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Listeria monocytogenes in bovine mastitis. Possible implication for human health
IWl OfFood~ International
ELSEVIER
Journal
Joumal
of
Food Microbiology 32 (1996) 209-216
Short communication
Listeria monocytogenes in bovine mastitis. Possible implication for human health Niels Einar Jensen”,*,
Frank Mdler Henrik Caspar
Aarestrup”, Wegenef
Johannes
Jensenb,
“Danish Veterinary Laboratory, Biilowsvej 27, DK-1790 Copenhagen V, Denmark bCattle Health Laboratory, Danish Dairy Board, DK-6650 Brerup, Denmark
Received
5 September
1995; revised
16 February
1996; accepted
I March
1996
Abstract During the 23-year period 1972 through 1994 quarter milk samples from 1 132958 cows originating from 36 199 herds were examined for the presence of Listeriu monocytogenes. Through the period the reference population amounted to 12 742 600 cow years and 401682 herd years. The percentage of cows infected with L. monocytogenes varied from 0.01 to 0.1% (mean 0.04%) and of herds with an infected cow from 0.2 to 4.2% (mean 1.2%) through the period, showing a low but constant level of infection. A comparison of 33 isolates from bovine mastitis and 27 human clinical isolates was made by sero- and ribotyping. Serotyping showed that all bovine and 17 (63%) of the human isolates belonged to serogroup 1, whereas 10 (37%) of the human isolates belonged to serogroup 4. Ribotyping using EcoRl as restriction enzyme divided the 60 isolates into 16 different types, 7 of which were found among both the bovine and human types. The combination of the typing methods showed that 26 (79%) bovine and 13 (48%) human isolates shared common types. This study showed that a low but constant percentage of Danish dairy herds have cows infected with L. monocytogenes and that some of the bovine types could be found among types causing human infections.
* Corresponding 0168-1605/96/$15.00
author.
Tel:
+ 45-35300100;
0 1996 Elsevier
PII SO168-1605(96)01105-l
Fax:
+ 45-35300301,
Science B.V. All rights
reserved
210
N.E. Jensen et ul. /ht.
Keywords: Mastitis;
J. Food Microbiology
Listeriu monocytogenes;
Typing;
32 (1996) 209-216
Human
health
1. Introduction Listeria monocytogenes causes sporadic cases and epidemic outbreaks of listeriosis in humans and a large number of animal species (Anonymous, 1991; Farber and Peterkin, 1991; Rocourt, 1994). L. monocytogenes is recognized as a zoonotic agent and listeriosis in humans is normally considered a foodborne disease but other modes of transmission may take place. High age, pregnancy and especially immunosuppression or underlying severe disease are significant risk factors (Anonymous, 1991). In a Danish study, unpasteurized milk was identified as a significant risk factor for sporadic listeriosis and an outbreak of epidemic listeriosis was claimed to be linked to consumption of a blue-mould cheese (7). L. monocytogenes is frequently isolated from raw milk (Anonymous, 1991; Lovett et al., 1987). A Danish study (Anonymous, 1990) revealed that 8.5% of 445 bulk milk samples were contaminated with L. monocytogenes in 1990. The first cases of bovine mastitis caused by L. monocytogenes in Denmark were reported in 1973 by Jensen and Larsen (1973). The possibility exists that L. monocytogenes infected quarters can serve as a source of human infections since the infection contributes to the overall presence of L. monocytogenes in raw milk. A possible connection between isolates causing bovine mastitis and isolates from human clinical infections can be elucidated by comparing the isolates of L. monocytogenes prevalent in the two reservoirs by epidemiological molecular typing methods such as ribotyping. In this study, the incidence of bovine L. monocytogenes mastitis cases in the period 1972 through 1994 is reported. Furthermore, a subset of the bovine strains isolated in 1993 was compared with L. monocytogenes strains isolated from human listeriosis cases from the same time period by means of sero- and ribotyping.
2. Materials and methods 2.1. Milk samples During the 23-year period 1972 through 1994 quarter milk samples from a total of 1 132 958 cows originating in 36 199 herds were examined bacteriologically (including L. monocytogenes) and cytologically in connection with the Danish mastitis control/eradication programme. Samples were restricted to one control area (Ladelund) during the first IO-year (1972- 1981) period, whereas during the remaining 13 years (1982- 1994) samples included the whole country. Through the period in question the reference population amounted to 12 742 600 cowyears and 401682 herdyears. The actual number of cows/herds examined is given by year in Table 1. The milk samples were collected aseptically as fore milk samples in
N.E. Jensen et al. /ht.
accordance California
J. Food Microbiology 32 (1996) 209-216
with standard recommendations Mastitis Test (CMT) (Klastrup
2.2. Bacteriological
examination
and examined cytologically and Madsen, 1974).
211
using the
of milk samples
Primary cultivation was performed by inoculation of 0.01 ml of milk onto the quarter of the surface of a blood agar plate (Columbia agar, CM 331, supplemented with 5% sterile bovine blood), supplemented with 0.1% aesculin. The plates were incubated aerobically and inspected for growth after 24 and 48 h. A tentative diagnosis of L. monocytogenes was based on characteristic colony morphology and colour, positive catalase test and the presence of a narrow j?-haemolytic zone. After sub-cultivation the final diagnosis was based on the presence of characteristic rods, motility in semi-solid agar, hydrolysis of aesculin and positive CAMP test with Staphylococcus aureus, negative CAMP test with Rhodococcus equi and fermentation of rhamnose, but not of xylose or mannitol (Swaminathan et al., 1995). 2.3. Bacteriological
examination
of human samples
L. monocytogenes was isolated from blood, liver, placenta or trachea of humans with clinical infections. The strains were supplied by Dr. W. Frederiksen, at Statens Seruminstitut. L. monocytogenes was identified on basis of characteristic colony morphology and colour, positive catalase test and the presence of a narrow B-haemolytic zone. After sub-cultivation the final diagnosis was based on the presence of characteristic rods in microscopy, motility, hydrolysis of aesculin, positive tellurite reaction and fermentation of rhamnose and glucose, but not of xylose or mannitol. A total of 27 sporadic human clinical infections caused by L. monocytogenes was diagnosed in Denmark in 1993. 2.4. Serological
typing und ribotyping
All 33 bovine (32 cows) and 27 human isolates of L. monocytogenes isolated in 1993, were investigated using sero- and ribotyping. Serological typing was performed by means of commercial antisera (Difco Lab. Detroit, MI, USA). The antisera employed can distinguish between serogroups 1 and 4. The isolates were ribotyped using EcoRI as restriction endonuclease, as described by Fussing and Wegener (1993).
3. Results and discussion The number of L. monocytogenes positive samples and number of herds and cows examined are given in Table 1. From 3 to 87 positive samples were found per year. In almost all herds where infections with L. monocytogenes occurred, only one quarter of one cow was infected. More than 95% of L. monocytogenes infected quarters yielded pure cultures. The actually excreted number of colony forming
N.E. Jensen et al. /hi.
212
J. Food Microbiology 32 (1996) 209-216
units (CFU) was not determined. A conservative estimate based on colony counting on primary plates from some cases indicate, however, that L. monocytogenes infected quarters excrete at least 10 000 CFU/ml of milk. The CMT reactions were as high as 4 or 5 in all cases indicating a significant inflammatory response in the range of 5 x lo6 somatic cells per ml. The percentage of L. monocytogenes positive herds and cows varied somewhat through the period of investigation, i.e. from 0.2 to 4.2 and from 0.01 to 0.1, respectively (Fig. 1). The variation may, to some degree, be accidental and caused by varying awareness of the L. monocytogenes problem. On an average through the 23-year period, L. monocytogenes was demonstrated in quarter milk samples from 1.2% of the herds and 0.04% of the cows examined. Of the isolates that were investigated by typing all 33 bovine isolates belonged to serogroup 1. This was the case for 17 (63%) of the human strains, too, whereas 10 (37%) of these belonged to serogroup 4 (Table 2). A total of 16 ribotype patterns Table 1 The number of herds and cows examined through a 23-year period Year
No. examined” Herds
1972 1973 1914 1975 1976 1977 1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994
748 879 871 961 998 811 973 817 881 668 2560 2614 2623 2551 2609 2134 2033 1459 825 1716 1816 2319 2213
Total
36 199
cows
Total
and the number
no. in Denmark”
Herds
of isolations
of L. monocytogenes by year
L. monocytogenes isolations
(cows)
cows
11 888 12 178 14 395 17 713 23 573 18 902 25811 20 762 25 584 18 932 72 610 78 785 79 894 76 446 80 053 69 333 73 157 54 609 32 706 65 989 70 885 99 521 89 232
9796 10 266 10 543 10 794 10 239 9833 9443 7929 7803 7583 32 214 31075 30 928 28 944 26 917 24 150 22 136 22 463 20 186 19 427 16 957 16 886 15 110
137 000 144 000 174 200 242 700 242 000 230 000 250 500 201 500 226 700 215000 999 000 1 003 000 951 000 896 000 864 000 81 I 000 774 000 759 000 753 000 742 000 720 000 707 000 700 000
3 4 5 7 6 4 5 8 10 7 4 3 7 38 87 46 42 30 35 22 20 32 23
1 132 958
401 682
12 742 600
448
“The first 10 years (1972-1981) of the period was restricted to one control remaining 13 years (1982- 1994) included the whole country.
area (Ladelund)
whereas
the
N.E. Jensen et al. / ht. J. Food Microbiology
70
75
85
80
213
32 (1996) 209-216
90
95
Year
0 70
75
80
85
90
95
Year Fig. 1. Percentage of the herds and percentage Denmark, by year.
of the the cows infected
with Listeriu monocytogenes in
were seen among the 60 strains typed (Table 2). Seven ribotypes including 79% (26) of the bovine strains and 48% (13) of the human strains were common for the two reservoirs. During the last decade, listeriosis has been recognized as an important foodborne illness. Considerable research has attempted to characterize L. monocytogenes, define the magnitude of the problem, identify the risk factors and suggest appropriate control strategies (Anonymous, 1991). Typing methods have also been reviewed (Farber and Peterkin, 1991; Wegener et al., 1993). In the USA, an annual incidence rate of listeriosis in the human population has been estimated at 0.7 cases per 100000 population (Schuchat et al., 1991). This figure is very close to the incidence rate in Denmark where 6-7 cases has been reported per 1 million inhabitants per year during recent years (Jensen et al., 1994).
214
N.E. Jensen et al. 1 ht.
J. Food Microbiology
32 (1996) 209-216
In Denmark a strain of serogroup 4, (referred to as the EPI strain) caused an epidemic outbreak of listeriosis in the years 1989 through 1990 (Jensen et al., 1994). Listeriosis is presently the alimentary infection associated with the highest lethality (20-30%) (Rocourt, 1994). In Denmark the mortality amounts to 26% for sporadic cases of listeriosis versus 47% for patients infected by the EPI strain (Jensen et al., 1994). Several reviews have dealt with the occurrence of L. monocytogenes as a commensal in healthy human and animal individuals. It is also well documented that L. monocytogenes exist and multiply as a saprophytic organism in the soil and on plants as well as in sewage and river water (Anonymous, 1991; Farber and Peterkin, 1991; Wegener et al., 1993). In Denmark L. monocytogenes has often been isolated from cattle food items such as beets (31%), straw (29%), grain (23%), and hay (17%) and L. monocytogenes was present in 18% of 44 examined faeces samples (Anonymous, 1990). Thus, it is obvious that a large reservoir of L. monocytogenes exists in and around milking cows. Consequently, it is not surprising that bulk milk may contain L. monocytogenes. From the USA up to 12”/ of milk deliveries have been reported L. monocytogenes positive (Lovett et al., 1987). In Northern Ireland 5.3% of bulk milk samples were reported positive (Harvey and Gilmour, 1992), while a study in Denmark revealed that 8.5% of 445 bulk milk samples contained the organism (Anonymous, 1990). Our study demonstrated that bulk milk, in addition to contamination from the environment, may be contaminated directly from infected udder quarters. It should be noted that our figures are prevalence rates. In 1994, 23 L. monocytogenes infections were diagnosed among 2213 herds Table 2 Serogroups and ribotypes among isolated in 1993 in Denmark
all 33 bovine
Serogroup
Ribotype
1
1 2 3 4 5 6 7 8 9 10 11 12 13 14
4
13 15 16
and all 27 human
Bovine
isolates
of Listeria
Human
1 3
4 4 2
monocytogmes
N.E. Jrmen ei al. ! Int. J. Food Micmhiolop
215
32 (1996) 209-216
examined. Among the 15 110 herds in the whole country a figure of 157 L. monocytogenes-diagnoses was to be expected, meaning that 157 milk deliveries (1.04%) theoretically were contaminated by L. monocytogenes each day of 1994. It is generally believed that bulk milk contain low numbers of L. monoqytogenes (Lovett et al., 1987). However, when contaminated directly the number of L. monocytogenes may be much higher since an infected quarter can contain at least 10 000 CFU/ml of milk. In addition, L. monocytogenes can multiply even in cooled milk. Based on the current information, the infection dose of L. monocytogenes for humans cannot be stated with precision. Small doses may infect immunocompromised hosts, but much larger doses are required for normal individuals (Anonymous, 1991). Jensen et al. (1994) were able to demonstrate a significant association between sporadic cases of listeriosis (non-EPI strain) and the consumption of raw milk. The odds ratio was calculated to be 8.6, but other factors such as immunosuppression and underlying diseases were much more important than the consumption of raw milk. These authors also reported an association between L. monocytogenes infections caused by the EPI strain and the consumption of a blue-mould and a hard cheese. To further investigate a possible association between the bovine reservoir and human infections, L. monocytogenes strains isolated in 1993 from all 33 bovine mastitis cases, and all 27 human isolates from 1993 were typed. All bovine isolates and 63%. (17) of the human isolates belonged to serogroup 1. Thus, isolates of serogroup 4, to which the EPI strain belonged, were detected among human isolates only. A total of 16 different ribotype patterns were recorded, i.e. 12 among the bovine isolates and 11 among the human strains. Five ribotypes were seen only among bovine strains and 4 only among human isolates. However, 7 ribotypes including 26 (79%) of the bovine and 13 (48%) of the human strains were shared by the two groups of isolates. These 39 strains were all of serogroup 1. Thus, based on the typing methods employed in this study, an epidemiological relationship between the bovine and the human reservoir cannot be excluded. Considering this information together with the low but constant level of contamination of bulk milk by L. monocytogenes infected cows, it seems likely that L. monocytogenes causing mastitis in cows may be transferred to humans by raw milk or milk that have not been correctly pasteurized or that have been contaminated postpasteurization with raw milk. Thus, it is important to maintain strict hygiene and practice pasteurization of raw milk in order to minimize the risks of human infection with L. monocytogenes.
References Anonymous (1990) Pilotprojekt vedrsrende integreret kontrol 201, Danish Dairy Organization. Anonymous (1991) Listeriu monocytogenes. Recommendations microbiological criteria for foods. Int. J. Food Microbial.
af m2elk og mejeriprodukter. by the national 14, 185-246.
advisory
Report committee
No. on
216
N.E. Jensen et al. / Int. J. Food Microbiology 32 (1996) 209-216
Farber, J.M. and Peterkin, PI. (1991) Listeria monocytogenes, a food-borne pathogen. Microbial. Rev. 55, 4766511. Fussing, V. and Wegener, H.C. (1993) Characterization of bovine Haemophilus somnus by biotyping, plasmid profiling, REA-patterns and ribotyping. Zentralbl. Bakteriol. 279, 60-74. Harvey, J. and Gilmour, A. (1992) Occurrence of Listeria species in raw milk and dairy products produced in Northern Ireland. J. Appl. Bacterial. 72, 119-125. Jensen, J. and Larsen, H.E. (1973) Listeria monocytogenes som arsag til 3 tilfrelde af mastitis hos kvreg. Nord. Vet. Med. 25, 3222329. Jensen, A., Frederiksen, W. and Gerner-Smidt, P. (1994) Risk factors for listeriosis in Denmark, 198991990. Stand. J. Infect. Dis. 26, 171-178. Klastrup, 0. and Madsen, P.S. (1974) Nordiske rekommendationer vedrerende mastitisunderssgelser af kirtelprover. Nord. Vet. Med. 26, 197-204. Lovett, J., Francis, D.W. and Hunt, J.M. (1987) Listeria monocytogenes in raw milk: Detection, incidence, and pathogenicity. J. Food Protect. 50, 188-192. Rocourt, J. (1994) Listeria monocytogenes: the state of the science. Dairy Food Environ. Sanitation 14, 70-82. Schuchat, A., Swaminathan, B. and Broome, C.V. (1991) Epidemiology of human listeriosis. Clin. Microbial. Rev. 4, 1699183. Swaminathan, B., Rocourt, J. and Bille, J. (1995) Listeriu. In: Murray, P.R., Baron, E.J., Pfaller, M.A., Tenover, F.C. and Yolken, R.H. (Eds.), Manual of Clinical Microbiology 6, American Society for Microbiology, Washington, DC, pp. 341-348. Wegener, H.C., Norrung, B., Jensen, A., Ben Embarek, K. and Tolstoy, A. (1993) Listeria monocytogenes - an overview, Danish Veterinary Journal, 76, 501-512. (In Danish: Listeria monocytogenes en oversigt, with English summary).
ELSEVIER
Journal
Joumal
of
Food Microbiology 32 (1996) 209-216
Short communication
Listeria monocytogenes in bovine mastitis. Possible implication for human health Niels Einar Jensen”,*,
Frank Mdler Henrik Caspar
Aarestrup”, Wegenef
Johannes
Jensenb,
“Danish Veterinary Laboratory, Biilowsvej 27, DK-1790 Copenhagen V, Denmark bCattle Health Laboratory, Danish Dairy Board, DK-6650 Brerup, Denmark
Received
5 September
1995; revised
16 February
1996; accepted
I March
1996
Abstract During the 23-year period 1972 through 1994 quarter milk samples from 1 132958 cows originating from 36 199 herds were examined for the presence of Listeriu monocytogenes. Through the period the reference population amounted to 12 742 600 cow years and 401682 herd years. The percentage of cows infected with L. monocytogenes varied from 0.01 to 0.1% (mean 0.04%) and of herds with an infected cow from 0.2 to 4.2% (mean 1.2%) through the period, showing a low but constant level of infection. A comparison of 33 isolates from bovine mastitis and 27 human clinical isolates was made by sero- and ribotyping. Serotyping showed that all bovine and 17 (63%) of the human isolates belonged to serogroup 1, whereas 10 (37%) of the human isolates belonged to serogroup 4. Ribotyping using EcoRl as restriction enzyme divided the 60 isolates into 16 different types, 7 of which were found among both the bovine and human types. The combination of the typing methods showed that 26 (79%) bovine and 13 (48%) human isolates shared common types. This study showed that a low but constant percentage of Danish dairy herds have cows infected with L. monocytogenes and that some of the bovine types could be found among types causing human infections.
* Corresponding 0168-1605/96/$15.00
author.
Tel:
+ 45-35300100;
0 1996 Elsevier
PII SO168-1605(96)01105-l
Fax:
+ 45-35300301,
Science B.V. All rights
reserved
210
N.E. Jensen et ul. /ht.
Keywords: Mastitis;
J. Food Microbiology
Listeriu monocytogenes;
Typing;
32 (1996) 209-216
Human
health
1. Introduction Listeria monocytogenes causes sporadic cases and epidemic outbreaks of listeriosis in humans and a large number of animal species (Anonymous, 1991; Farber and Peterkin, 1991; Rocourt, 1994). L. monocytogenes is recognized as a zoonotic agent and listeriosis in humans is normally considered a foodborne disease but other modes of transmission may take place. High age, pregnancy and especially immunosuppression or underlying severe disease are significant risk factors (Anonymous, 1991). In a Danish study, unpasteurized milk was identified as a significant risk factor for sporadic listeriosis and an outbreak of epidemic listeriosis was claimed to be linked to consumption of a blue-mould cheese (7). L. monocytogenes is frequently isolated from raw milk (Anonymous, 1991; Lovett et al., 1987). A Danish study (Anonymous, 1990) revealed that 8.5% of 445 bulk milk samples were contaminated with L. monocytogenes in 1990. The first cases of bovine mastitis caused by L. monocytogenes in Denmark were reported in 1973 by Jensen and Larsen (1973). The possibility exists that L. monocytogenes infected quarters can serve as a source of human infections since the infection contributes to the overall presence of L. monocytogenes in raw milk. A possible connection between isolates causing bovine mastitis and isolates from human clinical infections can be elucidated by comparing the isolates of L. monocytogenes prevalent in the two reservoirs by epidemiological molecular typing methods such as ribotyping. In this study, the incidence of bovine L. monocytogenes mastitis cases in the period 1972 through 1994 is reported. Furthermore, a subset of the bovine strains isolated in 1993 was compared with L. monocytogenes strains isolated from human listeriosis cases from the same time period by means of sero- and ribotyping.
2. Materials and methods 2.1. Milk samples During the 23-year period 1972 through 1994 quarter milk samples from a total of 1 132 958 cows originating in 36 199 herds were examined bacteriologically (including L. monocytogenes) and cytologically in connection with the Danish mastitis control/eradication programme. Samples were restricted to one control area (Ladelund) during the first IO-year (1972- 1981) period, whereas during the remaining 13 years (1982- 1994) samples included the whole country. Through the period in question the reference population amounted to 12 742 600 cowyears and 401682 herdyears. The actual number of cows/herds examined is given by year in Table 1. The milk samples were collected aseptically as fore milk samples in
N.E. Jensen et al. /ht.
accordance California
J. Food Microbiology 32 (1996) 209-216
with standard recommendations Mastitis Test (CMT) (Klastrup
2.2. Bacteriological
examination
and examined cytologically and Madsen, 1974).
211
using the
of milk samples
Primary cultivation was performed by inoculation of 0.01 ml of milk onto the quarter of the surface of a blood agar plate (Columbia agar, CM 331, supplemented with 5% sterile bovine blood), supplemented with 0.1% aesculin. The plates were incubated aerobically and inspected for growth after 24 and 48 h. A tentative diagnosis of L. monocytogenes was based on characteristic colony morphology and colour, positive catalase test and the presence of a narrow j?-haemolytic zone. After sub-cultivation the final diagnosis was based on the presence of characteristic rods, motility in semi-solid agar, hydrolysis of aesculin and positive CAMP test with Staphylococcus aureus, negative CAMP test with Rhodococcus equi and fermentation of rhamnose, but not of xylose or mannitol (Swaminathan et al., 1995). 2.3. Bacteriological
examination
of human samples
L. monocytogenes was isolated from blood, liver, placenta or trachea of humans with clinical infections. The strains were supplied by Dr. W. Frederiksen, at Statens Seruminstitut. L. monocytogenes was identified on basis of characteristic colony morphology and colour, positive catalase test and the presence of a narrow B-haemolytic zone. After sub-cultivation the final diagnosis was based on the presence of characteristic rods in microscopy, motility, hydrolysis of aesculin, positive tellurite reaction and fermentation of rhamnose and glucose, but not of xylose or mannitol. A total of 27 sporadic human clinical infections caused by L. monocytogenes was diagnosed in Denmark in 1993. 2.4. Serological
typing und ribotyping
All 33 bovine (32 cows) and 27 human isolates of L. monocytogenes isolated in 1993, were investigated using sero- and ribotyping. Serological typing was performed by means of commercial antisera (Difco Lab. Detroit, MI, USA). The antisera employed can distinguish between serogroups 1 and 4. The isolates were ribotyped using EcoRI as restriction endonuclease, as described by Fussing and Wegener (1993).
3. Results and discussion The number of L. monocytogenes positive samples and number of herds and cows examined are given in Table 1. From 3 to 87 positive samples were found per year. In almost all herds where infections with L. monocytogenes occurred, only one quarter of one cow was infected. More than 95% of L. monocytogenes infected quarters yielded pure cultures. The actually excreted number of colony forming
N.E. Jensen et al. /hi.
212
J. Food Microbiology 32 (1996) 209-216
units (CFU) was not determined. A conservative estimate based on colony counting on primary plates from some cases indicate, however, that L. monocytogenes infected quarters excrete at least 10 000 CFU/ml of milk. The CMT reactions were as high as 4 or 5 in all cases indicating a significant inflammatory response in the range of 5 x lo6 somatic cells per ml. The percentage of L. monocytogenes positive herds and cows varied somewhat through the period of investigation, i.e. from 0.2 to 4.2 and from 0.01 to 0.1, respectively (Fig. 1). The variation may, to some degree, be accidental and caused by varying awareness of the L. monocytogenes problem. On an average through the 23-year period, L. monocytogenes was demonstrated in quarter milk samples from 1.2% of the herds and 0.04% of the cows examined. Of the isolates that were investigated by typing all 33 bovine isolates belonged to serogroup 1. This was the case for 17 (63%) of the human strains, too, whereas 10 (37%) of these belonged to serogroup 4 (Table 2). A total of 16 ribotype patterns Table 1 The number of herds and cows examined through a 23-year period Year
No. examined” Herds
1972 1973 1914 1975 1976 1977 1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994
748 879 871 961 998 811 973 817 881 668 2560 2614 2623 2551 2609 2134 2033 1459 825 1716 1816 2319 2213
Total
36 199
cows
Total
and the number
no. in Denmark”
Herds
of isolations
of L. monocytogenes by year
L. monocytogenes isolations
(cows)
cows
11 888 12 178 14 395 17 713 23 573 18 902 25811 20 762 25 584 18 932 72 610 78 785 79 894 76 446 80 053 69 333 73 157 54 609 32 706 65 989 70 885 99 521 89 232
9796 10 266 10 543 10 794 10 239 9833 9443 7929 7803 7583 32 214 31075 30 928 28 944 26 917 24 150 22 136 22 463 20 186 19 427 16 957 16 886 15 110
137 000 144 000 174 200 242 700 242 000 230 000 250 500 201 500 226 700 215000 999 000 1 003 000 951 000 896 000 864 000 81 I 000 774 000 759 000 753 000 742 000 720 000 707 000 700 000
3 4 5 7 6 4 5 8 10 7 4 3 7 38 87 46 42 30 35 22 20 32 23
1 132 958
401 682
12 742 600
448
“The first 10 years (1972-1981) of the period was restricted to one control remaining 13 years (1982- 1994) included the whole country.
area (Ladelund)
whereas
the
N.E. Jensen et al. / ht. J. Food Microbiology
70
75
85
80
213
32 (1996) 209-216
90
95
Year
0 70
75
80
85
90
95
Year Fig. 1. Percentage of the herds and percentage Denmark, by year.
of the the cows infected
with Listeriu monocytogenes in
were seen among the 60 strains typed (Table 2). Seven ribotypes including 79% (26) of the bovine strains and 48% (13) of the human strains were common for the two reservoirs. During the last decade, listeriosis has been recognized as an important foodborne illness. Considerable research has attempted to characterize L. monocytogenes, define the magnitude of the problem, identify the risk factors and suggest appropriate control strategies (Anonymous, 1991). Typing methods have also been reviewed (Farber and Peterkin, 1991; Wegener et al., 1993). In the USA, an annual incidence rate of listeriosis in the human population has been estimated at 0.7 cases per 100000 population (Schuchat et al., 1991). This figure is very close to the incidence rate in Denmark where 6-7 cases has been reported per 1 million inhabitants per year during recent years (Jensen et al., 1994).
214
N.E. Jensen et al. 1 ht.
J. Food Microbiology
32 (1996) 209-216
In Denmark a strain of serogroup 4, (referred to as the EPI strain) caused an epidemic outbreak of listeriosis in the years 1989 through 1990 (Jensen et al., 1994). Listeriosis is presently the alimentary infection associated with the highest lethality (20-30%) (Rocourt, 1994). In Denmark the mortality amounts to 26% for sporadic cases of listeriosis versus 47% for patients infected by the EPI strain (Jensen et al., 1994). Several reviews have dealt with the occurrence of L. monocytogenes as a commensal in healthy human and animal individuals. It is also well documented that L. monocytogenes exist and multiply as a saprophytic organism in the soil and on plants as well as in sewage and river water (Anonymous, 1991; Farber and Peterkin, 1991; Wegener et al., 1993). In Denmark L. monocytogenes has often been isolated from cattle food items such as beets (31%), straw (29%), grain (23%), and hay (17%) and L. monocytogenes was present in 18% of 44 examined faeces samples (Anonymous, 1990). Thus, it is obvious that a large reservoir of L. monocytogenes exists in and around milking cows. Consequently, it is not surprising that bulk milk may contain L. monocytogenes. From the USA up to 12”/ of milk deliveries have been reported L. monocytogenes positive (Lovett et al., 1987). In Northern Ireland 5.3% of bulk milk samples were reported positive (Harvey and Gilmour, 1992), while a study in Denmark revealed that 8.5% of 445 bulk milk samples contained the organism (Anonymous, 1990). Our study demonstrated that bulk milk, in addition to contamination from the environment, may be contaminated directly from infected udder quarters. It should be noted that our figures are prevalence rates. In 1994, 23 L. monocytogenes infections were diagnosed among 2213 herds Table 2 Serogroups and ribotypes among isolated in 1993 in Denmark
all 33 bovine
Serogroup
Ribotype
1
1 2 3 4 5 6 7 8 9 10 11 12 13 14
4
13 15 16
and all 27 human
Bovine
isolates
of Listeria
Human
1 3
4 4 2
monocytogmes
N.E. Jrmen ei al. ! Int. J. Food Micmhiolop
215
32 (1996) 209-216
examined. Among the 15 110 herds in the whole country a figure of 157 L. monocytogenes-diagnoses was to be expected, meaning that 157 milk deliveries (1.04%) theoretically were contaminated by L. monocytogenes each day of 1994. It is generally believed that bulk milk contain low numbers of L. monoqytogenes (Lovett et al., 1987). However, when contaminated directly the number of L. monocytogenes may be much higher since an infected quarter can contain at least 10 000 CFU/ml of milk. In addition, L. monocytogenes can multiply even in cooled milk. Based on the current information, the infection dose of L. monocytogenes for humans cannot be stated with precision. Small doses may infect immunocompromised hosts, but much larger doses are required for normal individuals (Anonymous, 1991). Jensen et al. (1994) were able to demonstrate a significant association between sporadic cases of listeriosis (non-EPI strain) and the consumption of raw milk. The odds ratio was calculated to be 8.6, but other factors such as immunosuppression and underlying diseases were much more important than the consumption of raw milk. These authors also reported an association between L. monocytogenes infections caused by the EPI strain and the consumption of a blue-mould and a hard cheese. To further investigate a possible association between the bovine reservoir and human infections, L. monocytogenes strains isolated in 1993 from all 33 bovine mastitis cases, and all 27 human isolates from 1993 were typed. All bovine isolates and 63%. (17) of the human isolates belonged to serogroup 1. Thus, isolates of serogroup 4, to which the EPI strain belonged, were detected among human isolates only. A total of 16 different ribotype patterns were recorded, i.e. 12 among the bovine isolates and 11 among the human strains. Five ribotypes were seen only among bovine strains and 4 only among human isolates. However, 7 ribotypes including 26 (79%) of the bovine and 13 (48%) of the human strains were shared by the two groups of isolates. These 39 strains were all of serogroup 1. Thus, based on the typing methods employed in this study, an epidemiological relationship between the bovine and the human reservoir cannot be excluded. Considering this information together with the low but constant level of contamination of bulk milk by L. monocytogenes infected cows, it seems likely that L. monocytogenes causing mastitis in cows may be transferred to humans by raw milk or milk that have not been correctly pasteurized or that have been contaminated postpasteurization with raw milk. Thus, it is important to maintain strict hygiene and practice pasteurization of raw milk in order to minimize the risks of human infection with L. monocytogenes.
References Anonymous (1990) Pilotprojekt vedrsrende integreret kontrol 201, Danish Dairy Organization. Anonymous (1991) Listeriu monocytogenes. Recommendations microbiological criteria for foods. Int. J. Food Microbial.
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