Iranian Journal of Medical Microbiology

Sampling

This cross-sectional and descriptive-analytical study was performed on milk (227 samples), soft cheese (43 samples), and creams (21 samples) from traditional or non-pasteurized dairy supply centers in Hamadan, west of Iran (urban and rural areas), and industrial regions. Milk samples were collected from local unpasteurized dairy distributors in urban areas and industrial sites that passed all hygienic processes. Regarding cheese samples, half of them were collected from dairy product dispensaries with sources in villages, countries, and half-regular pasteurized cheese. The cream samples were taken from stores or rural houses prepared in a traditional and non-pasteurized manner. This study was approved by the ethics committee of Hamadan University of Medical Sciences (No: IRUMSHA. REC.1393.930222646).

Diagnosis and Identification of Brucella spp.

To detect Brucella spp. in samples, 100-200 mL milk was collected and centrifuged at 3000 rpm for 20 minutes, and the pellet was plated on Brucella agar medium supplemented with different antibiotics and incubated in a 10% CO₂ incubator at 35°C for at least seven days. One hundred grams of fresh cheese and cream were taken into sterilized tubes, homogenized in Brucella broth medium, and centrifuged at 3000 rpm for 20 minutes. The supernatant was discarded; then, 10 mL of Brucella broth medium was added to the pellet and incubated at 35°C for 24 hours. After the preliminary incubation by a sterile loop, inoculate the sample on a medium supplemented with different antibiotics. The following concentrations of antibiotics were added per liter of media to eliminate undesirable microorganisms: cycloheximide (100 mg), bacitracin (25000 units), polymyxin B sulfate (5000 units), vancomycin (20 mg), nalidixic acid (5 mg) and nystatin (100 000 units). The plates were then incubated in a 10% CO₂ incubator in a humidified atmosphere at 35°C for at least seven days. On culture, colonies appear small, convex, smooth, translucent, nonhemolytic, and slightly yellow and opalescent after at least 48 hours of incubation, and suspected colonies were identified by gram staining, CO₂ requirement, and biochemical tests such as H2S production, susceptibility to the aniline dyes thionine and basic fuchsin, urease, oxidase, and catalase (17, 18).

Data Analysis

The obtained data and values are entered into SPSS software 20 (SPSS Inc., Chicago, IL., USA), and the results were analyzed using descriptive statistics by performing Fischer's exact test.

The use of unpasteurized dairy products such as milk, cheese, cream, and whey is the source of some infections and diseases in humans. These diseases are classified as foodborne diseases and are mainly caused by bacteria such as Listeria monocytogenes, Salmonella spp., Escherichia coli, Staphylococcus aureus, Bacillus cereus, and Campylobacter spp. (17). In addition to being a zoonosis disease between humans and livestock, brucellosis is also transmitted through milk and dairy products (19, 20). In the present study, 12 samples (4.1%) of dairy products, including milk, cheese, and cream infected by Brucella spp. In a study by Bateni et al. in Zanjan, out of 299 milk and cheese samples examined by culture method, 5 samples (1.67%) were infected by Brucella (21). In other research reported by Movasagh et al. in the Parsabad-Moghan region of Ardabil province, Iran, the raw cattle milk contamination rate was 37.5% (22). In another study carried out by Akbarmehr et al. conducted on 1000 samples of cheese from Sarab city in Iran and its suburbs from 1999 to 2001, they found Brucella infection rate to be 2.2 %, which 0.7% and 1.5% of the cases were reported as B. melitensis and B. abortus respectively (23). Izadi et al. investigated the rate of Brucella infection in milk and dairy products using the Nested PCR technique in Tehran province, Iran. In 34 pasteurized milk samples, they reported 10 cases were PCR positive, from 28 pasteurized cheese samples, only 8 cases, from 23 traditional cheese 14 cases, and finally, from 33 samples of raw sheep milk, 19 cases were PCR positive (24). It seems that the differences between the results of the current study and previous ones are related to the methodology and the area of research (25, 26).
A study on 1028 brucellosis patients in Turkey showed that 63.6% had a history of raw dairy products and/or raw milk consumption. In Turkish studies, the level of reported human Brucella contaminations resulting from infected dairy products varies from 62.6% to 94.6%. Infected raw milk consumption was also accountable for 69% of brucellosis cases in Kuwait, 57.1% in Iran, and 63% in Oman. Recently in Qatar, an outbreak of B. melitensis and B. abortus infections has been accompanied by camel milk drinking. Eating unpasteurized raw milk and cheese has also been reported as an important source of human brucellosis in other Middle Eastern areas such as Saudi Arabia (27). Khalili et al. in Kerman city, Iran, reported that the rate of Brucella contamination in the delivery tank to one of the dairy factories by polymerase chain reaction (PCR) method was 3.8%, which is more than our findings (28). Movasagh et al. took 50 random samples of cow's milk and, by using the ELISA method (Milk ring test), reported that 10% of samples were positive for B. abortus (29). In a study by Silva et al. in Amazon areas on samples of cow's milk and cheese from buffalo milk, the Brucella infection rate was reported at 21% (14 samples out of 66), one of which was caused by the vaccine strain and in all of the other cases B. abortus isolated (30). In Egypt, the study of Gamal Wareth showed that from 215 bovine milk and milk products using indirect enzyme-linked immunosorbent assay (iELISA), anti-Brucella antibodies were detected in 34 samples (16%).
In contrast, the real-time PCR (RT-PCR) technique amplified Brucella-specific DNA from 17 milk samples (7.9%), which 16 of the RT-PCR-positive samples containing B. melitensis DNA; 1 RT-PCR-positive sample was identified as having B. abortus DNA (31). In another study in Turkey, fresh cheese from sheep milk, which was for sale in the central market of Cannakale, was investigated by Alper et al. by culture method, the rate of infection was found to be 0 % (32). Considering the high level of contamination in dairy products, it seems that using boiled milk to prepare cheese might be an effective way to combat the disease (33, 34).

 

This study showed that unpasteurized milk and dairy products are highly contaminated with Brucella spp. Therefore consumption of raw milk and unpasteurized dairy products is considered a serious public health hazard. The old belief about the usefulness of raw milk over pasteurized milk should be considered in light of current scientific information.
In general, education about the nature of the infection and how it is spread through raw milk and dairy products is essential to prevent infection or the spread of the disease.

 

The authors would like to acknowledge the Vice-chancellor of Research and Technology, Brucellosis Research Center of Hamadan University of Medical Sciences, Hamadan, Iran, and microbiology laboratory staff.
 

The Vice-chancellor of Research and Technology, Hamadan University of Medical Sciences, Hamadan, Iran, supported the study financially (Grant Number: 930222646).

 

Conflicts of Interest

The authors declare that they have no conflict of interests.