INFEcrION AND IMMUNrrY, Feb. 1978, p. 493-498
Vol. 19, No. 2
0019-9567/78/0019-0493$02.00/O
Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Staphylococcal Enterotoxin and Thermonuclease Production During Induced Bovine Mastitis and the Clinical Reaction of Enterotoxin in Udders AIMO NISKANEN,l LEO KOIRANEN,2 AND KAUKO ROINE3 Technical Research Centre of Finland,' Food Research Laboratory, Biologinkuja 1, SF-02150 Espoo 15, State Veterinary Medical Institute,2 Hameentie 57, SF-00550 Helsinki 55, and Veterinary College of Finland,3 Ambulatory Clinic, SF-04840 Hautjarvi, Finland
Received for publication 29 June 1977
Enterotoxin A- and C-producing strains of Staphylococcus aureus and partially and extensively purified enterotoxin A were inoculated into the udder quarters of cows. In the course of experimentally induced mastitis caused by the inoculated S. aureus strain, enterotoxin C but not A was detected in the infected udder. Enterotoxin C was observed in mastitic milk samples at very low S. aureus population levels (102 to 103 colony-forming units per ml). The results suggest that either the synthesis of enterotoxin C is stimulated in vitro or that growth of S. aureus cells in udders was, in fact, higher than the colony-forming unit values indicated. Thermonuclease was shown to be excreted into mastitic milk at a slower rate than was enterotoxin. An inoculation of 1 ,ug of enterotoxin A in autogenic milk returned to the udder caused clinical reactions (swelling, palpation sensitivity, and increase in the level of somatic cells) within 6 h.
Staphylococcal enterotoxin is a toxic extracellular metabolite produced by staphylococcal cell populations at all growth phases (8). Staphylococci have been shown in many countries to be the principal cause of both acute and chronic inflammations of udders in cows (7, 15, 23, 29) and also of mammary gland infections in humans (12, 31). Cases of staphylococcal food poisoning caused by milk and milk products have frequently been reported. Some outbreaks derived from dried milk products (2, 3, 14) and cheeses (1, 11, 36) have been rather widespread. Many smaller scale outbreaks derived from raw milk (32), butter (21), and confectionary products containing cream (6) have been reported. Enterotoxins from infected mother's milk (25) and mother's milk substitute have also been detected (20). Toxigenic Staphylococcus aureus strains isolated from milk samples have usually produced either enterotoxin C (SEC) or D, or both (21, 24), whereas strains isolated from cases of food poisoning have usually been producers of enterotoxin A (SEA) (4, 30). The production of enterotoxins in milk (9, 11, 34), cream, and butter has been investigated (19). The results of S. aureus cultivation in milk have shown that the growth of staphylococci was considerably slower in nonheated than in heat-treated milk because of competing flora in the non-heated milk. Enterotoxin was formed
in detectable levels only when staphylococci had multiplied to levels of 107 to 108 cells per ml (9, 32). In heat-treated milk or milk with low initial concentrations of bacterial cells, S. aureus was able to multiply, at suitable temperatures, to very high levels and produce detectable amounts of enterotoxins within 3 h, depending on the level of inoculation (11). The aim of this research was to investigate the capacity of enterotoxigenic staphylococci to cause clinical symptoms of udder inflammation in cows and also to study the production of enterotoxins at different stages of the infection. Thus, it was hoped that through these studies the public health hazard arising form both clinical and subclinical staphylococcal mastitis in cows and humans could be evaluated.
MATERIALS AND METHODS Experimental animals. The Ayrshire cows used in this research (10 specimens) belonged to the same herd, were 3 to 8 years old, and were in the last third of their lactation periods, producing milk at a rate of 8 to 14 kg/day. All of the cows were initially clinically healthy. The initial condition of the udder quarters was excellent in all the animals except no. 5, in which the left front (If) and left hind (Ih) quarters yielded milk containing 3.0 x 103 colony-forming units (CFU) of Streptococcus uberis per ml. The Californian mastitis test (CMT) values of the milk from all udder quarters of this animal were 4 to 5, whereas the CMT 493
494
INFECT. IMMUN.
NISKANEN, KOIRANEN, AND ROINE
values in all milk samples of the other animals were between 1 and 2. S. aureus strains. The staphylococcal strains used to induce mastitis are listed in Table 1, along with some of their more important properties. The SEAproducing strains (510 and 530) were isolated from food poisoning outbreaks, and the SEC-producing strains were isolated from clinical mastitic samples in Norway. Microbiological assays. The CFU of S. aureus present in each milk sample were counted from the calf blood agar. The inoculated S. aureus strains produced hemotoxins. All colonies surrounded by hemotoxic reaction after 24 h of incubation at 37°C and having typical shape, size, and color were considered as S. aureus according to method no. 66/1968 of the Nordic Committee on Food Analysis. Some typical colonies were investigated for coagulase production by the tube method, using rabbit plasma, coagulase plasma ethylenediaminetetraacetic acid (Difco). Induction of mastitis. Cell populations of S. aureus for inoculation were cultivated overnight in nutrient broth (Difco) (20 ml), after which the cells were centrifuged and washed twice with 100 ml of saline. The cells were then suspended in saline, giving an end volume of 100 ml for measuring the CFU by plating on calf blood agar. The suspension was stored at 4°C until the inoculum was prepared in the autogenic milk sample (100 ml), which was heat treated for 30 min at 60°C and cooled to 37°C before use. Infusions of 20 ml of milk containing S. aureus at the levels indicated in Table 2 were then inoculated into the udder quarters. Enterotoxins and antisera. SEA and SEC, together with their respective antisera, were obtained from M. S. Bergdoll, Food Research Institute, University of Wisconsin, Madison. Purified SEA was obtained from Serva Feinbiochemica GmbH and Co., Heidelberg, Germany. Enterotoxin detection. Enterotoxins were extracted and detected in mastitic milk samples (80 ml) using the method described by A. Niskanen and S. Lindroth (21a). The minimum level of enterotoxin detectable by using this method was 1.0 ug. When sample volumes were sufficient, a comparative detection by the method of Reiser et al. (16) was also employed. Thermonuclease detection. Thermonuclease was
extracted from milk samples (30 ml) by the method of Tatini et al. (35) and detected by using a partial modification of the metachromic well-agar-diffusion technique (17), in which the deoxyribonucleic acid agar described by Lachica et al. (18) was used as reported earlier (22). Inoculation of pure and crude SEA into udders. The SEA used was produced by the dialysis sac method of Donnelly et al. (10) by using S. aureus 196E and purified by the method described by Schantz et al. (28). The toxin was then inoculated into two udder quarters of two cows at inoculation levels of 10 and 40 gLg in 20 ml of autogenic milk, treated as described above; in addition, purified SEA (Serva, declared purity >99%) was inoculated to one udder quarter of one cow. In each animal, an inoculation of non-contaminated autogenic milk was made into one quarter, and the fourth, uninoculated quarter served as a negative control.
RESULTS
Clinical changes during induced mastitis. Test cows 1, 2, 5, 6, and 7 reacted within 24 h to the staphylococcal infusion (levels of S. aureus inoculation are presented in Table 2). Among the symptoms observed were loss of appetite, sweating, shaking, rise in body temperature (40.3 to 41.1°C), and, in one case, diarrhea. Typical inflammation symptoms (swelling and palpate sensitivity) developed in the udders within 9 to 48 h. The severity of clinical symptoms varied among different animals, and in the same animal among different udder quarters. Cows 3 and 4 showed no clinical reactions, although no. 3 received 104 CFU of S. aureus 530 to the right front (rf) udder quarter, and no. 4 received 103 CFU of strain 576 to the If and 103 CFU of strain 530 to the right hind (rh) udder quarter (data not presented). The clinical findings have been reported in detail elsewhere (16). S. aureus growth and enterotoxin production. Strains 510 and 530, both producers of SEA, were inoculated into two udder quarters of cow 1 and into one udder quarter of cows 6
TABLE 1. Properties of S. aureus strains used for inducing mastitis in cows S. aureus
Source
strain
510
ATCC 196 E
530
Food poisoning outbreak in Finland Mastitis case in Norway Mastitis case in Norway Mastitis case in Norway
575
576 581
Enterotoxin
clease
Hemo-
Phage
Phage
pattem
group
Type
pg/ml
(pg/ml)
M
A
4
18
,B
III
A
10
190
a
78
M78
C
5
5
78
M7
C
7
4
78
M78
C
8
10
6, 42E, 54, 81, 84, 107, 117 53
type
VOL. 19, 1978
STAPHYLOCOCCAL ENTEROTOXIN PRODUCTION
495
TABLE 2. Production of staphylococcal enterotoxin and thermonuclease during induced mastitis cow no.
1
2
Time after Log of CFU/ Enterotoxin CFU offin Udder CFUol ki- smpleteedcasdoculaition inoculation ml of mil de-
quarter um
(strain)
no.a
(h)
if
103 (510)
1
9
Vol. 19, No. 2
0019-9567/78/0019-0493$02.00/O
Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Staphylococcal Enterotoxin and Thermonuclease Production During Induced Bovine Mastitis and the Clinical Reaction of Enterotoxin in Udders AIMO NISKANEN,l LEO KOIRANEN,2 AND KAUKO ROINE3 Technical Research Centre of Finland,' Food Research Laboratory, Biologinkuja 1, SF-02150 Espoo 15, State Veterinary Medical Institute,2 Hameentie 57, SF-00550 Helsinki 55, and Veterinary College of Finland,3 Ambulatory Clinic, SF-04840 Hautjarvi, Finland
Received for publication 29 June 1977
Enterotoxin A- and C-producing strains of Staphylococcus aureus and partially and extensively purified enterotoxin A were inoculated into the udder quarters of cows. In the course of experimentally induced mastitis caused by the inoculated S. aureus strain, enterotoxin C but not A was detected in the infected udder. Enterotoxin C was observed in mastitic milk samples at very low S. aureus population levels (102 to 103 colony-forming units per ml). The results suggest that either the synthesis of enterotoxin C is stimulated in vitro or that growth of S. aureus cells in udders was, in fact, higher than the colony-forming unit values indicated. Thermonuclease was shown to be excreted into mastitic milk at a slower rate than was enterotoxin. An inoculation of 1 ,ug of enterotoxin A in autogenic milk returned to the udder caused clinical reactions (swelling, palpation sensitivity, and increase in the level of somatic cells) within 6 h.
Staphylococcal enterotoxin is a toxic extracellular metabolite produced by staphylococcal cell populations at all growth phases (8). Staphylococci have been shown in many countries to be the principal cause of both acute and chronic inflammations of udders in cows (7, 15, 23, 29) and also of mammary gland infections in humans (12, 31). Cases of staphylococcal food poisoning caused by milk and milk products have frequently been reported. Some outbreaks derived from dried milk products (2, 3, 14) and cheeses (1, 11, 36) have been rather widespread. Many smaller scale outbreaks derived from raw milk (32), butter (21), and confectionary products containing cream (6) have been reported. Enterotoxins from infected mother's milk (25) and mother's milk substitute have also been detected (20). Toxigenic Staphylococcus aureus strains isolated from milk samples have usually produced either enterotoxin C (SEC) or D, or both (21, 24), whereas strains isolated from cases of food poisoning have usually been producers of enterotoxin A (SEA) (4, 30). The production of enterotoxins in milk (9, 11, 34), cream, and butter has been investigated (19). The results of S. aureus cultivation in milk have shown that the growth of staphylococci was considerably slower in nonheated than in heat-treated milk because of competing flora in the non-heated milk. Enterotoxin was formed
in detectable levels only when staphylococci had multiplied to levels of 107 to 108 cells per ml (9, 32). In heat-treated milk or milk with low initial concentrations of bacterial cells, S. aureus was able to multiply, at suitable temperatures, to very high levels and produce detectable amounts of enterotoxins within 3 h, depending on the level of inoculation (11). The aim of this research was to investigate the capacity of enterotoxigenic staphylococci to cause clinical symptoms of udder inflammation in cows and also to study the production of enterotoxins at different stages of the infection. Thus, it was hoped that through these studies the public health hazard arising form both clinical and subclinical staphylococcal mastitis in cows and humans could be evaluated.
MATERIALS AND METHODS Experimental animals. The Ayrshire cows used in this research (10 specimens) belonged to the same herd, were 3 to 8 years old, and were in the last third of their lactation periods, producing milk at a rate of 8 to 14 kg/day. All of the cows were initially clinically healthy. The initial condition of the udder quarters was excellent in all the animals except no. 5, in which the left front (If) and left hind (Ih) quarters yielded milk containing 3.0 x 103 colony-forming units (CFU) of Streptococcus uberis per ml. The Californian mastitis test (CMT) values of the milk from all udder quarters of this animal were 4 to 5, whereas the CMT 493
494
INFECT. IMMUN.
NISKANEN, KOIRANEN, AND ROINE
values in all milk samples of the other animals were between 1 and 2. S. aureus strains. The staphylococcal strains used to induce mastitis are listed in Table 1, along with some of their more important properties. The SEAproducing strains (510 and 530) were isolated from food poisoning outbreaks, and the SEC-producing strains were isolated from clinical mastitic samples in Norway. Microbiological assays. The CFU of S. aureus present in each milk sample were counted from the calf blood agar. The inoculated S. aureus strains produced hemotoxins. All colonies surrounded by hemotoxic reaction after 24 h of incubation at 37°C and having typical shape, size, and color were considered as S. aureus according to method no. 66/1968 of the Nordic Committee on Food Analysis. Some typical colonies were investigated for coagulase production by the tube method, using rabbit plasma, coagulase plasma ethylenediaminetetraacetic acid (Difco). Induction of mastitis. Cell populations of S. aureus for inoculation were cultivated overnight in nutrient broth (Difco) (20 ml), after which the cells were centrifuged and washed twice with 100 ml of saline. The cells were then suspended in saline, giving an end volume of 100 ml for measuring the CFU by plating on calf blood agar. The suspension was stored at 4°C until the inoculum was prepared in the autogenic milk sample (100 ml), which was heat treated for 30 min at 60°C and cooled to 37°C before use. Infusions of 20 ml of milk containing S. aureus at the levels indicated in Table 2 were then inoculated into the udder quarters. Enterotoxins and antisera. SEA and SEC, together with their respective antisera, were obtained from M. S. Bergdoll, Food Research Institute, University of Wisconsin, Madison. Purified SEA was obtained from Serva Feinbiochemica GmbH and Co., Heidelberg, Germany. Enterotoxin detection. Enterotoxins were extracted and detected in mastitic milk samples (80 ml) using the method described by A. Niskanen and S. Lindroth (21a). The minimum level of enterotoxin detectable by using this method was 1.0 ug. When sample volumes were sufficient, a comparative detection by the method of Reiser et al. (16) was also employed. Thermonuclease detection. Thermonuclease was
extracted from milk samples (30 ml) by the method of Tatini et al. (35) and detected by using a partial modification of the metachromic well-agar-diffusion technique (17), in which the deoxyribonucleic acid agar described by Lachica et al. (18) was used as reported earlier (22). Inoculation of pure and crude SEA into udders. The SEA used was produced by the dialysis sac method of Donnelly et al. (10) by using S. aureus 196E and purified by the method described by Schantz et al. (28). The toxin was then inoculated into two udder quarters of two cows at inoculation levels of 10 and 40 gLg in 20 ml of autogenic milk, treated as described above; in addition, purified SEA (Serva, declared purity >99%) was inoculated to one udder quarter of one cow. In each animal, an inoculation of non-contaminated autogenic milk was made into one quarter, and the fourth, uninoculated quarter served as a negative control.
RESULTS
Clinical changes during induced mastitis. Test cows 1, 2, 5, 6, and 7 reacted within 24 h to the staphylococcal infusion (levels of S. aureus inoculation are presented in Table 2). Among the symptoms observed were loss of appetite, sweating, shaking, rise in body temperature (40.3 to 41.1°C), and, in one case, diarrhea. Typical inflammation symptoms (swelling and palpate sensitivity) developed in the udders within 9 to 48 h. The severity of clinical symptoms varied among different animals, and in the same animal among different udder quarters. Cows 3 and 4 showed no clinical reactions, although no. 3 received 104 CFU of S. aureus 530 to the right front (rf) udder quarter, and no. 4 received 103 CFU of strain 576 to the If and 103 CFU of strain 530 to the right hind (rh) udder quarter (data not presented). The clinical findings have been reported in detail elsewhere (16). S. aureus growth and enterotoxin production. Strains 510 and 530, both producers of SEA, were inoculated into two udder quarters of cow 1 and into one udder quarter of cows 6
TABLE 1. Properties of S. aureus strains used for inducing mastitis in cows S. aureus
Source
strain
510
ATCC 196 E
530
Food poisoning outbreak in Finland Mastitis case in Norway Mastitis case in Norway Mastitis case in Norway
575
576 581
Enterotoxin
clease
Hemo-
Phage
Phage
pattem
group
Type
pg/ml
(pg/ml)
M
A
4
18
,B
III
A
10
190
a
78
M78
C
5
5
78
M7
C
7
4
78
M78
C
8
10
6, 42E, 54, 81, 84, 107, 117 53
type
VOL. 19, 1978
STAPHYLOCOCCAL ENTEROTOXIN PRODUCTION
495
TABLE 2. Production of staphylococcal enterotoxin and thermonuclease during induced mastitis cow no.
1
2
Time after Log of CFU/ Enterotoxin CFU offin Udder CFUol ki- smpleteedcasdoculaition inoculation ml of mil de-
quarter um
(strain)
no.a
(h)
if
103 (510)
1
9
