A total of 100 raw milk samples were obtained using stratified random sampling method from different milk collection centers (MCC) in northeast of Iran, from January 2017 until July 2017. Milk thoroughly mixed before sampling and then 50 ml of milk was obtained in sterilized tubes and transport to the laboratory in the ice box, immediately. For enrichment, 10 ml of each milk sample were aseptically transferred to 90 ml of Peptone Sorbitol Broth (PSB) (Sigma-Aldrich, Germany) mixed for 5 min and incubated for 48 hours at 25 °C in a shaker incubator. Amount of 0.5 ml of each enriched sample mixed well with 4.5 ml of KOH 0.25% and then streaked onto CIN agar plates (Merck, Darmstadt, Germany) supplemented with Cefsulodin-irgasan-novobiocin antibiotics (Merck, Darmstadt, Germany) [14]. Agar plates were incubated at 30 °C for 24–48 h. Small (1–2 mm diameter) colonies having deep red center with sharp border surrounded by clear colorless zone with entire edge in CIN agar plates were selected. Gram-negative bacilli with oxidase negative, catalase and urease-positive tests were presumptively regarded as Yersinia spp and selected for DNA extraction.
The DNA extracted from purified presumptive colonies using the boiling method [15]. PCR assay for amplification of 16s rRNA was carried out in final volume of 20 μl, containing 1 μl (10 picomol) of forward (5´-AATACCGCATAACGTCTTCG-3´) and reverse (5´-CTTCTTCTGCGAGTAACGTC-3´) primer (Macrogen, South Korea), 2 μl DNA template, 10 μl of master mix (Ampliqon, Denmark) and 6 μl Nuclease-free deionized distilled water. Thermal cycler program is presented in Table 1. Yersinia enterocolitica (ATCC 9610) and nuclease-free deionized water were used as the positive and negative control, respectively. PCR products were separated on a 1.5% agarose gel which was pre-stained by green viewer and photo documented under UV illumination.
For determination the biotype of isolates, esculin hydrolysis, indole production and lipase activity were investigated. Also, tests for fermentation of salicin, trehalose, sorbose and xylose were carried out [16].
According to the conventional culture method 35 out of 100 (35%) raw milk samples were contaminated by Yersinia spp. Further, by molecular detection, 16s rRNA gene specific to Yersinia enterocolitica was amplified in 33 out of 35 contaminated samples. In other words, 33% of raw milk samples were detected to be contaminated by Yersinia enterocolitica in northeast of Iran. Figure 1 shows the amplification of the 16s rRNA gene in raw milk samples. Among these 33 positive isolates, 26 isolates belonged to 1A biotype (26%), 6 isolates belonged to 1B (6%) and 1 isolate belonged to 5 (1%) biotypes.
Milk contains various proteins, minerals and vitamins that made it a complete food especially for children as well as a rich medium for growth of microorganism. Therefore, hygienic quality and safety of this product is very important. One of the most prevalent health issues is food-borne diseases caused by a variety of bacterial, viral and parasitic micro-organisms. The most common cause of the bacterial food-borne disease is animal origin’s food [17].
To our knowledge, such a high percentage of positive results (33%) have never been reported in Iran. In another study, 1.6% of raw milk, but no pasteurized milk samples, were positive for Y. enterocolitica [5], which was lower than our results. Another study in Iran showed that 9.4% of milk and dairy products were contaminated with Yersinia spp. and 5.07% were contaminated with Y. enterocolitica in culture method. Although in 16srRNA gene amplification, 4.34% of the samples were contaminated with this organism [18]. In another study, Y. enterocolitica was detected in 5.8% of raw cow’s milk [2]. These studies used different enrichment medium.
Alavi et al. [19] investigated the raw milk of sheep and goat regarding the presence of Y. enterocolitica. According to the reported results, 9 out of 84 sheep milk samples were contaminated with Y. enterocolitica in culture assay and all of the isolates confirmed through PCR assay, but the organism was not detected in 16 goat milk samples [19]. Hanifian and Khani [6] determined the prevalence of virulent Yersinia enterocolitica in raw milk and cheese samples. According to their results 7.62% of bulk raw milk samples were contaminated with Y. enterocolitica [6].
In Mexico City, Yersinia spp. was isolated from 454 out of 1300 raw cow’s milk samples which were collected from stables. From the total isolated Yersinia genus, 44.25% were diagnosed as Y. enterocolitica [20]. In Turkey, Y. enterocolitica was the most common isolated species from raw milk; it was recovered from 47.3% of raw milk samples [21]. Alkali treatment was used in their isolation procedure prior to streaking on CIN agar. The reported rates of above studies are higher than the present study. According to a review, some studies suggested that alkali treatment may increase the rate of isolation of Y. enterocolitica but some others didn’t propose it [13]. These observed variations in reported rate of contamination may be due to the isolation procedure. In the present study, enrichment in PSB and culture on CIN agar were used. Also, PCR confirmation of organism helps to rapid identification of Y. enterocolitica.
In the present study, most of the isolates (26 out of 33) belonged to 1A biotype. The predominance of this biotype was also reported in the other studies [2,5,21]. Two pathogenic biotypes including 1B and 5, were isolated in the present study. Jamali et al. [2] reported that 15.8% of isolated Y. enterocolitica were belonged to 1B biotype which was lower than the present study. Other studies were also isolated this biotype from animal and animal food origin [22,23]. Some researchers reported the other pathogenic biotypes in milk [20].
Contamination of raw milk with Y. enterocolitica is likely to be associated with inadequate hygienic practice in milking. Implementing of an effective sanitary milking management is the only way of controlling this pathogen at the farm level. The main sources of growth, propagation and survival of Y. enterocolitica are consumption of raw unpasteurized milk and use of dairy products produced from raw milk or with non-hygienic methods. Thus, sanitary milking, use of pasteurized milk for consumption and preparation of dairy products and protecting milk and dairy produce from dust and insects are the best ways to prevent Y. enterocolitica infections [18].
The conventional culture based method in detection of Y. enterocolitica is labor intensive, and require 3 to 4 days to reach the preliminary result. For confirmation, it needs more time and use of numerous phenotypic tests. Moreover, they need a sufficient number of organisms to distinguish the contamination while molecular confirmation method not only is very specific and sensitive but also is rapid and distinguish the viable cells in comparison with direct molecular test that can not differentiate the dead cells from viable ones [13].
In Conclusion, the recovery of potentially pathogenic biotypes of Y. enterocolitica from raw milk samples showed that consumption of raw milk and dairy products which are made from raw milk may impose a potential risk and raise a public health concern for food safety agencies. As detection of this pathogen is not performed routinely, further research on the prevalence of Y. enterocolitica in pasteurized milk and other foods with the animal origin is needed.
Limitations
One of the main limitations of this study is the lack of resources and funding to study the isolates with more detail.