Comparative contamination of Listeria monocytogenes in traditional dairy products in Esfahan Province, Iran

Listeria monocytogenes, a primary human pathogen, has been found in different places in the environment of dairy, and the bacterium may survive for a long time in a dairy. L. monocytogenes is a major concern for the food industry, as it can cause listeriosis in humans. Listeriosis is one of the most important infections in Europe and the United States. It may cause fever, muscle aches and gastroenteritis; but does not usually cause septicaemia in healthy non-pregnant individuals. In pregnant women, it may cause abortion or neonatal death. From June 2010 to April 2012, a total of 420 samples were collected from different places in Esfahan Province. Samples were collected and analyzed based on the International Organization for Standardization using cultural method and biochemical test. Totally, in 57 samples (13.57%) of 420 dairy product samples L. monocytogenes was isolated. Based on biochemical observation, out of 210 raw milk samples, 42 (20%) were contaminated by Listeria monocytogenes. In traditional butter samples, 9 samples were contaminated by L. monocytogenes (12.85%); in traditional cheese samples, 5 (7.14%) samples were contaminated by L. monocytogenes and in traditional curd samples, 1 sample was contaminated by L. monocytogenes (1.43%). According to our finding, using traditional dairy products is dangerous, and it must be controlled by the ministry of health organization or other related organizations.


INTRODUCTION
Listeria monocytogenes is a rod shaped, gram positive, facultative anaerobic, non-spore forming bacterium with a low C+G content. L. monocytogenes is the primary human pathogen, although there have been rates of illnesses caused by Listeria selegeri, Listeria ivanovii and Listeria innocua (Jeyaletchumi et al., 2010). L. monocytogenes has been found in different places in the environment of dairy plants (Menendez et al., 1997), and the bacterium may survive for a long time in a dairy (Unnerstad et al., 1996). L. monocytogenes is a major concern for the food industry, as it can cause listeriosis in humans (Kathariou 2002). Listeriosis is one of the most important infections in Europe (European Food Safety Authority-European Centre for Disease Prevention and Control, 2007) and United States (Mead et al., 1999). Exposure to food borne L. monocytogenes may cause fever, muscle aches and gastroenteritis Riedo et al., 1994), (but does not usually cause septicaemia in healthy *Corresponding author. E-mail: ams_756@yahoo.com. Tel: (+98) 9132728458. Fax: (+98) 311-3601493.
Abbreviations: Listeria monocytogenes, Listeria innocua.  (Riedo et al., 1994). In pregnant women, it may cause abortion (Linnan et al., 1988;Riedo et al., 1994) or neonatal death (Linnan et al., 1988). Listeria is ubiquitous in dairy farms (Nightingale et al., 2004), and it is isolated from milk of cows (Jayarao and Henning 2001;Van Kessel et al., 2004;Arimi et al., 1197;Margolles and Reyes-Gavilan 1998;Unnerstad et al., 1996). L. monocytogenes in raw milk can be killed if heated at 71.7°C for 15 s (Bradshaw et al., 1985). L. monocytogenes has been detected in pasteurized whole milk, non-fat milk and chocolate milk produced in the United States (Frye and Donnelly 2005;Jayarao et al., 2006). The objective of this study was to determine contamination rate of Listeria in 420 different dairy product samples using cultural method and biochemical tests.  . This information confirms that there is a significant difference between contaminations of raw milk and dairy products. In addition, it is seen that there is no significant difference between various kinds of raw milk samples. There are significant differences between various kinds of dairy products: each three kinds of dairy products have significant difference between each other.

DISCUSSION
Human infections primarily result from eating contaminated food and may lead to serious and potentially life-threatening listeriosis (EI-Malek et al., 2010). Listeriosis has been recognized as one of the emerging zoonotic diseases during the last two decades and is contracted mainly from the consumption of contaminated foods and food products (Farber, 2000;Low and Donachie, 1997). Increasing evidence suggests that substantial portions of cases of human listeriosis are attributable to the food borne transmission of L. monocytogenes (Low and Donachie, 1997). According to preview studies, milk was contaminated by Listeria spp., especially L. monocytogenes in different rates; for example, 23% of 172 samples contain Listeria spp. in which L. monocytogenes was in 19.7% (Latorre et al., 2009). L. monocytogenes was isolated in 4.6% and 6.5% of bulk tank milk samples (Jayarao and Henning 2001;Van Kessel et al., 2004) and also found in 1.0%. The incidence of L. monocytogenes in the dairy silo milk was 19.6% (Waak et al., 2002); also incidence of L. monocytogenes was reported as 33.3% (54.0% for Listeria spp.), by Harvey and Gilmour (1992). Our result is similar to that of Waak et al. (2002), who confirmed that prevalence of L. monocytogenes is high in raw milk samples. This information is sufficient to warn ranchers about their farming. The difference between our finding in raw milk samples and others may be due to method of identification, season of sampling, source of food, geographic location, kinds of media employed, cross contamination and hygiene during milking. Cross contamination and hygiene during milking means that workers during milking have to clean the teats carefully so that the feces attached to the teats do not transfer to the milk and milking machine. There are some studies which showed Listeria spp. in fecal sample that may infest milk, leading to septicaemia. For example, Lattore et al. (2009) showed that 25% of fecal samples were infested by Listeria spp. and in 7.1% samples, L. monocytogenes was isolated. In addition, they reported that approximately the source of infestation is environmental and fecal. Arimi et al. (1997) stated that diversity of Listeria ribotypes is isolated from different farm and dairy-related environments. They suggested that the raw milk is contaminated by numerous Listeria ribotypes endemic to the farm environment. Our findings show varied range of contaminations in traditional dairy products from 1.43 to 12.85%. This difference may be due to their nature and processing. It means that curd samples enriched with salt and low moisture have lowest contamination rate between different products. This product is commonly used by women, because it is rich in calcium and other mineral elements. According to our finding, widespread usage of traditional curd of Iran cannot be a serious problem, but we suggest that pregnant women should the commercial type because it is prepared in a hygienic condition; and there is not any literature about contamination of commercial curd. This obtained information confirms previous study of Mojtahedi et al. (2004) on curd samples, where they mentioned there was no contamination of curd sample. Perhaps the little difference between our results and theirs is the size of the samples.
In this study, we isolated 12.85% of contamination in traditional butter samples. This almost high prevalence is due lack of heating during processing stages or due to contamination of instruments used for L. monocytogenes. According to Dole et al. (1987), L. monocytogenes was isolated from milk heated at 72.2°C for 16.4 s. The organism was not detected in the few trials of milk heated at 76.4 to 77.8°C for 15.4 s. In another study, Beckers et al. (1987) found out that L. monocytogenes inoculated at a level of 1.8 × 10 4 /ml did not survive heating at 67°C for 20 s or more. This information and our finding showed that heating in a good manner can reduce the contamination rate of L. monocytogenes, because in the traditional processing of dairy products, heating is not in the main stage. Beckers et al. (1987) stated that soft cheese samples (10.14 5%) were contaminated by L. monocytogenes. In other studies, Mojtahedi et al. (2004) isolated 2.5% and Delgado da Silva et al. (1998) recovered L. monocytogenes from seven out of 17 samples (41%) of cheese made from raw milk and from one of 33 samples (3%) of cheese made from pasteurized milk. In another example, Carvalho et al. (2007) recovered L. monocytogenes from three out of 93 samples (3%) of cheese made with pasteurized milk. However, according to our findings, L. monocytogenes was isolated in five out of 70 samples (7.14%).
Traditional cheese was prepared from raw milk, so we expected more contamination rate of L. monocytogenes in cheese samples. As a result, processing stages and storing of traditional cheese in Iran need much salty solution, which may lead to low prevalence than expected. It is noteworthy that contamination in cheese prepared from raw milk is almost equal in different studies.
According to our finding, using of traditional dairy products is dangerous and it must be controlled by Ministry of Health Organization or other related organizations.