Listeria spp. in raw milk was lowest in summer and highest in winter. The seasonal variation in the occurrence of Listeria spp. in milk was important enough to limit the selection period for new cases from September to December. From September to December 1989, we detected 40 herds with bulk milk contaminated by L. monocytogenes (cases); 24 other cases were selected during the same period in 1990. After selecting the first 40 herds, we engaged in a preliminary study comparing those herds with 80 other herds for which milk was not contaminated by Listeria spp. The group of 80 herds was selected randomly from herds with no contaminated bulk tank milk. These two groups were compared for housing type, milk yield, and herd size. selection of Controls

Using the results on the monitoring program, a control was defined as a herd for which milk was not contaminated by Listeria spp. The group of controls was selected randomly, and constraints were used to obtain the same distributions of housing type, milk yield, and herd size in both groups (case and control). Dat8 Collection

Management practices often are interrelated; therefore, a categorization often is subjective. Nevertheless, we divided the potential risk factors into categories: 1) general management, including herd size, number of replacement heifers, and other breeding activities; 2) milk quality data, including milk yield and bulk milk SCC and total bacterial count data for the 6 mo before the study; 3) health status, including prevalence of clinical mastitis, number of abortions not related to Brucella. and calf mortality; 4) feeds and feeding of cows, including the quality and distribution of silage and source of water; 5) housing conditions of

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USTERIA MONOCITOGENES IN RAW MILK

TABLE 1. Comparison between cases and controls on probability of herd size, milk yield and housing type being a predisposition for the presence of Listeria monocytogenes in the raw milk. Variable

Herds

Herd size. no. of cows Cases I Controls 2

(no.)

40 80

34 22

17 16

10 5

80 75

.0003

Milk yield. kglyr per cow Cases Controls

40 80

4240 3600

1270 1140

2000 1600

7600 7000

0070

Housing type Cases Controls

X

SO

Tie stall 13 53

Minimum

Maximum

Loose housing 27 27

P

<.0001

IFanns producing milk containing Listeria monocytogenes. 2Selected randomly from herds with no contaminated milk.

cows, including type of housing, bam size, cubicles size, loose house area, and bedding material; 6) cleaning procedures, including cleaning of the bam, cleaning of the cubicles, and bedding replacement and cleaning; 7) hygienic conditions of facilities and cows, including collected information on the cleanliness of cows and bedding and average cleanliness record of cow udders, thighs, and anal regions was calculated for each farm; 8) milking procedures, including number of milkers, udder preparation, postdipping, and management of cows with mastitis; and 9) milking machine factors, including frequency of machine checking, replacement of liners, and cleaning procedures. The data were collected on the farm by questionnaire, verified, and completed by a

tour of the farm. The 128 sample farms (64 cases and 64 controls) were investigated. The data were not included in the analysis when the answers were homogeneous (for example, all of the farmers replied no to the question concerning mortality and nervous symptoms). All farms were visited in January. Data Analysis

The data were verified and analyzed using SAS (21). Pooled variance Student's t tests for continuous variables and chi-square analysis for categorical variables were performed to measure the simple association between the hypothetical risk factors and raw milk contamination by L. monocytogenes. Only varia-

TABLE 2. Comparison of total bacterial counts and SCC in bulk milk between cases and controls.

X

Variance within herds

Variance between herds

Intraherd correlation coefficient I

64 64

4.713 4.651

.0359 .0267

.0370 .0308

.507 .536

64 64

5.497 5.485

.0340 .0394

.0308 .0463

.475 .540

Variable

Herds

Bacterial count 2 Case s3 Controls4 SCC5 Cases Controls

(no.)

IIntraherd correlation coefficient (P); all p were significant at P < .0001. 2Mean of natural logarithm of the last 6 bacterial countslml of milk prior to January 1990 or 1991. 3Farms producing milk containing Listeria monocytogenes. 4Selected randomly from herds with no contaminated milk. sMean of natural logarithm of the last 6 SCClml of milk prior to January 1990 or 1991. Journal of Dairy Science Vol. 76. No. 10. 1993

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SANAA ET AL.

TABLE 3. Variables associated with contamination of milIc in 128 cases and controls farms. Description of variables Silage pH of silage. 15 cm deep in silo <4.0 >4.0 pH of silage in the top and side-edges of silos <4.0 >4.0 Weight on the plastic covering sheet Sufficient Insufficient Distribution of silage Trough Self service Removal of spoiled material Yes No Removal of unconsumed silage Yes No Cow housing Cleanliness sco~ of the cows Clean Dirty Frequency of ~moval of bedding material Adequate Inadequate Quantity of litter per cow per day Sufficient Insufficient Frequency of cleaning exercise area Adequate Inadequate Bedding area per cow Sufficient Insufficient Milking Sufficient lighting Yes No Cleaning of the waiting area Sufficient Insufficient Cleanliness of milking bams and parlors Sufficient Insufficient Use of individual cotton towel Yes No Teats correctly dried Yes No Use of soap to clean teats Yes No Towel disinfected between milIcings Yes No

pa

Cases I

Controls 2 OR3

95%

39 23

44 9

2.88

1.21

6.90

9 53

27 26

6.10

2.62

14.29

29 33

39 14

3.17

1.45

6.90

29 35

37 27

1.65

.82

3.33

NS

53 9

47 6

1.33

.44

4.03

NS

47 15

41 12

1.09

.46

2.60

NS

8 56

29 35

5.81

2.49

38

26

43 21

1.40

.68

2.89

NS

42 22

22 21

1.07

.51

2.24

NS

15 49

41 23

5.81

2.75

12.35

28 13

34 6

2.63

.90

7.69

39 25

56 8

4.48

1.90

19 45

30 34

2.10

1.01

40 24

57 7

4.88

2.01

20 44

32 32

2.20

1.07

4.50

.031

.37

CI4

13.5

.017

<10-"

.004

<10-"

<10-5

.07

.001

10.6

4.31

11.9

34

39

30

2S

1.38

.68

2.79

33 31

31 33

.88

.44

8.06

32 32

43 21

2.05

1.01

4.18

.045 <10-3

NS

.048 (continued)

Journal of Dairy Science Vol. 76. No. 10. 1993

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USTERIA MONOCYfOGENES IN RAW MILK

TABLE 3. (continued) Variables associated with contamination of milk in 128 cases and controls farms. Description of variables Postmilking disinfection test Yes No Frequency of machine function tests Once yearly More than once yearly Frequency of changing liners Once yearly More than once yearly

pa

Casesl

Controls2 OR3

28 36

32 32

1.29

.64

2.58

NS

49 IS

54 10

1.65

.68

4.02

NS

49 15

56 8

2.14

.85

5.43

95%

Cl4

.10

INo significant association (P > .10). (Farms producing milk containing Listeria monocytogenes. 2Selected randomly from herds with no contaminated milk. 30R = Odds ratio. 4CI = Confidence interval of OR at 95%.

bles with P S .10 were considered for further analysis. A multivariate ANOVA model (21) was used to test the relationship between the milk quality results and the contamination of the bulk milk by L. monocytogenes. Stepwise multivariate logistic regression (21) was used to evaluate the effect of each risk factor controlling the effect of other risk factors. Variables were entered into the logistic model if they explained a significant portion of the variation (P S .05) in the likelihood ratio test. For removal of variables from the model, P was > .05. Type of cow housing was included in every model. Two-way interactions were tested between all main effects remaining in the model. RESULTS Preliminary Study

The results of the preliminary study comparing the housing type, milk yield, and herd size between the first 40 selected cases and the 80 controls selected randomly without constraints are presented in Table 1. The risk of milk contamination by L monocytogenes was significantly associated with the herd size, milk yield, and housing type. Association with Quality Results

The data for total bacteria and sec in bulk milk are summarized in Table 2. Compared with regional averages, the means in Table 2

show that the herds included in this study had high milk quality with low bacterial counts and sec. No marginal difference existed between control and case groups for the means of total bacterial counts and sec. Two simultaneous analyses of the natural logarithm of the last six bacterial counts performed on a milliliter of bulk tank milk and the last six sec performed on a milliliter of milk prior to January 1990 or 1991 (depending on the selection year) tested the relationship between milk quality results and contamination of bulk milk by L monocytogenes. For multivariate ANOVA test criteria and F statistics (Wilks' lambda, Pillai's trace, HotellingLawley trace, and Roy's Greatest root) (21) for the hypotheses of no overall difference between cases and controls, P was >.10. RIsk Factors

In the first step of the analysis, 12 variables had a significant bivariate relation with the milk contamination. Associations between each variable of interest and the occurrence of L monocytogenes in bulk tank milk are presented in Table 3. For example, the OR of contamination of milk by L monocytogenes were 2.88 times greater on farms for which the silage sample, taken at a depth of 15 cm in the bulk, had pH >4.0 than on farms with lower silage pH. pH >4.0 at the top and at the edges of the silo increased the OR of the occurrence of L monocytogenes in bulk milk by a factor of 6.10 (P < 10-4). Journal of Dairy Science Vol. 76, No. 10. 1993

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SANAA ET AL.

Variable Type of housing pH of silage in the top and side edges of silos Cleanness of the cows Frequency of cleaning the exercise area Lighting of milking barns and parlors Towel disinfected between rnilkings

-1.1528 1.7294 1.7766 1.3548 1.5197 1.0754

OR3

95%

.32 5.64 5.91 3.88 4.57 2.93

2.140 1.880 1.465 1.454 1.093

.096

=

=

CI4

p

1.042 14.85 18.57 10.16 14.37 7.859

.0580 .0005 .0024 .0059 .0093 .0326

=

'Criteria for assessing the final model: likelihood ratio test 59.5358 with 6 df (J' .00(1). residual chi-square 3.5358 with 6 df (P = .7392). and likelihood ratio test of two-way interactions = 39.3814 with 35 df (P = .28(1). 2Logistic regression parameter estimate. 30R 4CI

= Odds ratio = In(B). = Confidence interval of OR

at 95%.

After the second step in the analysis, only 5 variables were significantly (P < .05) associated with milk contamination. TIle variables in the final model for prediction of the likelihood of a raw milk contamination by L monocytogenes are listed in Table 4. Stepwise logistic regression selected 5 variables from among those for which P was <.10 (fable 3). Type of cow housing was included in all models. The OR indicated that, with poor quality of silage (pH >4.0) fed to the herd, the fann would be 5.64 times more likely (OR =5.64; P = .0005) to have milk contaminated by L. monocytogenes. The mean score for cow cleanliness in the herd (OR = 5.91; P = .(024), inadequate frequency of cleaning the exercise area (OR = 3.88; P = .0059), insufficient lighting of milking barns and parlors (OR = 4.57; P = .0093), and incorrect disinfection of towels between milkings (OR = 2.93; P = .0326) also increased the odds of the milk containing Listeria. The final model is summarized in Table 4. The criteria for assessing the likelihood ratio and score statistic of the final model (P < .00(1) indicate that the model was appropriate for the data. The CATMOD procedure (21), used to compare the final model with the unrestricted model, including two-way interactions among main effects remaining in the model, indicated that the predicted values fitted the data well (P = .2801). DISCUSSION

In observational studies, the epidemiologist observes but does not attempt to influence or to control the independent or dependent variaJournal of Dairy Science Vol. 76. No. 10. 1993

bles under study. The conclusions are not in terms of causal relations between observed risk factors and the disease. The observed risk factors that are significantly associated with raw milk contamination by L monocytogenes (fable 4) are not necessarily causally related. A set of criteria assesses whether the observed risk factors are likely to be causally associated (18). The most important criteria are time sequence, strength of association, biologic gradient, coherence, and consistency and specificity of association. Time sequence refers to the fact that the cause precedes contamination. Biologic gradient refers to a relationship between dose and effect. Coherence refers to the biological plausibility given the current state of knowledge, whereas consistency refers to the presence of similar findings in different studies under different conditions. The risk factors presented in the current study should be interpreted via those criteria. Seven of the variables that were initially associated with the risk of milk contamination were removed from the final model. partially because of the relatively small number of observations (n 128) and because of the strong interdependence among management practices. The occurrence of L monocytogenes in the raw milk from individual dairy fanns was sporadic. Except for one limited descriptive study (I), the overall incidence worldwide of L. monocytogenes in raw milk appears to be around 2.2% (2). In the present study area, the overall incidence rate was around 3%. In agreement with the previous study (2), variation in the prevalence of L monocytogenes and nonpathogenic Listeria spp. was seasonal.

=

USTERIA MONOCITOGENES IN RAW MILK

Feeding practices, such as the use of spoiled silage, have been suggested (3) to affect seasonal variation in the occurrence of Listeria spp. in raw milk. The contamination by L. monocytogenes of feces of healthy animals was related to feeding practices such as feeding spoiled silage (15, 20, 24). Fecal material is a potential source of exogenous contamination of raw milk (12). In an observational epidemiologic study on the occurrence of L. monocytogenes in the feces of dairy cattle, Husu (12) reported that the incidence of Listeria spp. in raw milk was associated with the prevalence of these bacteria in fecal samples. The risk of fecal contamination of raw milk by Listeria spp. seems to increase during the indoor season when the number of fecal excreters is highest because cows are grouped together and fed silage. Other surveys (5, 7) revealed that L. monocytogenes can be present in considerable numbers in poor quality silage. Pockets of silage that spoil and do not develop normal acidic pH (around 4.0) may allow the growth of Listeria spp. which can then reach infective concentrations. The pH of silage seems to be a good indicator of Listeria spp. growth potential in the silo. The pH check, a quick procedure to assess quality of the silage, is widely used in France in programs to control contamination of milk by spores of Clostridium tyrobutyricum. In the present study, the pH of silage was strongly related to the occurrence of L. monocytogenes in raw milk. In the final logistic regression model, the pH of silage taken at the top and at the edges of the silo increases the occurrence of L. monocytogenes in milk by 5.64 (P = .0005). Contamination of silage appears to be highest on the edges of the silo; within some samples, concentration is > 106 viable Listerialg of silage (6). The pH of silage at a depth of 15 cm in the bulk and the weight applied to the plastic sheet that covered the silo were not present in the final model. This absence could be explained by their close relationship with the pH taken at the top and at the edges of the silo. The cleanliness score of the cows was related to the occurrence of L. monocytogenes in milk, the frequency of cleaning the exercise area, and the removal and quantity of bedding material and in the bedding area. In studies about mastitis (11, 19), the rate of mastitis was associated with the microbiological contamina-

2897

tion of the bedding, and dirty cubicles may have increased the exposure of the cow to coliform microorganisms. Apparently, insufficient hygiene in cow housing increases environmental exposure to pathogens, including L. monocytogenes, and constitutes an important environmental factor in the incidence of L. monocytogenes in milk. The OR in the final model (5.91 for the cleanliness of the cows and 3.88 for the frequency of cleaning the exerCIse area) supports this association. The milking procedure potentially introduces contamination. Poor hygiene adds to the microbial contamination of milk. Microorganisms, including Listeria spp.. can contaminate milk from unclean udders. teats, and equipment used for milking. However, proper washing and drying of the udder before milking seems to be important for the elimination of Listeria spp. (13). In the present study, the lighting of milking barns and parlors was related to the occurrence of L. monocytogenes in raw milk. This factor should be interpreted as a risk indicator. Insufficient lighting of milking barns and parlors may reflect general negligence of the farmer in milking hygiene. The use of individual cotton towels and their disinfection between milkings were both related to a lower incidence of L. monocytogenes in raw milk. These two practices seem to be equally important in reducing the contamination of teats by Listeria spp. and to prevent the transfer of bacteria among cows. Cleanliness of milking barns and parlors seems to be an important risk factor associated with milk contamination by L. monocytogenes, but it is not identified in the final model, which could be explained by a bias in collecting the data. We may have overestimated the cleanliness of milking barns and parlors when the lighting was low, but this bias in the model was taken into account. The risk of contamination of milk by L. monocytogenes on dairy farms increases with poor quality silage and insufficient housing and milking hygiene. This microorganism is widely distributed in nature. and a quality program for products made with raw milk should not allow the presence of L. monocytogenes in milk. To reduce the risk of L. monocytogenes contamination, the farmer should pay more attention to preparation of the silage. thus reducing the incidence of L. monocytogenes on the farm. Journal of Dairy Science Vol. 76. No. 10, 1993

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SANAA ET AL. ACKNOWLEDGMENTS

The authors acknowledge the financial support of Ministere de la Recherche et de l'Espace, Centre National Interprofessionnel de l'Economie Laitiere, and Office National de l'Industrie Laitiere. REFERENCES 1 Dominguez-Rodriguez, L., J. F. FernandezGarayzabal, J. A. Vazquez-Boland, E. RodriguezFerri, and G. Suarez-Fernandez. 1985. Isolement de micro-organismes du genre Listeria A partir du lait cru destin6 A la consornmation humaine. Can. 1. Microbiol. 31 :938. 2 Farber, J. M., and P. I. Peterkin. 1991. Listeria monocytogenes, a food-borne pathogen. Microbiol. Rev. 55:476. 3 Farber, J. M., G. W. Sanders, and S. A. Malcolm. 1988. The presence of Listeria spp. in raw milk in Ontario. Can. J. Microbiol. 34:95. 4 Fedio, W. M., M. Schoonderwoerd, R Shute, and H. Jackson. 1990. A case of bovine mastitis caused by Listeria monocytogenes. Can. Vet. J. 31:773. 5 Fenlon, D. R 1986. Growth of naturally occurring Listeria spp. in silage: a comparative study of laboratory and farm ensiled grass. Grass Forage Sci. 41:375. 6 Fenlon, D. R. 1986. Rapid quantitative assessment of the distribution of Listeria in silage implicated in a suspected outbreak of listeriosis in calves. Vet. Rec. 118:240. 7 Fenlon, D. R., and 1. Wilson. 1989. The incidence of Listeria monocytogenes in raw milk from farm bulk tanks in North-East Scotland. J. Appl. Bacteriol. 66: 191. 8 Fernandez-Garayzahal, J. F., L. Dominguez, A. Vazquez, E. Gomez-Lucia, E. R. Rodriguez-Ferri, and G. Suarez. 1987. Occurrence of Listeria monocylogenes in raw milk. Vet. Rec. 120:258. 9 Gitter, M., R Bradley, and P. H. Blampied. 1980. Listeria monocytogenes infection in bovine mastitis. Vet. Rec. 107:390. 10 Griffiths, M. W. 1989. Listeria monocytogenes: its inponance in the dairy industry. J. Sci. Food Agric. ~7:133.

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Journal of Dairy Science Vol. 76, No. 10, 1993

mastitis in low somatic cell count herds. J. Dairy Sci. 72:1547. 12 Husu, J. R 1990. Epidemiological studies on the occurrence of Listeria monocytogenes in the feces of dairy cattle. J. Vet. Med. Ser. B 37:276. 13 Husu, J. R, 1. T. Seppiinen, S. K. Sivelii, and A. L. Rauramaa. 1990. Contamination of raw milk by Listeria monocytogenes on dairy farms. 1. Vet. Med. Ser. B. 37:268. 14 Jensen, J., and H. E. Larsen. 1973. Listeria monocytogenes som arsag til 3 tilfaelde af mastitis hos kvaeg. Nord. Veterinaermed. 25:322. 15 Kampelmacher, E. H., and L. M. Van Noorle Jansen. 1972. Further studies in the isolation of Listeria monocytogenes in clinically healthy individuals. Zentralbl. Bakteriol. Hyg. A221:70. 16 Lovett, 1., D. W. Francis, and J. M. Hunt. 1987. Listeria monocylogenes in raw milk: detection, incidence and pathogenicity. 1. Food Prot. 50:188. 17 Mackey, B. M., and N. Bratchell. 1989. The heat Iesistance of Listeria monocytogenes. Lett. Appl. Microbiol. 9:89. 18 Martin, S. W., A. H. Meek, and P. Willeberg. 1987. Veterinary Epidemiology. Principles and Methods. Iowa State Univ. Press, Ames. 190z, H. H., R. J. Fransworth, and V. L. Larson. 1985. Environmental mastitis. Vet. Bun. 55:829. 20 Ralovich, B. 1984. Listeriosis Research-Present Situation and Perspective. Akademiai Kiado, Budapest, Hun~ary.

21 SAS User's Guide: Statistics, Version 6.03 Edition. 1988. SAS Inst., Inc., Cary, NC. 22 Schwartz, D., R Flarnnant, and J. Lellouch. 1970. L'essai tMrapeutique chez I'homme. Aarnrnarion M6d. Sci., Paris, France. 23 Sharp, M. W. 1989. Bovine mastitis and Listeria monocytogenes. Vet. Rec. 125:512. 24 Skovaard, N., and C. A. Morgen. 1988. Detection of Listeria spp. in faeces from animals, in feeds and in raw foods of animal origin. Int. J. Food Microbial. 6: 229. 25 Van Netten, P., I. Perales, and D.A.A. Mossel. 1988. A selective and diagnostic medium for isolation and counting of Listeria spp. in heavily contaminated foods. Lett. Appl. Microbiol. 7:17. 26 Van Netten, P., A. Van de Ven, I. Perales, and D.A.A. Mossel. 1988. A selective and diagnostic medium for use in the enumeration of Listeria spp. in foods. Int. J. Food Microbiol. 6:187.