Aims and background: Yersinia is a cocobacilliary gram-negative bacteria which can cause yersiniosis in all age groups. This study was carried out to investigate the presence of Yersinia pseudotuberculosis and Yersinia enterocolitica in raw milk of livestock farms in Tehran.

Materials and methods: Three hundred and sixty raw milk samples were collected from livestock farms in Tehran during 6 months. Y. pseudotuberculosis and Y. enterocolitica were isolated and identified according to the FDA guidelines. After treatment with low temperature (10°C for 10 days) and alkaline treatment (using KOH), the samples were cultured in the selective medium named CIN agar at 25°C. After incubation, colonies similar to the bull eye were chosen and characterized using phenotypic tests and ultimately confirmed by the API-20E strip test (Biomerieux, France) and detection of ail and inv genes by PCR. Serotyping and bio typing tests carried out on Y. enterocolitica isolates.

Results: Of 360 samples, a total of four isolates were identified as Yersinia species, including three Y. enterocolitica and one Y. pseudotuberculosis.

Conclusion: Due to the presence of Y. pseudotuberculosis and Y. enterocolitica in raw milk, it is suggested that the food and drug control laboratories, milk factories and other researchers pay more attention to these bacteria.

Keywords: raw milk, Yersinia enterocolitica, Yersinia pseudotuberculosis, cow, alkaline

Due to the increasing population, providing adequate and safe food is one of the most important issues in different countries of the world. In addition to the food deficiency, inappropriate sanitation in different steps of production to consumption process of these substances leads to a reduction in the amount of food. So, not only the providing of food and its products in terms of quantity, but also the providing of healthy and safe food is very important.¹ Milk is one of the most consuming dairy products and essential food. Healthy and safe milk will help the health of the community, hence, ignoring this point may endanger the community health as well as the health economy. Due to the importance of this subject, rapid and accurate identification of contamination of raw milk is important.² The contamination of both raw and pasteurized milk is one of the problems that may occur for many reasons even in advanced countries. In addition to the total number of bacteria as an indicator for health of milk, detection of pathogenic bacteria in milk is important.^3,4 Raw milk and its products which are contaminated by microorganisms, can cause important diseases. Among the microbial contaminants of milk, various bacteria such as Coxiella burnetii, Escherichia coli, Listeria monocytogenes, Mycobacterium paratuberculosis and Yersinia enterocolitica can be mentioned.^5–7 The genus Yersinia is particularly important due to the ability to grow at low temperature (4°C). Yersinia after Salmonella, Campylobacter and Shigella is considered as the most common pathogen isolated from human feces. Yersinia cause various clinical disorders such as enterocolitis, Pseudo-appendicitis, extra-intestinal infections, and bacteremia.^8,9 Yersinia is a psychrotrophic gram negative coccobacillus bacterium which contains three main species including Yersinia pestis, Y. enterocolitica, and Y. pseudotuberculosis.^10,11 Due to the growth at 4°C, Yersinia is a potential risk factor for food cold chain.^11,12 Unpasteurized milk and dairy products are potentially contaminated with this bacterium which can be transmitted to humans.⁵ Due to the lack of sufficient study on cow milk contamination by Yersinia spp., especially in Iran, the aim of this study was to isolation and identification of Y. enterocolitica and Y. pseudotuberculosis from raw milk.

Sample collection and phenotypic identification

Three hundred and sixty samples of raw milk were collected from livestock farms around Tehran during cold seasons of 2015–2016 (autumn and winter). The samples were transported to microbiology laboratory of School of Public Health of Tehran University of Medical Sciences in closed containers under sterile and cold conditions according to the National Standards of Iran No. 3549 (milk protection conditions after milking)¹³ and No. 164 (milk and its products- raw milk- characteristics and testing methods).¹⁴ The samples were enriched in phosphate buffered saline (PBS), and 25ml of milk samples was added to 225 ml of PBS and placed in a refrigerator for 10 days. After cold treatment, alkaline treatment was carried out in order to eliminate KOH susceptible bacteria especially normal flora of the milk. To performing such a treatment, 500µl of samples was mixed with 500µl of 0.5% KOH solution and 0.5% NaCl for 30 s. Subsequently, streak culture was carried out on selective medium CIN agar (Merck, Germany) and incubated for 24–48 h at 25°C.^5,11 After incubation, suspicious colonies (colonies with red center and white margin or bulls eye appearance) were chosen. Desired colonies were surveyed by phenotypic and biochemical tests such as O-nitrophenyl-β-D-galactopyranoside (ONPG), ornithine decarboxylase (ODC), urease and motility. For final approval, the API-20E (BioMerieux, France) strip test was used.¹² To determine the biotypes of Y. enterocolitica, fermentation of xylose and trehalose as well as lipase, bile-esculin, Voges–Proskauer (VP) and indole tests were evaluated. Furthermore, serotyping was carried out using anti-serum (MAST, UK) according to the manufacturer's instructions.¹⁵

PCR assay

The DNA of Yersinia isolates was extracted using boiling method from purified cultures.¹⁶ In short, using sterile loop a few colonies of bacteria were suspended in sterile distilled water in 1.5 ml micro tube, was placed in a boiling water container for 20 min. After centrifugation for 5 min, supernatant was removed and finally concentration of DNA was measured with Nano Drop 3300 (Thermo Fisher Scientific, USA). The primers which used for the ail and inv genes are listed in Table 1. The PCR reaction was performed in a total volume of 25μl containing 12.5μl of Master Mix, 8.5μl distilled water, 2μl DNA, 1μl forward primer and 1μl reverse primer. PCR reactions were carried out in a Master cycler gradient (Eppendorf, Germany). Table 2 shows appropriate time and temperature for PCR steps. Y. pseudotuberculosis PTTC 1244 and Y. entrocolitica ATTC 11010 were used as positive control strains. After amplification, to analyze the PCR products, electrophoresis was carried out using a 1% agarose gel containing safe stain and a TBE 0.5× for electrophoresis buffer as well as a 100-bp ladder and a voltage of 80–100 V (Table 1&2).

Phenotypic evaluation

Of 360 samples, a total of four isolates were identified as Yersinia species (1.1%), including three Y. enterocolitica (0.83%) and one Y. pseudotuberculosis (0.27%). Figure 1&2 shows the result of the API-20E strip test which was used for confirmation of both species. Two isolates of Y. entrocolitica were belonged to 1A biotype and one was 3 (Figure 1&2), (Table 3).

Figure 1 API-20E biochemical tests for Y. enterocolitica.

Figure 2 API-20E biochemical tests for Y. pseudotuberculosis.

Also, result of serotyping of Y. enterocolitica isolates showed that two isolates were belonged to O:5 serotype and one was O:9.

PCR assay

PCR amplification products indicated that Y. pseudotuberculosis included inv gene, while only one isolate of Y. enterocolitica included ail gene (Figure 3&4).

Figure 3 Amplification products from PCR assay for the detection of inv in Y. pseudotuberculosis (product size of 183bp).

Figure 4 Amplification products from PCR assay for the detection of ail in Y. enterocolitica (product size of 351bp).

Of 360 raw milk samples taken during the cold seasons of 2015-2016, three isolates of Y. enterocolitica and one isolate of Y. pseudotuberculosis were obtained. A similar study was carried out  in Finland in 2014 by Parn et al. during which an outbreak of Y. pseudotuberculosis serotype O:1 occurred following consumption of raw milk which created fever and gastroenteritis in 44 persons.¹⁷ A similar study was carried out in Finland in 2014;¹⁷ during which, an outbreak of Y. pseudotuberculosis serotype O:1 occurred following consumption of raw milk which created fever and gastroenteritis in 44 people. In 2015, Jamali et al,¹⁸ carried out a study on 446 raw milk samples of cattle, sheep and goats in the Varamin area. Twenty- nine isolates of Yersinia were obtained, of which 65.5% were Y. entrocolitica, 31% Y. frederiksenii, and 3.4% Y. kristensenii. Nineteen isolates of Y. enterocolitica were belonged to 1A/O:9, 1B/O:8 and 4/O:3 serotypes, no isolates of pseudotuberculosis were obtained.¹⁸ In 2015, Jamali et al. carried out a study on 446 raw milk samples   of cattle, sheep and goats in the Varamin area. Its results showed  that, 29 isolates of Yersinia were obtained, of which 65.5% were Y. entrocolitica, 31% Y. frederiksenii, and 3.4% Y. kristensenii. Nineteen isolates of Y. enterocolitica were belonged to 1A/O:9, 1B/O:8 and 4/O:3 serotypes, no isolates of pseudotuberculosis were achieved.¹⁸ In the present study, Y. pseudotuberculosis was isolated from raw milk. The first report of contamination of raw milk with Y. enterocolitica in Iran was presented by Soltan Dallal et al. on 310 raw milk samples in Behshahr town.⁵ The first report of contamination of raw milk with Y. enterocolitica in Iran was presented by Soltan Dallal et al,⁵ on 310 raw milk samples in Behshahr City. During this study, the prevalence of Y. enterocolitica was 1.6%, which is close to prevalence of present study. In another study, Sharifzadeh et al,¹⁹ isolated six Y. enterocolitica isolates from 400 samples of raw and pasteurized milk in 2004. Their findings and prevalence were similar to our study.¹⁹ In another study, six Y. enterocolitica isolates were isolated from 400 samples of raw and pasteurized milk in 2004.¹⁹ Their findings and prevalence were similar to the present study. The physiological characteristics of Yersinia are effective in growth and expression of virulent genes and bacterial toxin.²⁰ Although Yersinia is sensitive to pasteurization heat, but in the study of Pagan et al. in 1999, six strains of Y. enterocolitica was inoculated to the milk, after pasteurization three strains of them were survived.²⁰ Although Yersinia is sensitive to pasteurization heat, but in another study,²¹ six strains of Y. enterocolitica was inoculated to the milk, after pasteurization three strains of them were survived. In another study, Soltan Dallal et al,²⁰ showed that both pH and temperature influence growth but only temperature affects virulence.²¹ In other study,²² the obtained results showed that both pH and temperature of Yersinia influence growth but only temperature affects virulence. The heat-resistant enterotoxin is produced at 4°C and 25°C, but it is not possible to produce at 37°C. These results emphasize the importance of temperature on the expression of virulence and enterotoxin genes. Yersinia contamination occurs primarily by lactating animal and secondary by environment and water. However, livestock as a source of Yersinia has an important role, but water as carrier is important and can lead to the transfer of Yersinia species to milk and dairy products, especially in rural and nomadic areas. Cheyne reported   the presence of Y. entrocolitica and other atypical Yersinia species, such as Y. frederiksenii, Y. kristensenii, and Y. intermedia in surface water.²³ Soltan Dallal & Salmanian²² isolated Y. entrocolitica strains from drinking and surface water in Kan region of Tehran.²⁴ Milk is a nutritious and popular food; therefore, bacteriological quality control of raw and pasteurized milk at production, collection and processing stages is necessary. The microbial and chemical changes of raw milk can interfere with processing stages which reduces shelf-life of dairy products, and consequently endanger the health of consumers and community. Presentation of Yersinia as one of the most important food-borne agents along with ability to grow at room and refrigerator temperatures and production of enterotoxin, makes it an important issue which needs to be paid more attention to improving food safety.^2,22

This study recommends the importance of scientific follow-up in routine laboratory experiments to assess dairy samples. Furthermore, milk samples must bacteriologically be tested by food and drug control laboratories. Results of the current study indicate the importance of milk pasteurization in dairy industries due to the presence of Y. pseudotculosis and Y. enterocolitica as indicated by this study.

This study was supported by a grant from the Food Microbiology Research Center, Tehran University of Medical Sciences (contract No. 34878).

Author declares that there are no conflicts of interest.

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