- Email: [email protected]
Classical enterotoxigenic characteristics of Staphylococcus aureus strains isolated from bovine subclinical mastitis in Van, Turkey
International Journal of Food Microbiology 125 (2008) 209–211
Contents lists available at ScienceDirect
International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j f o o d m i c r o
Short communication
Classical enterotoxigenic characteristics of Staphylococcus aureus strains isolated from bovine subclinical mastitis in Van, Turkey Banur Boynukara a,⁎, Timur Gulhan a, Mustafa Alisarli b, Kemal Gurturk a, Hasan Solmaz a a b
Department of Microbiology, Faculty of Veterinary Medicine, University of Yuzuncu Yil, 65080, Van, Turkey Department of Food Hygiene and Technology, Faculty of Veterinary Medicine, University of Yuzuncu Yil, 65080, Van, Turkey
A R T I C L E
I N F O
Article history: Received 30 July 2007 Received in revised form 11 March 2008 Accepted 24 March 2008 Keywords: S. aureus Enterotoxin Subclinic mastitis Bovine RPLA
A B S T R A C T The aim of the present study was to investigate classical enterotoxigenic properties of Staphylococcus aureus strains isolated from cows with subclinical mastitis. For this purpose, 480 milk samples from cows with subclinical mastitis raised in different villages neighbouring Van city Center were collected. A total of 106 S. aureus strains were isolated. Twenty seven isolates (25.5%) were found to be enterotoxigenic by reverse passive latex agglutination (RPLA). Of these, 25 (23.6%) were positive for staphylococcal enterotoxin A (SEA), 2 (1.9%) for SEB. None of the isolates was positive for SEC or SED. This study showed that most S. aureus strains isolated from bovine subclinical mastitis produced SEA compared to other SEs. © 2008 Elsevier B.V. All rights reserved.
Staphylococcus aureus is the cause of a wide spectrum of infections in humans and different animal species. It has been frequently isolated from bovine mastitis (Hata et al., 2006). Cows are probably the main source of contamination of raw milk with S. aureus (da Silva et al., 2005). In particular, cows with subclinical S. aureus mastitis can shed large numbers of S. aureus in their milk. However, contamination of raw milk and raw milk products from human handling or from the environment during manufacture is also possible. The ability to cause a wide range of diseases may be associated with its production of a large number of extracellular toxins and other virulence factors. The staphylococcal enterotoxins are recognized as being agents of intoxication such as staphylococcal food poisoning in man and they may be involved in other types of infections. S. aureus produces a wide variety of toxic proteins such as toxic shock syndrome toxin 1, exfoliative toxins, and enterotoxins (SEs). In addition to the five classical major antigenic types of SEs (SEA, SEB, SEC, SED, and SEE), four additional SEs (SEG, SEH, SEI, SEJ) have been reported, and their corresponding genes have been described (Stephan et al., 2001; Kuzma et al., 2003). Recently many new types of staphylococcal enterotoxins (SEK, SEL, SEM, SEN, SEO, SEQ, SER and SET) have been described (Zschöck et al., 2005). There is little information concerning the occurrence and significance of these novel enterotoxins (SEG – SER, SEU) in S. aureus strains from animal infections (Adwan et al., 2005). ⁎ Corresponding author. Yuzuncu Yıl University, Faculty of Veterinary Medicine, Department of Microbiology, Van, Turkey. E-mail addresses: [email protected], [email protected] (B. Boynukara). 0168-1605/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2008.03.024
Some of these SEs may contribute to the persistence of S. aureus in subclinical mastitis. Because of the putative significance of these enterotoxins for public health and food safety, more information about their occurrence and an efficient means of screening for their genes are needed (Smyth et al., 2005). Development in food processing and retailing over recent decades have the potential to introduce toxigenic strains of S. aureus of animal origin into the food chain (Serraino et al., 2004). Since several of these SEs have been discovered in the last 5 years, there is reason to believe that additional enterotoxins will be described as research on pathogenic S. aureus isolates continues. The purpose of the present study was to investigate classical enterotoxigenic properties of S. aureus strains isolated from milk from cows with subclinical mastitis in Van, Turkey. 1. Materials and Methods In this study, milk samples were collected in the season from April to June, from 10 different villages neighbouring Van city center. The number of the cows raised in each village varied from 100 to 150. At first, lactating cows in these villages were checked for subclinic mastitis by California Mastitis Test (CMT) and 480 milk samples were found to be positive for subclinic mastitis. Raw milk samples were collected only from cows with subclinic mastitis aseptically into sterilized screw cap sample bottles at 10 ml after disinfection of mammary glands. Cows had no symptoms for clinical mastitis, and the udders were without clinical abnormalities and giving apparently normal milk. Subclinical mastitis was diagnosed by CMT, which
210
B. Boynukara et al. / International Journal of Food Microbiology 125 (2008) 209–211
Table 1 Enterotoxin types of S. aureus strains and some enzymatic activities (number of strains positive for each test) S. aureus (n = 106)
%
Caseinase
99 93.4
DNase
106 100
TNase
48 45.3
Lecithinase
75 70.7
Types of enterotoxin A
B
C
D
25 23.6
2 1.9
0 0
0 0
detects increased somatic cells in milk (Tollersrud et al., 2000). A total of 480 raw milk samples from individual cows with subclinic mastitis was included for bacteriological examination. For this, each milk sample (30 μl) was streaked on nutrient agar (Oxoid) that was widely used to cultivate S. aureus from clinical specimens and then agar plates were incubated at 37 °C for 24 – 48 h under aerobic conditions (Cenci - Goga et al., 2003). Ten colonies were randomly selected and transferred to individual tubes of nutrient broth. After incubation for 24 h under the same condition a loopfull of bacteria suspension was streaked on mannitol salt agar (MSA, Oxoid). Yellow colored colonies which were mannitol positive, suspected as S. aureus on MSA were selected and subcultured on 5% sheep blood agar (Difco) to make pure culture of staphylococci isolates. Gram stain, culture characteristics, catalase test, and coagulase test using fresh rabbit plasma (tube method) were used for the presumptive identification of all isolates. All coagulase - positive isolates were further tested using the API STAPH IDENT, 32 Staph (bioMerieux SA, 69280 Marcy - l'Etoile, France) to confirm identification of S. aureus isolates. The following S. aureus reference strains were kindly provided by Dr. B. Holmes, NCTC (National Collection of Type Cultures Public Health Laboratory Service, London): SEA 10652 FDA 196E, SEB 10654 FDA 243, SEC 10655 137 and SED 10656 494. The other reference strain (S. epidermis - 33) was obtained from our own culture collection. Caseinase activity was detected using casein medium according to the procedures described by Koneman et al. (1997). Caseinase positive colonies on the casein medium were surrounded by a transparent halo after 2 days of incubation at 37 °C. For the detection of DNase activity, staphylococci was cultivated on DNase agar (Oxoid, UK) supplemented with 0.5% yeast extract (Koneman et al., 1997). Thermonuclease (TNase) activity was detected using Toluidine Blue DNA agar (TBDA) according to the procedures described by Lachia et al. (1971). Lecithinase activity was examined on Baird - Parker medium containing 5% egg yolk emulsion. Inoculated plates were incubated for 48 h at 35 °C and thereafter for 24 h at room temperature. The lecithinase activity was recognized by an opaque zone of precipitate in the medium around the growth (Koneman et al., 1997). To detect staphylococcal enterotoxins S. aureus isolates were cultured in 10 ml Nutrient broth at 37 °C aerobically. Overnight culture supernatants of the isolates were used to detect S. aureus enterotoxins. Staphylococcal enterotoxins SEA, SEB, SEC and SED were detected by a reversed passive latex agglutination (RPLA) toxin detection kit (SET - RPLA; Oxoid, Basingstoke, UK) according to the manufacturer's instructions. S. aureus and S. epidermis strains mentioned above were used as positive and negative control strain for each test. 2. Results and Discussion From 480 milk samples examined bacteriologically, 106 S. aureus were isolated and used for the detection of classical types of SEs. Among the 106 S. aureus isolates, 27 (25.5%) were found to be enterotoxigenic. Twenty five (23.6%) were positive for SEA and 2 (1.9%) were positive for SEB. None of the isolates was positive for SEC or SED (Table 1). None of the strains produced more than one type of enterotoxins. Results of further characterization of 106 S. aureus such as caseinase, DNase, TNase and lecithinase activities are presented in Table 1.
Although the high ratio of enterotoxin - forming strains as the main factor of pathogenesis of S. aureus bacteria indicates the significant role of enterotoxins in pathogenesis of mastitis, the role of S. aureus enterotoxins in pathogenesis of bovine mastitis is not yet fully clarified (Joffe and Baranovičs, 2006). Production of SEA, SEB, SEC, SED by S. aureus strains associated with subclinical and/or clinical bovine mastitis has already been described by many authors (Tollersrud et al., 2000; Stephan et al., 2001; Joffe and Baranovičs, 2006; Cenci - Goga et al., 2003; Alisarlı and Solmaz, 2003; Serraino et al., 2004). Data from references related to the classical enterotoxigenic types of S. aureus isolated from cows with subclinical and/or clinic mastitis are summarized in Table 2. The predominant enterotoxin types produced by S. aureus strains from bovine milk with/without mastitis could be variable. A recent study (Kuzma et al., 2003) investigating the S. aureus isolates for toxin formation showed that in addition to enterotoxins A, C, and D, the newly described enterotoxins G, H, and I seemed to predominate in enterotoxins of S. aureus isolated from cows with mastitis. In the present study, SEA - producing strains were found to be most common in S. aureus strains (23.6%) from subclinical bovine mastitis. The conflicting results from our study and of other investigations concerning occurrence of superantigenic exotoxins among S. aureus isolates from bovine milk samples might be due to differences in the reservoir of the various countries or the ecological origin of strains, the sensitivity of detection methods, number and kinds of examined samples in these studies. Recently, new superantigenic enterotoxins (SEG - SEU) have also been described (Zschöck et al., 2005). We could not screen these enterotoxins in the present study, since commercial RPLA test kits for detecting these new enterotoxins are not available. In addition, S. aureus may also be able to produce unidentified superantigenic exotoxins which cannot be determined at present (Smyth et al., 2005). Nevertheless, a relative high percentage of classical enterotoxin - forming S. aureus strains from bovine subclinical mastitis found in our study is in agreement with other publications (Kuzma et al., 2003; Serraino et al., 2004; da Silva et al., 2005). It should be noted that enterotoxin A - producing S. aureus is one of the main causes of food - borne disease SEA also predominates in milk from cows with mastitis and milk products (Tollersrud et al., 2000; Stephan et al., 2001). Hence contamination of dairy products with S. aureus may be due to the presence of this pathogen in raw milk. This is very important, especially in countries producing large amounts of milk products such as cheese. In villages neighbouring Van city, where this study was performed, cheese is mostly prepared from unpasteurized cow and ewe milk and therefore may contribute to the sources of staphylococcal food poisoning. The results of this study showed that the rates of enterotoxin production were high (25.5%) in S. aureus strains isolated from bovine subclinical mastitis. Our results also showed that SEA - producing strains were predominant (23.6%). We have also determined in another study (Boynukara et al., 2007) that S. aureus strains isolated from various clinical human specimens were highly enterotoxigenic (56.8%) and SEA was also found to be predominant enterotoxin type (38.1%). Further epidemiological studies are needed to examine enterotoxigenic S. aureus
Table 2 References and reported data on classical enterotoxigenic types of S. aureus strains isolated from raw cow-milk samples References
Tollersrud et al. (2000) Stephan et al. (2001) Cenci-Goga et al. (2003) Alisarlı and Solmaz (2003) Serraino et al. (2004) Joffe and Baranovičs (2006)
Enterotoxigenic types and it's combinations (%) A
B
C
D
A+C
A+D
– 12.7 13.7 16.1 24.6 45.4
– – – – – 10.9
6.8 36.5 33.8 25.8 – 21
– 4.8 – 3.2 9.8 –
3.4
– 12.7 – – 24.6 –
– – – –
B. Boynukara et al. / International Journal of Food Microbiology 125 (2008) 209–211
isolates or their toxins in food, and investigations should also be performed to find the relationship between the presence of this pathogen in food and its ability to cause disease in human. Acknowledgements We would like to thank Dr. Janet E.L. Corry University of Bristol, UK 184 for her invaluable contribution. References Adwan, G., Abu - Shanab, B., Adwan, K., 2005. Enterotoxigenic Staphylococcus aureus in raw milk in the North of Palestine. Turkish Journal of Biology 29, 229–232. Alisarlı, M., Solmaz, H., 2003. The pathogenic properties and the varied antibiotic sensitivities of S. aureus isolated from teat skin and raw milk of dairy cows. Journal of the Faculty of Agriculture University of Ataturk 34, 333–339. Boynukara, B., Gulhan, T., Gurturk, K., Alisarlı, M., Ogun, E., 2007. Evolution of slime production by coagulase - negative staphylococci and enterotoxigenic characteristics of Staphylococcus aureus strains isolated from various clinical specimens of human. Journal of Medical Microbiology 56 (10), 1296–1300. Cenci - Goga, B.T., Karama, M., Rossitto, P.V., Morgante, R.A., Cullor, J.S., 2003. Enterotoxin production by Staphylococcus aureus isolated from mastitic cows. Journal of Food Protection 66, 1693–1696. da Silva, E.R., do Carmo, L.S., da Silva, N., 2005. Detection of the enterotoxins A, B, and C genes in Staphylococcus aureus from goat and bovine mastitis in Brazilian dairy herds. Veterinary Microbiology 106, 103–107. Hata, E., Katsuda, K., Kobayashi, H., Ogawai, T., Endo, T., Eguchi, M., 2006. Characteristics and epidemiologic genotyping of Staphylococcus aureus isolates from bovine mastitic milk in Hokkaido, Japan. Journal of Veterinary Medicine Science 68, 165–170.
211
Joffe, R., Baranovičs, E., 2006. Bovine mastitis as the primary contamination source of milk and milk products with S. aureus enterotoxins. Veterinarija Ir Zootechnika T 36, 21–26. Koneman, E.W., Allen, S.D., Janda, W.M., Schreckenberger, P.C., Winn, W.C., 1997. Color Atlas and Textbook of Diagnostic Microbiology, 5th ed. Lippincot, Philadelphia, PA, pp. 1347–1348. Kuzma, K., Malinowski, E., Lassa, H., Klossowska, A., 2003. Detection of genes for enterotoxins and toxic shock syndrome toxin-1in Staphylococcus aureus isolated from bovine mastitis. Bulletin of the Veterinary Research Institute in Pulawy 47, 419–426. Lachia, R.V.F., Genigeorgis, C., Hoeprich, P.D., 1971. Metachromic agar - diffusion methods for detecting staphylococcal nuclease activity. Applied Microbiology 21, 585–587. Serraino, A., Alberghini, L., Fontana, M.C., Annemüller, C., Lämmler, C., Rosmini, R., 2004. Occurrence of enterotoxin genes and macrorestriction analysis of Staphylococcus aureus isolated from bovine mastitis and bulk - tank milk samples in Italy. An epidemiological study. Italian Journal of Animal Science 3, 47–53. Smyth, D.S., Hartigan, P.J., Meaney, W.J., Fitzgerald, J.R., Deobald, C.F., Bohach, G.A., Smyth, C.J., 2005. Superantigen genes encoded by the egc cluster and SaPIbov are predominant among Staphylococcus aureus isolates from cows, goats, sheep, rabbits and poultry. Journal of Medical Microbiology 54, 401–411. Stephan, R., Annemüller, C., Hassan, A.A., Lämmler, Ch., 2001. Characterization of enterotoxigenic Staphylococcus aureus strains isolated from bovine mastitis in north - east Switzerland. Veterinary Microbiology 78, 373–382. Tollersrud, T., Kenny, K., Caugant, D.A., Lund, A., 2000. Characterization of isolates of Staphylococcus aureus from acute, chronic and subclinical mastitis in cows in Norway. Acta Pathologica, Microbiologica et Immunologica Scandinavica 108, 565–572. Zschöck, M., Kloppert, B., Wolter, W., Hamann, H.P., Lämmler, Ch., 2005. Pattern of enterotoxin genes seg, seh, sei and sej positive Staphylococcus aureus isolated from bovine mastitis. Veterinary Microbiology 108, 243–249.
Contents lists available at ScienceDirect
International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j f o o d m i c r o
Short communication
Classical enterotoxigenic characteristics of Staphylococcus aureus strains isolated from bovine subclinical mastitis in Van, Turkey Banur Boynukara a,⁎, Timur Gulhan a, Mustafa Alisarli b, Kemal Gurturk a, Hasan Solmaz a a b
Department of Microbiology, Faculty of Veterinary Medicine, University of Yuzuncu Yil, 65080, Van, Turkey Department of Food Hygiene and Technology, Faculty of Veterinary Medicine, University of Yuzuncu Yil, 65080, Van, Turkey
A R T I C L E
I N F O
Article history: Received 30 July 2007 Received in revised form 11 March 2008 Accepted 24 March 2008 Keywords: S. aureus Enterotoxin Subclinic mastitis Bovine RPLA
A B S T R A C T The aim of the present study was to investigate classical enterotoxigenic properties of Staphylococcus aureus strains isolated from cows with subclinical mastitis. For this purpose, 480 milk samples from cows with subclinical mastitis raised in different villages neighbouring Van city Center were collected. A total of 106 S. aureus strains were isolated. Twenty seven isolates (25.5%) were found to be enterotoxigenic by reverse passive latex agglutination (RPLA). Of these, 25 (23.6%) were positive for staphylococcal enterotoxin A (SEA), 2 (1.9%) for SEB. None of the isolates was positive for SEC or SED. This study showed that most S. aureus strains isolated from bovine subclinical mastitis produced SEA compared to other SEs. © 2008 Elsevier B.V. All rights reserved.
Staphylococcus aureus is the cause of a wide spectrum of infections in humans and different animal species. It has been frequently isolated from bovine mastitis (Hata et al., 2006). Cows are probably the main source of contamination of raw milk with S. aureus (da Silva et al., 2005). In particular, cows with subclinical S. aureus mastitis can shed large numbers of S. aureus in their milk. However, contamination of raw milk and raw milk products from human handling or from the environment during manufacture is also possible. The ability to cause a wide range of diseases may be associated with its production of a large number of extracellular toxins and other virulence factors. The staphylococcal enterotoxins are recognized as being agents of intoxication such as staphylococcal food poisoning in man and they may be involved in other types of infections. S. aureus produces a wide variety of toxic proteins such as toxic shock syndrome toxin 1, exfoliative toxins, and enterotoxins (SEs). In addition to the five classical major antigenic types of SEs (SEA, SEB, SEC, SED, and SEE), four additional SEs (SEG, SEH, SEI, SEJ) have been reported, and their corresponding genes have been described (Stephan et al., 2001; Kuzma et al., 2003). Recently many new types of staphylococcal enterotoxins (SEK, SEL, SEM, SEN, SEO, SEQ, SER and SET) have been described (Zschöck et al., 2005). There is little information concerning the occurrence and significance of these novel enterotoxins (SEG – SER, SEU) in S. aureus strains from animal infections (Adwan et al., 2005). ⁎ Corresponding author. Yuzuncu Yıl University, Faculty of Veterinary Medicine, Department of Microbiology, Van, Turkey. E-mail addresses: [email protected], [email protected] (B. Boynukara). 0168-1605/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2008.03.024
Some of these SEs may contribute to the persistence of S. aureus in subclinical mastitis. Because of the putative significance of these enterotoxins for public health and food safety, more information about their occurrence and an efficient means of screening for their genes are needed (Smyth et al., 2005). Development in food processing and retailing over recent decades have the potential to introduce toxigenic strains of S. aureus of animal origin into the food chain (Serraino et al., 2004). Since several of these SEs have been discovered in the last 5 years, there is reason to believe that additional enterotoxins will be described as research on pathogenic S. aureus isolates continues. The purpose of the present study was to investigate classical enterotoxigenic properties of S. aureus strains isolated from milk from cows with subclinical mastitis in Van, Turkey. 1. Materials and Methods In this study, milk samples were collected in the season from April to June, from 10 different villages neighbouring Van city center. The number of the cows raised in each village varied from 100 to 150. At first, lactating cows in these villages were checked for subclinic mastitis by California Mastitis Test (CMT) and 480 milk samples were found to be positive for subclinic mastitis. Raw milk samples were collected only from cows with subclinic mastitis aseptically into sterilized screw cap sample bottles at 10 ml after disinfection of mammary glands. Cows had no symptoms for clinical mastitis, and the udders were without clinical abnormalities and giving apparently normal milk. Subclinical mastitis was diagnosed by CMT, which
210
B. Boynukara et al. / International Journal of Food Microbiology 125 (2008) 209–211
Table 1 Enterotoxin types of S. aureus strains and some enzymatic activities (number of strains positive for each test) S. aureus (n = 106)
%
Caseinase
99 93.4
DNase
106 100
TNase
48 45.3
Lecithinase
75 70.7
Types of enterotoxin A
B
C
D
25 23.6
2 1.9
0 0
0 0
detects increased somatic cells in milk (Tollersrud et al., 2000). A total of 480 raw milk samples from individual cows with subclinic mastitis was included for bacteriological examination. For this, each milk sample (30 μl) was streaked on nutrient agar (Oxoid) that was widely used to cultivate S. aureus from clinical specimens and then agar plates were incubated at 37 °C for 24 – 48 h under aerobic conditions (Cenci - Goga et al., 2003). Ten colonies were randomly selected and transferred to individual tubes of nutrient broth. After incubation for 24 h under the same condition a loopfull of bacteria suspension was streaked on mannitol salt agar (MSA, Oxoid). Yellow colored colonies which were mannitol positive, suspected as S. aureus on MSA were selected and subcultured on 5% sheep blood agar (Difco) to make pure culture of staphylococci isolates. Gram stain, culture characteristics, catalase test, and coagulase test using fresh rabbit plasma (tube method) were used for the presumptive identification of all isolates. All coagulase - positive isolates were further tested using the API STAPH IDENT, 32 Staph (bioMerieux SA, 69280 Marcy - l'Etoile, France) to confirm identification of S. aureus isolates. The following S. aureus reference strains were kindly provided by Dr. B. Holmes, NCTC (National Collection of Type Cultures Public Health Laboratory Service, London): SEA 10652 FDA 196E, SEB 10654 FDA 243, SEC 10655 137 and SED 10656 494. The other reference strain (S. epidermis - 33) was obtained from our own culture collection. Caseinase activity was detected using casein medium according to the procedures described by Koneman et al. (1997). Caseinase positive colonies on the casein medium were surrounded by a transparent halo after 2 days of incubation at 37 °C. For the detection of DNase activity, staphylococci was cultivated on DNase agar (Oxoid, UK) supplemented with 0.5% yeast extract (Koneman et al., 1997). Thermonuclease (TNase) activity was detected using Toluidine Blue DNA agar (TBDA) according to the procedures described by Lachia et al. (1971). Lecithinase activity was examined on Baird - Parker medium containing 5% egg yolk emulsion. Inoculated plates were incubated for 48 h at 35 °C and thereafter for 24 h at room temperature. The lecithinase activity was recognized by an opaque zone of precipitate in the medium around the growth (Koneman et al., 1997). To detect staphylococcal enterotoxins S. aureus isolates were cultured in 10 ml Nutrient broth at 37 °C aerobically. Overnight culture supernatants of the isolates were used to detect S. aureus enterotoxins. Staphylococcal enterotoxins SEA, SEB, SEC and SED were detected by a reversed passive latex agglutination (RPLA) toxin detection kit (SET - RPLA; Oxoid, Basingstoke, UK) according to the manufacturer's instructions. S. aureus and S. epidermis strains mentioned above were used as positive and negative control strain for each test. 2. Results and Discussion From 480 milk samples examined bacteriologically, 106 S. aureus were isolated and used for the detection of classical types of SEs. Among the 106 S. aureus isolates, 27 (25.5%) were found to be enterotoxigenic. Twenty five (23.6%) were positive for SEA and 2 (1.9%) were positive for SEB. None of the isolates was positive for SEC or SED (Table 1). None of the strains produced more than one type of enterotoxins. Results of further characterization of 106 S. aureus such as caseinase, DNase, TNase and lecithinase activities are presented in Table 1.
Although the high ratio of enterotoxin - forming strains as the main factor of pathogenesis of S. aureus bacteria indicates the significant role of enterotoxins in pathogenesis of mastitis, the role of S. aureus enterotoxins in pathogenesis of bovine mastitis is not yet fully clarified (Joffe and Baranovičs, 2006). Production of SEA, SEB, SEC, SED by S. aureus strains associated with subclinical and/or clinical bovine mastitis has already been described by many authors (Tollersrud et al., 2000; Stephan et al., 2001; Joffe and Baranovičs, 2006; Cenci - Goga et al., 2003; Alisarlı and Solmaz, 2003; Serraino et al., 2004). Data from references related to the classical enterotoxigenic types of S. aureus isolated from cows with subclinical and/or clinic mastitis are summarized in Table 2. The predominant enterotoxin types produced by S. aureus strains from bovine milk with/without mastitis could be variable. A recent study (Kuzma et al., 2003) investigating the S. aureus isolates for toxin formation showed that in addition to enterotoxins A, C, and D, the newly described enterotoxins G, H, and I seemed to predominate in enterotoxins of S. aureus isolated from cows with mastitis. In the present study, SEA - producing strains were found to be most common in S. aureus strains (23.6%) from subclinical bovine mastitis. The conflicting results from our study and of other investigations concerning occurrence of superantigenic exotoxins among S. aureus isolates from bovine milk samples might be due to differences in the reservoir of the various countries or the ecological origin of strains, the sensitivity of detection methods, number and kinds of examined samples in these studies. Recently, new superantigenic enterotoxins (SEG - SEU) have also been described (Zschöck et al., 2005). We could not screen these enterotoxins in the present study, since commercial RPLA test kits for detecting these new enterotoxins are not available. In addition, S. aureus may also be able to produce unidentified superantigenic exotoxins which cannot be determined at present (Smyth et al., 2005). Nevertheless, a relative high percentage of classical enterotoxin - forming S. aureus strains from bovine subclinical mastitis found in our study is in agreement with other publications (Kuzma et al., 2003; Serraino et al., 2004; da Silva et al., 2005). It should be noted that enterotoxin A - producing S. aureus is one of the main causes of food - borne disease SEA also predominates in milk from cows with mastitis and milk products (Tollersrud et al., 2000; Stephan et al., 2001). Hence contamination of dairy products with S. aureus may be due to the presence of this pathogen in raw milk. This is very important, especially in countries producing large amounts of milk products such as cheese. In villages neighbouring Van city, where this study was performed, cheese is mostly prepared from unpasteurized cow and ewe milk and therefore may contribute to the sources of staphylococcal food poisoning. The results of this study showed that the rates of enterotoxin production were high (25.5%) in S. aureus strains isolated from bovine subclinical mastitis. Our results also showed that SEA - producing strains were predominant (23.6%). We have also determined in another study (Boynukara et al., 2007) that S. aureus strains isolated from various clinical human specimens were highly enterotoxigenic (56.8%) and SEA was also found to be predominant enterotoxin type (38.1%). Further epidemiological studies are needed to examine enterotoxigenic S. aureus
Table 2 References and reported data on classical enterotoxigenic types of S. aureus strains isolated from raw cow-milk samples References
Tollersrud et al. (2000) Stephan et al. (2001) Cenci-Goga et al. (2003) Alisarlı and Solmaz (2003) Serraino et al. (2004) Joffe and Baranovičs (2006)
Enterotoxigenic types and it's combinations (%) A
B
C
D
A+C
A+D
– 12.7 13.7 16.1 24.6 45.4
– – – – – 10.9
6.8 36.5 33.8 25.8 – 21
– 4.8 – 3.2 9.8 –
3.4
– 12.7 – – 24.6 –
– – – –
B. Boynukara et al. / International Journal of Food Microbiology 125 (2008) 209–211
isolates or their toxins in food, and investigations should also be performed to find the relationship between the presence of this pathogen in food and its ability to cause disease in human. Acknowledgements We would like to thank Dr. Janet E.L. Corry University of Bristol, UK 184 for her invaluable contribution. References Adwan, G., Abu - Shanab, B., Adwan, K., 2005. Enterotoxigenic Staphylococcus aureus in raw milk in the North of Palestine. Turkish Journal of Biology 29, 229–232. Alisarlı, M., Solmaz, H., 2003. The pathogenic properties and the varied antibiotic sensitivities of S. aureus isolated from teat skin and raw milk of dairy cows. Journal of the Faculty of Agriculture University of Ataturk 34, 333–339. Boynukara, B., Gulhan, T., Gurturk, K., Alisarlı, M., Ogun, E., 2007. Evolution of slime production by coagulase - negative staphylococci and enterotoxigenic characteristics of Staphylococcus aureus strains isolated from various clinical specimens of human. Journal of Medical Microbiology 56 (10), 1296–1300. Cenci - Goga, B.T., Karama, M., Rossitto, P.V., Morgante, R.A., Cullor, J.S., 2003. Enterotoxin production by Staphylococcus aureus isolated from mastitic cows. Journal of Food Protection 66, 1693–1696. da Silva, E.R., do Carmo, L.S., da Silva, N., 2005. Detection of the enterotoxins A, B, and C genes in Staphylococcus aureus from goat and bovine mastitis in Brazilian dairy herds. Veterinary Microbiology 106, 103–107. Hata, E., Katsuda, K., Kobayashi, H., Ogawai, T., Endo, T., Eguchi, M., 2006. Characteristics and epidemiologic genotyping of Staphylococcus aureus isolates from bovine mastitic milk in Hokkaido, Japan. Journal of Veterinary Medicine Science 68, 165–170.
211
Joffe, R., Baranovičs, E., 2006. Bovine mastitis as the primary contamination source of milk and milk products with S. aureus enterotoxins. Veterinarija Ir Zootechnika T 36, 21–26. Koneman, E.W., Allen, S.D., Janda, W.M., Schreckenberger, P.C., Winn, W.C., 1997. Color Atlas and Textbook of Diagnostic Microbiology, 5th ed. Lippincot, Philadelphia, PA, pp. 1347–1348. Kuzma, K., Malinowski, E., Lassa, H., Klossowska, A., 2003. Detection of genes for enterotoxins and toxic shock syndrome toxin-1in Staphylococcus aureus isolated from bovine mastitis. Bulletin of the Veterinary Research Institute in Pulawy 47, 419–426. Lachia, R.V.F., Genigeorgis, C., Hoeprich, P.D., 1971. Metachromic agar - diffusion methods for detecting staphylococcal nuclease activity. Applied Microbiology 21, 585–587. Serraino, A., Alberghini, L., Fontana, M.C., Annemüller, C., Lämmler, C., Rosmini, R., 2004. Occurrence of enterotoxin genes and macrorestriction analysis of Staphylococcus aureus isolated from bovine mastitis and bulk - tank milk samples in Italy. An epidemiological study. Italian Journal of Animal Science 3, 47–53. Smyth, D.S., Hartigan, P.J., Meaney, W.J., Fitzgerald, J.R., Deobald, C.F., Bohach, G.A., Smyth, C.J., 2005. Superantigen genes encoded by the egc cluster and SaPIbov are predominant among Staphylococcus aureus isolates from cows, goats, sheep, rabbits and poultry. Journal of Medical Microbiology 54, 401–411. Stephan, R., Annemüller, C., Hassan, A.A., Lämmler, Ch., 2001. Characterization of enterotoxigenic Staphylococcus aureus strains isolated from bovine mastitis in north - east Switzerland. Veterinary Microbiology 78, 373–382. Tollersrud, T., Kenny, K., Caugant, D.A., Lund, A., 2000. Characterization of isolates of Staphylococcus aureus from acute, chronic and subclinical mastitis in cows in Norway. Acta Pathologica, Microbiologica et Immunologica Scandinavica 108, 565–572. Zschöck, M., Kloppert, B., Wolter, W., Hamann, H.P., Lämmler, Ch., 2005. Pattern of enterotoxin genes seg, seh, sei and sej positive Staphylococcus aureus isolated from bovine mastitis. Veterinary Microbiology 108, 243–249.