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Isolation and identification of Brucella spp. in ice cream
Food Control 15 (2004) 511–514 www.elsevier.com/locate/foodcont
Isolation and identification of Brucella spp. in ice cream O. Kuplulu *, B. Sarimehmetoglu Faculty of Veterinary Medicine, Department of Food Hygiene and Technology, Ankara University, Ankara, Turkey Received 16 March 2003; received in revised form 1 August 2003; accepted 5 August 2003
Abstract In this study, a total of 217 samples consisting of 80 vanilla flavoured, 75 chocolate flavoured and 62 fruit flavoured ice cream, were examined for their level of Brucella spp. contamination and species distribution. Whilst the isolation and identification of Brucella spp. in the ice cream samples was done according to the method described by Farrell, the level of Brucella spp. was determined by the MPN technique. Whereas no Brucella spp. was isolated in the chocolate and fruit flavoured ice cream samples taken for analysis, B. abortus was isolated in 6.25% of the vanilla flavoured ice cream samples. B. abortus was found to be present in ice cream samples at the level of 5.4 · 102 MPN/g. In conclusion, the results of this study have established ice cream produced in Turkey as a public health threat in view of its potential risk of brucellosis. It is in view of this fact that we deem it necessary that, to curb the spread of brucellosis in this community, establishing critical control measures in ice cream production units and provision of the necessary hygienic measures with regular supervision be made mandatory. 2003 Published by Elsevier Ltd. Keywords: Brucella spp.; Brucella abortus; Ice cream
1. Introduction Brucellosis is an important zoonotic disease that has serious implications on the health of humans, as well as enormous economic losses in animals. Despite eradication efforts, to date, the disease still maintains its importance all over the world, especially in the Mediterranean countries (Anonymous, 1997; Manes, 1984; Roux, 1989). The causative organism of the disease was first isolated from the spleen of a soldier by Sir David Bruce in 1887 in an island in Malta. Among the Brucella genus are six types namely; B. abortus, B. melitensis, B. suis, B. canis, B. ovis and B. neotomae. B. abortus occurs in cattle, B. melitensis in sheep and goats, B. suis in swine, B. canis in dogs, B. ovis in sheep, B. neotomae causes abortion and/or reproductive diseases in desert rats. In humans, the most pathogenic type is B. melitensis followed in descending order by B. suis and B. abortus. Brucellosis, also known as Malta fever or Mediterranean fever, causes undulating fever in hu-
*
Corresponding author. Tel.: +90-312-317-0315/0355; fax: +90-312317-0010. E-mail address: [email protected] (O. Kuplulu). 0956-7135/$ - see front matter 2003 Published by Elsevier Ltd. doi:10.1016/j.foodcont.2003.08.002
mans, whereas in animals it causes abortus, infertility, mastitis, orchitis and arthritis (Bisping & Amtsberg, 1988; Garin-Bastuji & Verger, 1994; Mikolich & Boyce, 1990). Cattle, sheep and goats with brucellosis after abortion or normal delivery continue to shed the organism in their milk. Brucella spp. found in milk is inactivated by pasteurisation with heat. For this reason, milk and milk products produced with milk pasteurized in conformity with the heat-time parameters have no risk of brucellosis (Bryan, 1983; Eyles, 1992). Transmission of Brucella spp. in humans is by consumption of contaminated foods, direct contact with animals or fresh animal products, or through inhalation. The risk of transmission through direct contact with contaminated material is highest among veterinarians, farmers, personnel involved in the production of milk and in abattoir workers. Transmission through inhalation, however, is more frequent among laboratory workers. The risk of transmission in people not in these high risk group occupations is usually by the alimentary route. In this connection, consumption of contaminated fresh milk or dairy products leads to transmission of the disease among humans. Epidemiological studies conducted have reported fresh milk and milk products as the cause of brucellosis. Brucellosis is also defined as a
512
O. Kuplulu, B. Sarimehmetoglu / Food Control 15 (2004) 511–514
zoonotic infection of milk origin (Garin-Bastuji & Verger, 1994; Herr & Roux, 1981; Jawetz, Melnick, & Adelberg, 1987). In addition to dairy products like cheese made from fresh milk, cream, and butter, ice cream has also been reported to carry the risk of brucellosis. When the fresh materials are contaminated with Brucella spp., the end product will inevitably carry this risk if regulations on ice cream production technology are not followed. In the ice cream technology, mixing of the fresh material is done at 50–60 C after which it is pasteurized (at 70 C for 10–30 min or at 83–85 C for 15 min). After the pasteurization procedure, flavours and colouring agents like fruits and hazelnut are added to the ice cream mixture which has been cooled at 4 C and left for aging. Following the freezing procedure at )5 C the packaged ice cream products are then left to harden at )34 C and stored at )18 C. The use of unpasteurized milk in the production of ice cream or non-compliance with pasteurization procedures during production, production under unhygienic conditions, lack of proper cleansing and disinfection of tools and equipment, and other factors like insufficient freezing and long storage times that allow the thawed ice cream to refreeze for long periods, lead to contamination with the pathogen and hence an increase in the microbial load. The contamination of ice cream by Brucella spp., however, is reported to be solely dependent on the use of contaminated fresh milk (Anonymous, 2002a; Bryan, 1983; Eyles, 1992; Keren, 1999; Tekinsßen, 1997). It has been reported that whereas ice cream manufactured by the industrial type large production plants and packaged under adequate hygienic conditions do not constitute any risk of infections and intoxications, those produced by small scale manufacturers usually made under poor hygienic conditions may be contaminated with various food pathogens (Erg€ un & Cıvar, 1984). In a study based on samples from small scale manufacturers in Turkey (Akol & U gur, 1984; Erg€ un & Cıvar, 1984; Erol, K€ upl€ ul€ u, Sırıken, & C ß elik, 1998; Omurtag, Ceran, & Akın, 1977), it has been reported that the quality of hygienic condition under which the ice cream is produced is poor, and pathogenic microorganisms like Salmonella, coagulase positive staphylococci and E. coli may abound, apparently the result of the use of unpasteurized milk, non-adherence to proper hygienic conditions, as well as poor personal hygienic practices of the production personnel themselves. Considering the fact that most of the ice cream produced in Turkey is by the small scale producers who use fresh materials that are hygienically substandard, the magnitude of the risk of brucellosis that ice cream poses to the consumer cannot be underestimated. Review of the literature revealed no study on Brucella spp. in ice cream. This study was therefore conducted with the aim of determining whether or not consumption of ice cream
carries the risk of brucellosis in all age groups, especially among children.
2. Materials and methods 2.1. Materials In this study, a total of 217 ice cream samples consisting of 80 vanilla flavoured, 75 chocolate flavoured and 62 fruit flavoured samples obtained from various small scale producers within the Ankara district were used as the study material. The ice cream samples were transported to the laboratory in ice bags under aseptic conditions and analysed the same day. 2.2. Methods 2.2.1. Isolation and identification of Brucella spp., from ice cream samples Isolation and identification methods were as described by Farrell (1974). According to this procedure, 10 g of each ice cream sample was taken and placed in a stomacher bag to which 90 ml of Farrell’s broth was added and homogenized with a stomacher (Lab. Lemco 400) for 1–2 min. Further dilutions were prepared from the ice cream samples by adding 1–9 ml of the Farrell’s broth. One of the dilutions, prepared in duplicate was incubated under aerobic conditions whilst the other series was incubated under 10% CO2 at 37 C for 5 days. The Farrell’s broth was prepared by adding 5% inactivated horse serum (Oxoid SR35), 10 g/l glucose (Merck 1.08346.1000) and 1 vial/500 ml of Brucella selective supplement (Oxoid SR83) to the Brucella broth (BBL 4311088). After incubation, 0.1 ml aliquot of each enriched homogenate was inoculated by the spread plating technique onto the Farrell’s agar. The plates from the aerobically enriched homogenate were incubated at 37 C for 5 days under aerobic conditions whereas those from the CO2 medium were incubated for the same time and temperature under 10% CO2 conditions. The Farrell’s agar was prepared by adding 5% inactivated horse serum, 1 vial/500 ml of Brucella selective supplement to the Brucella Medium Base (Oxoid CM169). After incubation, colonies of suspect Brucella spp. were identified by their characteristic 1–2 mm diameter convex, entirely round edges, with translucent and pale yellow appearance and the colonies Gram stained, and examined for H2 S formation, urease, and catalase activities, and agglutination of antiserum using slide agglutination tests (Brucella abortus antisera Difco 2871-47-7, B. melitensis antisera Difco 2889-47-7) and sensitivity staining. The 3tube MPN technique was used to determine the level of contamination of Brucella spp. in samples found positive. In this respect, four ice cream samples (1 g each) were taken and placed in 9 ml of the Farrel’s broth. One
O. Kuplulu, B. Sarimehmetoglu / Food Control 15 (2004) 511–514
the four tubes were used for dilution (Preeler, Houghtby, & Rainosek, 1992).
3. Results and discussion Of the 75 chocolate flavoured and the 62 fruit flavoured ice cream samples analysed, no Brucella spp. was isolated. In contrast, Brucella spp. were isolated in 5 (6.25%) of the 80 vanilla flavoured ice cream samples. All the isolates of Brucella spp. in the five samples were identified as Brucella abortus. The B. abortus level in the positive samples is presented as MPN/g in Table 1. Between 1.1 · 102 and 2.3 · 103 MPN/g, with a mean of 5.4 · 102 MPN/g, of B. abortus was isolated from the positive samples. Because no studies on the incidence of Brucella spp. are available we could not compare the findings of our study. In an experimental study B. abortus has been reported to remain viable in ice cream for up to one month (Garin-Bastuji & Verger, 1994). Under this condition, the use of Brucella spp. contaminated fresh milk or insufficiently pasteurised milk in production makes ice cream a risk for brucellosis. Brucellosis has been reported after consumption of ice cream (Anonymous, 2002b). In this study, the mean B. abortus level was determined to be 5.4 · 102 MPN/g in the vanilla flavoured ice cream samples. There is no definite knowledge regarding the minimum level of Brucella that is needed to cause infection. Nevertheless, for brucellosis to occur through the inhalation route, it has been reported that between 10 and 100 bacteria need to be inhaled. Thus, a minimal infective dose has been established, as seen for other infectious pathogens. Therefore the presence of even 1 cfu/g or ml in foods is of considerable importance (Anonymous, 2002a; Jawetz et al., 1987). The incidence of B. abortus and B. melitensis in cattle, sheep and goats in Turkey has been reported to be rather high (Anonymous, 1997). In studies conducted, milk and dairy products in Turkey have been found to carry the risk of brucellosis. Akbulut and Kavas (1993) in a study conducted on street milk samples in the dis_ trict of Izmir found 95 out of 101 milk samples to be contaminated with Brucella spp., and reported that samples in the months of summer carry an even much Table 1 The MPN levels of Brucella abortus in positive ice cream samples Positive samples (n ¼ 5)
Brucella abortus (MPN/g)
A B C D E
2.3 · 103 1.1 · 102 9.3 · 102 1.1 · 102 1.5 · 103
513
higher risk of brucellosis. Ilhan, Keskin, Sareyy€ upo glu, €, and Akan (1999) in their study on a cattle ranch, K€ okcßu have reported the isolation of B. abortus in 4 (8%) of 50 fresh milk samples examined. There are several studies also investigating Brucella spp. in cheese. Mert (1984), in a study on 150 Turkish white cheese produced from fresh milk in Ankara, showed 19.3% to be contaminated with Brucella spp. and that in the positive samples whilst 90% of the isolates were B. melitensis, 10% were B. abortus. Tuncßbilek (1992) in his analysis of 100 Turkish white cheese samples in Ankara, found three of the four samples produced from fresh milk to be contaminated with B. melitensis and the other with B. abortus. Sancak, Boynukara, and Yardımcı (1993) in their analysis of 40 otlu cheese (local brand of cheese) samples found 7 of them to be contaminated with Brucella spp.; six B. melitensis and one B. abortus. These studies demonstrate clearly the contamination of fresh milk used in the production of cheese with Brucella spp. The isolation of B. abortus in 6.25% of the vanilla flavoured ice cream samples in this study reveals the potential risk that ice cream carries for brucellosis. To maintain public health safety, therefore, we are of the opinion that in addition to pasteurizing the fresh milk used in the production of ice cream, maintenance of good hygienic standards, strict adherence to the Hazard Analysis Critical Control Point (HACCP) system protocol, as well as provision of adequate hygienic conditions is mandatory.
References Anonymous (1997). Brucellosis: an Overview. 1st International Conference on Emerging Zoonoses Jerusalem, Israel. Emerging Infectious Disease, 3(2), 213–221. Anonymous (2002a). Brucellosis. Available: http://www.nbc-med.org/ SiteContent/MedRef/FieldManualsmedman/Brucellosis.htm. Anonymous (2002b). Public Health Fact sheet-Brucellosis. Available: http://www.state.ma.us/dph/cdc/factsheets/brucellosis.htm. _ Akbulut, N., & Kavas, G. (1993). Izmir ilinde satılan sokak s€ utlerinin € niversitesi €zellikleri u €zerinde bir arasßtırma. Ege U mikrobiyolojik o Ziraat Fak€ultesi Dergisi, 30(1–2), 89–96. _ Akol, N., & ve Ugur, M. (1984). Istanbul piyasasında satılmakta olan €zerine arasßtırmalar. kaymaklı dondurmaların hijyenik kalitesi u _ € niversitesi Veteriner Fak€ultesi Dergisi, 10(1), 53–59. Istanbul U Bisping, W., & Amtsberg, G. (1988). Colour atlas for the diagnosis of bacterial pathogens in animals. Hamburg: Paul Parey Scientific Publishers (pp. 246–262). Bryan, F. L. (1983). Epidemiology of milkborne diseases. Journal of Food Protection, 46(7), 637–649. € & ve Cıvar, E. (1984). Zur bakteriologischen qualitat von in Erg€ un, O., _ Istanbul angelbotenen einheimischen und importierten Speisseeis (Fertiggepackle und konditories). Veterinarium, 3(1), 29–31. € Sırıken, B., & C _ K€ Erol, I., upl€ ul€ u, O., ß elik, T. H. (1998). Ankara’daki ßcesßitli pastanelere ait dondurmaların mikrobiyolojik kalitelerinin belirlenmesi. DOGA, Turkish Journal of Veterinary and Animal Science, 22(4), 345–352. Eyles, M. (1992). Fresh milk cheese: the issues. The Australian Journal of Dairy Technology, 47, 102–105.
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Farrel, I. D. (1974). The development of a new selective medium for the isolation of Brucella melitensis from contaminated sources. Research Veterinary Science, 16, 280–286. Garin-Bastuji, B., & Verger, J. M. (1994). Brucella abortus and Brucella melitensis. The Significance of Pathogenic Microorganism in Fresh milk. International Dairy Federation, No: 9405. Herr, S., & Roux, D. (1981). The efficacy of bacteriological procedures for the isolation of Brucella abortus from abattoir material. Journal of Veterinary Research, 48, 7–12. €, L., & Akan, M. Ilhan, Z., Keskin, O., Sareyy€ upoglu, B., K€ okcßu (1999). Bir sı gırcılık isßletmesinde Brucella salgını. Ankara Universitesi. Veteriner Fak€ ultesi Dergisi, 46(2–3), 257–262. Jawetz, E., Melnick, J. L., & Adelberg, E. A. (1987). Review of medical microbiology. California: Appleton & Lange (pp. 265–267). Keren, G. (1999). Brucellosis: clinical manifestations and treatment. Available: http://www.fao.org/ag/aga/agah/id/brunet/public_sub2_ pl.html. Manes, G. (1984). Epidemiological stuation of brucellosis in the medirranean countries. Development of Biological Standards, 56, 739–747. Mert, A. (1984). Ankara y€oresinde pazarlanan taze beyaz peynirlerde Brucella’ların varlı gı €uzerinde arasßtırmalar. Doktora Tezi, Ankara € Universitesi. Sa glıkBilimleri Enstit€ us€ u.
Mikolich, D. J., & Boyce, J. M. (1990). Brucella species. In G. L. Mandell, R. G. Douglas, & J. E. Bennett (Eds.), Principle and practice of infectious diseases (pp. 1735–1742). London: Churchill Livingstone. Omurtag, A. C., Ceran, G., & Akın, A. (1977). Denizli ilinde satılan €zerinde arasßtırmalar. kaymaklı dondurmaların hijyenik kaliteleri u T€urk Veteriner Hekimli gi Derne gi Dergisi, 47(1), 40–47. Preeler, J. T., Houghtby, G. A., & Rainosek, A. P. (1992). The most probable number technique. In C. Vanderzant, & D. F. Splittistoesser (Eds.), Compendium of methods for the microbiological examination of foods (pp. 106–117). Washington: American public Health Association. € nemi. Roux, J. (1989). Brucellosis hastalıgının halk saglıgı y€ on€ unden o In Uluslararasi Brucella sempozyumu. Hayvan hastalıkları Merkez Arasßtırma Enstit€ us€ u Yayınları, No: 9, Pendik. Istanbul. Sancak, Y. C., Boynukara, B., & Yardımcı, H. (1993). The existence and lifetime of Brucella species in Van herby cheese. Veterinarium, 4, 1–3. € unleri Teknolojisi (pp. 257–316). Selcßuk Tekinsßen, O. C. (1997). S€ut Ur€ € € Universitesi Veteriner Fak€ ultesi Yayın Unitesi, Konya. Tucßbilek, M. (1992). Ankara piyasasında satılan taze beyaz peynirlerin brucellosis riski y€ on€ unden incelenmesi. Y€uksek Lisans Tezi. Ankara € Universiresi Saglık Bilimleri Enstit€ us€ u.
Isolation and identification of Brucella spp. in ice cream O. Kuplulu *, B. Sarimehmetoglu Faculty of Veterinary Medicine, Department of Food Hygiene and Technology, Ankara University, Ankara, Turkey Received 16 March 2003; received in revised form 1 August 2003; accepted 5 August 2003
Abstract In this study, a total of 217 samples consisting of 80 vanilla flavoured, 75 chocolate flavoured and 62 fruit flavoured ice cream, were examined for their level of Brucella spp. contamination and species distribution. Whilst the isolation and identification of Brucella spp. in the ice cream samples was done according to the method described by Farrell, the level of Brucella spp. was determined by the MPN technique. Whereas no Brucella spp. was isolated in the chocolate and fruit flavoured ice cream samples taken for analysis, B. abortus was isolated in 6.25% of the vanilla flavoured ice cream samples. B. abortus was found to be present in ice cream samples at the level of 5.4 · 102 MPN/g. In conclusion, the results of this study have established ice cream produced in Turkey as a public health threat in view of its potential risk of brucellosis. It is in view of this fact that we deem it necessary that, to curb the spread of brucellosis in this community, establishing critical control measures in ice cream production units and provision of the necessary hygienic measures with regular supervision be made mandatory. 2003 Published by Elsevier Ltd. Keywords: Brucella spp.; Brucella abortus; Ice cream
1. Introduction Brucellosis is an important zoonotic disease that has serious implications on the health of humans, as well as enormous economic losses in animals. Despite eradication efforts, to date, the disease still maintains its importance all over the world, especially in the Mediterranean countries (Anonymous, 1997; Manes, 1984; Roux, 1989). The causative organism of the disease was first isolated from the spleen of a soldier by Sir David Bruce in 1887 in an island in Malta. Among the Brucella genus are six types namely; B. abortus, B. melitensis, B. suis, B. canis, B. ovis and B. neotomae. B. abortus occurs in cattle, B. melitensis in sheep and goats, B. suis in swine, B. canis in dogs, B. ovis in sheep, B. neotomae causes abortion and/or reproductive diseases in desert rats. In humans, the most pathogenic type is B. melitensis followed in descending order by B. suis and B. abortus. Brucellosis, also known as Malta fever or Mediterranean fever, causes undulating fever in hu-
*
Corresponding author. Tel.: +90-312-317-0315/0355; fax: +90-312317-0010. E-mail address: [email protected] (O. Kuplulu). 0956-7135/$ - see front matter 2003 Published by Elsevier Ltd. doi:10.1016/j.foodcont.2003.08.002
mans, whereas in animals it causes abortus, infertility, mastitis, orchitis and arthritis (Bisping & Amtsberg, 1988; Garin-Bastuji & Verger, 1994; Mikolich & Boyce, 1990). Cattle, sheep and goats with brucellosis after abortion or normal delivery continue to shed the organism in their milk. Brucella spp. found in milk is inactivated by pasteurisation with heat. For this reason, milk and milk products produced with milk pasteurized in conformity with the heat-time parameters have no risk of brucellosis (Bryan, 1983; Eyles, 1992). Transmission of Brucella spp. in humans is by consumption of contaminated foods, direct contact with animals or fresh animal products, or through inhalation. The risk of transmission through direct contact with contaminated material is highest among veterinarians, farmers, personnel involved in the production of milk and in abattoir workers. Transmission through inhalation, however, is more frequent among laboratory workers. The risk of transmission in people not in these high risk group occupations is usually by the alimentary route. In this connection, consumption of contaminated fresh milk or dairy products leads to transmission of the disease among humans. Epidemiological studies conducted have reported fresh milk and milk products as the cause of brucellosis. Brucellosis is also defined as a
512
O. Kuplulu, B. Sarimehmetoglu / Food Control 15 (2004) 511–514
zoonotic infection of milk origin (Garin-Bastuji & Verger, 1994; Herr & Roux, 1981; Jawetz, Melnick, & Adelberg, 1987). In addition to dairy products like cheese made from fresh milk, cream, and butter, ice cream has also been reported to carry the risk of brucellosis. When the fresh materials are contaminated with Brucella spp., the end product will inevitably carry this risk if regulations on ice cream production technology are not followed. In the ice cream technology, mixing of the fresh material is done at 50–60 C after which it is pasteurized (at 70 C for 10–30 min or at 83–85 C for 15 min). After the pasteurization procedure, flavours and colouring agents like fruits and hazelnut are added to the ice cream mixture which has been cooled at 4 C and left for aging. Following the freezing procedure at )5 C the packaged ice cream products are then left to harden at )34 C and stored at )18 C. The use of unpasteurized milk in the production of ice cream or non-compliance with pasteurization procedures during production, production under unhygienic conditions, lack of proper cleansing and disinfection of tools and equipment, and other factors like insufficient freezing and long storage times that allow the thawed ice cream to refreeze for long periods, lead to contamination with the pathogen and hence an increase in the microbial load. The contamination of ice cream by Brucella spp., however, is reported to be solely dependent on the use of contaminated fresh milk (Anonymous, 2002a; Bryan, 1983; Eyles, 1992; Keren, 1999; Tekinsßen, 1997). It has been reported that whereas ice cream manufactured by the industrial type large production plants and packaged under adequate hygienic conditions do not constitute any risk of infections and intoxications, those produced by small scale manufacturers usually made under poor hygienic conditions may be contaminated with various food pathogens (Erg€ un & Cıvar, 1984). In a study based on samples from small scale manufacturers in Turkey (Akol & U gur, 1984; Erg€ un & Cıvar, 1984; Erol, K€ upl€ ul€ u, Sırıken, & C ß elik, 1998; Omurtag, Ceran, & Akın, 1977), it has been reported that the quality of hygienic condition under which the ice cream is produced is poor, and pathogenic microorganisms like Salmonella, coagulase positive staphylococci and E. coli may abound, apparently the result of the use of unpasteurized milk, non-adherence to proper hygienic conditions, as well as poor personal hygienic practices of the production personnel themselves. Considering the fact that most of the ice cream produced in Turkey is by the small scale producers who use fresh materials that are hygienically substandard, the magnitude of the risk of brucellosis that ice cream poses to the consumer cannot be underestimated. Review of the literature revealed no study on Brucella spp. in ice cream. This study was therefore conducted with the aim of determining whether or not consumption of ice cream
carries the risk of brucellosis in all age groups, especially among children.
2. Materials and methods 2.1. Materials In this study, a total of 217 ice cream samples consisting of 80 vanilla flavoured, 75 chocolate flavoured and 62 fruit flavoured samples obtained from various small scale producers within the Ankara district were used as the study material. The ice cream samples were transported to the laboratory in ice bags under aseptic conditions and analysed the same day. 2.2. Methods 2.2.1. Isolation and identification of Brucella spp., from ice cream samples Isolation and identification methods were as described by Farrell (1974). According to this procedure, 10 g of each ice cream sample was taken and placed in a stomacher bag to which 90 ml of Farrell’s broth was added and homogenized with a stomacher (Lab. Lemco 400) for 1–2 min. Further dilutions were prepared from the ice cream samples by adding 1–9 ml of the Farrell’s broth. One of the dilutions, prepared in duplicate was incubated under aerobic conditions whilst the other series was incubated under 10% CO2 at 37 C for 5 days. The Farrell’s broth was prepared by adding 5% inactivated horse serum (Oxoid SR35), 10 g/l glucose (Merck 1.08346.1000) and 1 vial/500 ml of Brucella selective supplement (Oxoid SR83) to the Brucella broth (BBL 4311088). After incubation, 0.1 ml aliquot of each enriched homogenate was inoculated by the spread plating technique onto the Farrell’s agar. The plates from the aerobically enriched homogenate were incubated at 37 C for 5 days under aerobic conditions whereas those from the CO2 medium were incubated for the same time and temperature under 10% CO2 conditions. The Farrell’s agar was prepared by adding 5% inactivated horse serum, 1 vial/500 ml of Brucella selective supplement to the Brucella Medium Base (Oxoid CM169). After incubation, colonies of suspect Brucella spp. were identified by their characteristic 1–2 mm diameter convex, entirely round edges, with translucent and pale yellow appearance and the colonies Gram stained, and examined for H2 S formation, urease, and catalase activities, and agglutination of antiserum using slide agglutination tests (Brucella abortus antisera Difco 2871-47-7, B. melitensis antisera Difco 2889-47-7) and sensitivity staining. The 3tube MPN technique was used to determine the level of contamination of Brucella spp. in samples found positive. In this respect, four ice cream samples (1 g each) were taken and placed in 9 ml of the Farrel’s broth. One
O. Kuplulu, B. Sarimehmetoglu / Food Control 15 (2004) 511–514
the four tubes were used for dilution (Preeler, Houghtby, & Rainosek, 1992).
3. Results and discussion Of the 75 chocolate flavoured and the 62 fruit flavoured ice cream samples analysed, no Brucella spp. was isolated. In contrast, Brucella spp. were isolated in 5 (6.25%) of the 80 vanilla flavoured ice cream samples. All the isolates of Brucella spp. in the five samples were identified as Brucella abortus. The B. abortus level in the positive samples is presented as MPN/g in Table 1. Between 1.1 · 102 and 2.3 · 103 MPN/g, with a mean of 5.4 · 102 MPN/g, of B. abortus was isolated from the positive samples. Because no studies on the incidence of Brucella spp. are available we could not compare the findings of our study. In an experimental study B. abortus has been reported to remain viable in ice cream for up to one month (Garin-Bastuji & Verger, 1994). Under this condition, the use of Brucella spp. contaminated fresh milk or insufficiently pasteurised milk in production makes ice cream a risk for brucellosis. Brucellosis has been reported after consumption of ice cream (Anonymous, 2002b). In this study, the mean B. abortus level was determined to be 5.4 · 102 MPN/g in the vanilla flavoured ice cream samples. There is no definite knowledge regarding the minimum level of Brucella that is needed to cause infection. Nevertheless, for brucellosis to occur through the inhalation route, it has been reported that between 10 and 100 bacteria need to be inhaled. Thus, a minimal infective dose has been established, as seen for other infectious pathogens. Therefore the presence of even 1 cfu/g or ml in foods is of considerable importance (Anonymous, 2002a; Jawetz et al., 1987). The incidence of B. abortus and B. melitensis in cattle, sheep and goats in Turkey has been reported to be rather high (Anonymous, 1997). In studies conducted, milk and dairy products in Turkey have been found to carry the risk of brucellosis. Akbulut and Kavas (1993) in a study conducted on street milk samples in the dis_ trict of Izmir found 95 out of 101 milk samples to be contaminated with Brucella spp., and reported that samples in the months of summer carry an even much Table 1 The MPN levels of Brucella abortus in positive ice cream samples Positive samples (n ¼ 5)
Brucella abortus (MPN/g)
A B C D E
2.3 · 103 1.1 · 102 9.3 · 102 1.1 · 102 1.5 · 103
513
higher risk of brucellosis. Ilhan, Keskin, Sareyy€ upo glu, €, and Akan (1999) in their study on a cattle ranch, K€ okcßu have reported the isolation of B. abortus in 4 (8%) of 50 fresh milk samples examined. There are several studies also investigating Brucella spp. in cheese. Mert (1984), in a study on 150 Turkish white cheese produced from fresh milk in Ankara, showed 19.3% to be contaminated with Brucella spp. and that in the positive samples whilst 90% of the isolates were B. melitensis, 10% were B. abortus. Tuncßbilek (1992) in his analysis of 100 Turkish white cheese samples in Ankara, found three of the four samples produced from fresh milk to be contaminated with B. melitensis and the other with B. abortus. Sancak, Boynukara, and Yardımcı (1993) in their analysis of 40 otlu cheese (local brand of cheese) samples found 7 of them to be contaminated with Brucella spp.; six B. melitensis and one B. abortus. These studies demonstrate clearly the contamination of fresh milk used in the production of cheese with Brucella spp. The isolation of B. abortus in 6.25% of the vanilla flavoured ice cream samples in this study reveals the potential risk that ice cream carries for brucellosis. To maintain public health safety, therefore, we are of the opinion that in addition to pasteurizing the fresh milk used in the production of ice cream, maintenance of good hygienic standards, strict adherence to the Hazard Analysis Critical Control Point (HACCP) system protocol, as well as provision of adequate hygienic conditions is mandatory.
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