Behavior of Brucella abortus and Brucella melitensis in Raw Meatball

In the present study, we aimed to determine the survival and reproducibility of the said pathogenic microorganisms during the storage period (24 h) of raw meatball samples contaminated with reference strains of Brucella abortus and Brucella melitensis at levels of 10 and 10 cfu/g. Brucella abortus (NCTC 11363) and Brucella melitensis (NCTC 10094) strains were incubated in sterile full-cream milk with 12 % fat at 37°C for 48 hours under a 6% CO2 aerobic atmosphere. Subsequent to the incubation, culture counts were performed on Brucella Agar Base using tryptose soy broth. As a result during our analysis at the 12th hour of the storage, it was noted that while pH remained 5.2, aw 0.96 and moisture %52.16, population of B. abortus in-group A2 rose from 10 cfu/gr to 10 cfu/gr (P<0.05). At hour 24, pH was found 5.1, aw 0.92 and moisture % 49.07 whereas population of B. abortus decreased to 10 cfu/gr in-group A1 and to 10 cfu/gr in-group A2 (P<0.05). In-group B1, there was no change in the number of B. melitensis. It was also observed at hour 24 population of B. melitensis in-group B2 increased to 10 cfu/gr (P<0.05).


Introduction
Çiğ köfte (raw meatball) is a traditional meat dish/delicacy that is usually made and served as an appetizer in Turkey, particularly in Southeastern Anatolia, on special occasions and at communal events (Yıldırım et al, 2005).Since no standards exist for the preparation of çiğ köfte, the ingredients and amount of content vary depending on preference.It is a meat product that doesn't undergo any thermal treatment and is consumed raw.It is produced by kneading finely ground lean mutton or beef, with a mixture of bulgur (fine wheat groats), onion, garlic, paste, parsley, and spices (powdered isot, red pepper, black pepper, cinnamon, clove, cumin, mint) with water or ice (Uzunlu & Yıldırım, 2003;Yıldırım et al, 2005).
Studies conducted in Turkey on raw meatballs (Sagun et al., 1997a;Kuplulu & Sarımehmetoglu, 2003;Cetin et al., 2008), have revealed that microbiological quality of the food appears hardly acceptable for human consumption and that it poses a significant threat to public health.
Brucellosis is a zoonotic infection, which has spread across many countries and remains endemic in developed ones, too (WHO, 2006).It is reported by the World Health Organisation (WHO) that an estimated 500,000 new human cases occur annually worldwide (Pappas et al., 2006).Brucellosis is endemic almost in all regions of the world, including the Mediterranean countries such as Spain, Portugal, Southern France, Italy, Greece, Turkey; North Africa, the Near East, India, Mexico, and Central and South America (Yüce & Çavuş, 2006).For humans, the contraction of the disease arises from intake of raw or unpasteurized infected milk or dairy products.However inhalation of contaminated dust, and contact with infected carcasses, and uterine contents and discharges (Chahota et al., 2003), and via consumption of meat and meat products (Robinson et al., 2000).
In the present study, we aimed to determine the survival and reproducibility of the said pathogenic microorganisms during the storage period (24 h) of raw meatball samples contaminated with reference strains of Brucella abortus (NCTC 11363) and Brucella melitensis (NCTC 10094) at levels of 10 4 and 10 6 CFU/g, and to discover whether they pose a threat to public health or not.

Preparation of raw meatballs
Raw Meatballs were made by mixing and kneading ground beef and bulghur, each ingredient in equal amounts of 2 kgs, powdered cumin, red pepper, black pepper, garlic, onion, parsley, tomato paste, and salt, based on the method as prescribed by Durmaz et al. (2007).The mixture was hand shaped into small balls each weighting approximately 25 g and stored at 4 o C for 24 hours.All çiğ köfte samples were analyzed in duplicate after storage for 0, 6, 12 and 24 h.

Preparation and Inoculation of Test Strains
Brucella abortus (NCTC 11363) and Brucella melitensis (NCTC 10094) strains were obtained from Refik Saydam National Publich Health Agency and incubated in sterile full-cream milk with 12 % fat at 37°C for 48 hours under a 6% CO 2 aerobic atmosphere.Subsequent to the incubation, culture counts were performed on Farrell's Agar plates (Oxoid CM 169; Brucella Selective Supplement Oxoid SR 83) using tryptose soy broth as diluent in the range up to 10 -9.The amount derived from the initial solutions inoculated showed that it could contaminate 1 g of raw meatball at levels of 10 4 cfu/g and 10 6 cfu/g.After the samples were inspected for any presence of Brucella abortus and Brucella melitensis, they were contaminated with strains of B. abortus and B. melitensis at levels of 10 4 cfu/g and 10 6 cfu/g (Estrada et al., 2005).The prepared samples (A1: 10 6 cfu/g B. abortus; A2: 10 4 cfu/g B. abortus; B1: 10 6 cfu/g B. melitensis; B2: 10 4 cfu/g B. melitensis) were then stored at +4 o C for 24 hours.

Sampling and Preparation of Dilutions
Throughout the storage period, samples were taken from all raw meatballs at hours 0, 6, 12, and 24. 10 g specimens were weighed out into sterile Stomacher bags, and 90 ml of sterile peptone saline (% 0,85 NaCl + % 0,1 peptone) was added to each container.The samples were immediately homogenized in the stomacher (Interscience, UK) for 2 minutes and were diluted ten-fold.Following the homogenization, serial decimal dilutions were performed until reaching 10 -7 (Pichhardt, 1993).

Enumeration of Total Mesophilic Aerobic Bacteria and Lactic Acid Bacteria
The dilutions prepared were planted in Plate Count Agar (PCA, Oxoid, CM325 -at 32 o C for 48 h) to count the population of mesophilic aerobic bacteria, and in Man Rogasa Sharpe Agar (MRS, Oxoid, CM 361 -pH:5.7-at35 o C for 48 h, anaerobic) to count lactic acid bacteria (Pichhardt, 1993).

Enumeration of Brucella abortus ve Brucella melitensis
25 g samples were weighed out from each raw meatball specimen under aseptic conditions and placed into sterile stomacher bags.225 ml of BPW (Buffered Peptone Water, Oxoid CM0509) was added and homogenized.After being blended in the stomacher, the decimal dilutions obtained from homogenized samples were diluted ten-fold in BPW, being prepared until reaching 10 -9.The samples were plated onto Farrell's agar (Oxoid CM 169; Brucella Selective Supplement Oxoid SR 83) by means of spread plate technique.The petri dishes planted were subjected to incubation at 37°C for 48 hours under a % 6 CO 2 environment.At the end of the incubation, colonies with typical Brucella were enumerated and justified through agglutination tests (B.abortus antisera Difco 2871-47-7, B. melitensis antisera Difco 2889-47-7) (Estrada et al., 2005).

Chemical analysis
pH values of the sampled raw meatballs were measured by means of pH metre (InoLab pH 720 model, Germany), based on method as prescribed by Troller and Scott (1992).Determination of moisture was achieved by AOAC (1990), and water activity (aw) values were analyzed according to the method by Rodel et al. (1975).

Statistics analysis
The data obtained from two replications were analysed by ANOVA using the SPSS statistical package program and differences among the means were compared using Duncan's Multiple Range test.

Result
Survival and reproducibility of the pathogenic microorganisms in raw meatball samples (A1: 10 6 cfu/g B. abortus, A2: 10 4 cfu/g B. abortus, B1: 10 6 cfu/g B. melitensis, B2: 10 4 cfu/g B. melitensis) contaminated with reference strains of Brucella abortus (NCTC 11363) and Brucella melitensis (NCTC 10094) at levels of 10 4 and 10 6 CFU/g can be seen in Table 1.During our analysis at the 6th hour of the storage, no change was recorded in the values of pH 5.3, aw 0.98 and moisture 64.10% as well as in numbers of B. abortus and B. Melitensis in all four groups.However, at the 12th hour of the storage, it was noted that while pH remained 5.2, aw 0.96 and moisture 52.16%, population of B. abortus in-group A2 rose from 10 4 cfu/gr to 10 5 cfu/gr (P<0,05).At hour 24, pH was found 5.1, aw 0.92 and moisture 49.07%whereas population of B. abortus decreased to 10 5 cfu/gr in group A1 and to 10 4 cfu/gr in group A2.In group B1, there was no change in the number of B. melitensis.It was also observed at hour 24 that although values for pH (5.1), aw (0.92) and moisture (%49.07)dropped, population of B. melitensis in group B2 increased to 10 5 cfu/gr (P<0,05).

Discussion
In the present study, we aimed to determine the survival and reproducibility of the said pathogenic microorganisms during the storage period (24 h) of çiğ köfte samples contaminated with reference strains of Brucella abortus (NCTC 11363) and Brucella melitensis (NCTC 10094) at levels of 10 4 and 10 6 CFU/g, and to discover whether they pose a threat to public health or not.
Studies carried out have showed that 54.7% of total food poisoning cases are caused by the consumption of meat and meat products (Farber et al., 1989).A major epidemiological factor in contracting the disease lies in the fact that in certain societies people are in the habit of consuming raw meat, eg raw liver or other offal with spices.(Syrjamaki et al., 1984).Following slaughter, micro-organisms, which cause foodborne infection and intoxication in humans, are transmitted to the end product as a result of crosss-contamination during carcass boning/cutting, processing, packing, and storage, thereby posing a threat to consumer health.Researchers have reported that infected lymph nodes are particularly noted for their being the most likely source for endogenic contamination of ground meat (Erol, 1999a).Due to its texture and processing techniques, ground meat turns out to be the leading meat product suitable for microbial contamination (Sinell, 1992).
Under the present study, in our analysis on the sample raw meatballs at the 6th hour of the storage, the original values of pH 5.3, aw 0.98 and moisture % 64,10 showed no change.Also, numbers of B. abortus and B. Melitensis didn't alter in any of the four groups: A1, A2, B1 and B2.At hour 12 of the storage, however, it was noted that while pH remained 5.2, aw 0.96 and moisture 52.16%, population of B. abortus in group A2 rose from 10 4 cfu/gr to 10 5 cfu/gr.Our measurement at the 24th hour revealed pH 5.1, aw 0.92 and moisture % 49.07, whereas population of B. abortus decreased to 10 5 cfu/gr in group A1 and to 10 4 cfu/gr in that of A2.In group B1, no change was noted in the number of B. melitensis.It was also observed that although values for pH (5.1), aw (0.92) and moisture (49.07%) dropped, population of B. melitensis in group B2 increased to 10 5 cfu/gr.Optimum pH value for the reproduction of Brucella spp. is 6,6-7,4, with maximum 8.7 and minimum 5.8 (Frobisher, 1968).However, various studies have yielded results which exhibit that Brucella spp can somehow survive even at lower pH values in different foods.Robinson et al. (2000), reported that Brucella abortus managed to survive for 34 days in the milk set at pH 5.0-5.8 by means of lactic acid, and for 2 days at pH 3.9.In the study carried out by Estrada et al. (2005), sterile skim milk was inoculated with Brucella abortus at 10 5 cfu/g with a yoghurt starter culture of lactic acid bacteria, and was incubated at +4 o C. Their results demonstrated that after 10 days of storage at 4 degrees C, B. abortus was recovered in fermented milk at a level of 10 5 cfu/g , despite the low pH value below 4.0.Ozturk and Nazli (1996), have reported that they experimentally contaminated sheep cheese and cow's cheese stuffed into sheepskin bags with Brucella melitensis at 10 9 cfu/g , and that the pathogen decreased to 10 2 cfu/g in both cheese varieties on the 21st day of the maturation.However, Brucella melitensis couldn't be isolated at pH 5.0 in either samples on the 30th day of the maturation.Researchers have also documented that following contamination, Brucella spp.managed to survive for 14 days in chilled meat (Taşkın, 2007), and for several years in frozen tissues and organs (Altekruse et al., 1998).
This experimental study demonstrated that while levels of reference strains B. abortus and B. melitensis in raw meatball samples stored for 24 hours at +4 o C did not change significantly, the total population of mesophilic aerobic bacteria (10 7 cfu/g) increased (Table 1).When the literature was screened, no similar experimental study related to meat and meat products occurred.As to experimental studies on milk and dairy products, while Ozturk and Nazlı (1996) observed that brucellae in cheese were inhibited at pH 5.0, Robinson et al. (2000) and Estrada et al. (2005), found that they were not inhibited even at lower pH levels (3.9 and 4.0).In the present study, at 24-hour storage period, pH ranged between 5.3 -5.1, and B. abortus and B. melitensis were not inhibited at pH 5.1.The fact that B. abortus and B. melitensis survive for different time periods or is inhibited at different pH levels may depend on products' composition; amount of moisture, protein and fat content; their texture; and storage period and conditions.
Çiğ köfte is a traditional and popular meat dish/delicacy throughout Turkey; however, since it is consumed raw, it gives rise to the spread of various pathogenic bacteria as well as Brucella outbreaks and poses a major threat to public health.Indeed, it has been documented by different investigators in Turkey that sanitary quality of ground meat, which is the basic ingredient for making çiğ , köfte (Tekinşen et al., 1980;Yetim, 1985;Akın & Kaya, 1988;Sancak et al., 1993;Ciftcioglu & Ugur, 1992;Guven et al., 1997;Erol, 1999b;Sireli & Erol, 1999), as well as the spices used (Sagun et al., 1997b;Erol et al., 1999;Vural, 2004) is hazardous for public health.
Antimicrobial effect of spices added to çiğ köfte is not powerful enough to eliminate the risk of pathogenic microorganisms.Because the antibacterial effect of spices such as isot and black pepper used in the preparation of çiğ köfte is limited, population of microorganisms doesn't undergo any significant change under different temperatures and periods (Uzunlu & Yıldırım, 2003).Experimental studies have demonstrated that many pathogenic bacteria can sustain their survival and are not inhibited in çiğ köfte environments (Sagun et al., 2003;Uzunlu & Yıldırım, 2003;Sireli et al., 2008).Therefore, the folk belief that added spices will thoroughly kill the pathogenic microorganisms in raw meatballs remains only a myth.
Brucellosis remains a major public health issue in Turkey.Varying rates of Brucella prevalance among humans have been noted by researchers in our country (Durmaz et al., 1997;Altındiş, 2001;Sumer et al., 2003;Atmaca et al., 2004;Cetinkaya et al., 2005;Demirturk et al., 2008).In Turkey, brucellosis is a particularly common problem in dairy cattle (Gokcen and Eskiizmirliler, 1998;Iyisan et al., 2000;Solmaz et al., 2002;Ceylan et al., 2003) and in sheep (Gokcen & Eskiizmirliler, 1998;Muz et al., 1999;Iyisan et al., 2000;Ongor et al., 2001;Ceylan et al., 2003).It is unfortunate that sale and slaughtering of animals suffering from brucella still continues in abattoirs due to negligence and lack of proper inspection at livestock markets and slaughterhouses.Meat and ground meat obtained from such animals may end up at points of sale, posing a risk for public health.Fearing possible health risks, some consumers have, in recent years, turned to çiğ köfte completely made with bulghur as a replacement for ground meat.We suggest that that a more pro-active and pre-emptive policy should be adopted to eradicate brucella infections, which is especially widespread in dairy cattle and sheep in Turkey; that procedures and practices like HACCP and GMP should be rigorously implemented during the production phase of meat and meat products by following the "from farm to table" continuum; that all the community working in the field of animal husbandry should be trained; that alternative methods should be taken up for making this traditional meat product, such as preparing çiğ köfte without adding any ground meat, or exposing it to thermal treatment in a way that will not spoil its characteristics; that different methods, such as food irradiation techniques should be developed to enhance the microbiological quality of çiğ köfte; and that training courses and seminars should be organized for çiğ köfte producers so that awareness could be raised among them.WHO. (2006).Brucellosis in humans and animals.WHO Press, World Health Organization. Geneva: Switzerland. Yetim, H. (1985).Erzurum piyasasında tüketime sunulan sığır kıymalarının bazı saprofit ve bir kısım patojen bakteriler yönünden incelenmesi, Atatürk Üniversitesi, Fen Bilimleri Enstitüsü, Yüksek Lisans Tezi.Erzurum.Yıldırım, İ., Uzunlu, S., & Topuz, A. (2005).Effect of gamma irradiation on some principle microbiological and chemical quality parameters of raw Turkish meat ball.Food Control, 16, 363-367. http://dx.doi.org/10.1016/j.foodcont.2004.04.001. Yüce, A., & Çavuş, S.A. (2006).Türkiye'de Bruselloz: Genel Bakış.Klinik Dergisi, 19, 87-97.