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Sharifi Yazdi S, Mobasseri B, Torabi Bonab P, Sharifi Yazdi S, Mirbagheri Z, Soltan Dallal M M. Prevalence and Characteristics of Yersinia Enterocolitica and Yersinia Pseudotuberculosis from Raw Milk Supplied in Tehran. JNFS 2023; 8 (1) :114-121
URL: http://jnfs.ssu.ac.ir/article-1-529-en.html
URL: http://jnfs.ssu.ac.ir/article-1-529-en.html
Sara Sharifi Yazdi * 
, Behzad Mobasseri , Parisa Torabi Bonab , Sina Sharifi Yazdi , Zohre Mirbagheri , Mohammad Mehdi Soltan Dallal
, Behzad Mobasseri , Parisa Torabi Bonab , Sina Sharifi Yazdi , Zohre Mirbagheri , Mohammad Mehdi Soltan Dallal
School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
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Sara Sharifi Yazdi; MD 1, Behzad Mobasseri; MSc 2, Parisa Torabi Bonab; MSc 3, Sina Sharifi Yazdi 4, Zohre Mirbagheri; PhD 2 & Mohammad Mehdi Soltan Dallal; PhD* 2,3
1 School of Medicine, Tehran University of Medical Sciences, Tehran, Iran; 2 Division of Food Microbiology, Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran; 3 Food Microbiology Research Center, Tehran University of Medical Sciences, Tehran, Iran; 4 Medical Student, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
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Prevalence and Characteristics of Yersinia Enterocolitica and Yersinia Pseudotuberculosis from Raw Milk Supplied in Tehran
Sara Sharifi Yazdi; MD 1, Behzad Mobasseri; MSc 2, Parisa Torabi Bonab; MSc 3, Sina Sharifi Yazdi 4, Zohre Mirbagheri; PhD 2 & Mohammad Mehdi Soltan Dallal; PhD* 2,3
1 School of Medicine, Tehran University of Medical Sciences, Tehran, Iran; 2 Division of Food Microbiology, Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran; 3 Food Microbiology Research Center, Tehran University of Medical Sciences, Tehran, Iran; 4 Medical Student, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
| ARTICLE INFO | ABSTRACT | |
| ORIGINAL ARTICLE | Background: Foodborne diseases are caused by eating contaminated food. Yersinia species are among the bacteria involved in food contamination, such as meat, poultry, vegetables, and milk. This study aims to investigate the prevalence and characteristics of Yersinia enterocolitica and Yersinia pseudotuberculosis from raw milk in Tehran. Methods: In this study, 360 samples of raw milk were collected from farms around Tehran and examined according to the FDA method. Then, 25 ml of milk samples were added to 225 ml of Peptone Sorbitol Bile Broth and kept for 10 days at 4 °C for enrichment. After 10 days, 0.5 ml of the sample was added to 0.5 ml of 0.72% KOH solution and 0.54% NaCl. After 30 sec, it was cultured in the selected Cefsulodin-Irgasan-Novobiocin agar (CINagar) medium to remove the normal flora. The plates were stored for 48 h at 28 °C. The suspected Bull's eye colonies were purified and phenotyping tests were carried out on the selected colony. The 20 E API kits were used for confirming identification. Results: From 360 raw milk samples, 4 Yersinia isolates (1.1%) including one Y. pseudotuberculosis (0.27%) and three Y. enterocolitica (0.83%) were isolated. In addition to Yersinia, other bacteria such as Klebsiella, Serratia, Citrobacter, and Providencia were isolated from milk samples. Conclusion: The findings showed that clean tap water and healthy cattle in livestock can be effective in preventing Yersinia contamination. Lack of personal and environmental hygiene could cause food contamination by Yersinia and other intestinal bacteria leading to gastrointestinal infections. Keywords: Yersinia enterocolitica; Yersinia pseudotuberculosis; Milk; Diarrhea |
|
| Article history: Received: 27 Nov 2021 Revised: 6 Feb 2022 Accepted: 15 Mar 2022 |
||
| *Corresponding author: msoltandallal@gmail.com Food Microbiology Research Center/Division of Food Microbiology, Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. Postal code: 6446-14155 Tel: +98 21 42933082 |
Introduction
Milk is mainly made up of water in which a wide range of nutrients including vitamins, proteins, fats, and carbohydrates are suspended. These rich nutritional contents and production and processing methods in commercial milk production make it susceptible to contamination by pathogenic microbes that can cause disease in humans. Therefore, milk is known as an effective means of transmitting disease-causing agents to humans (Muehlhoff et al., 2013). Bacterial agents may contaminate milk at various stages of preparation, processing, and distribution. Bacterial contamination may be caused by cow's udder, storage, milk collection materials, various additives added to dairy products, and livestock workers. Staphylococcus aureus, Salmonella spp., Listeria monocytogenes, Escherichia coli O157: H7, Campylobacter, and Yersinia spp. are the main microbiological hazards associated with raw milk consumption (McAuley et al., 2014, Owusu-Kwarteng et al., 2020). Preventing the growth of contaminated bacteria in milk includes limiting the level of contamination, cooling immediately after milking, and maintaining a refrigerated storage temperature. Bacterial constraints mainly include cleaning, disinfecting, and drying the cow's udder and udder before milking, and using healthy milking equipment. Removing solid milk from milk containers is very important in controlling psychrophilic bacteria (van den Brom et al., 2020). Milk pasteurization has been used as the most effective method to reduce the risk of infection and the spread of disease. Although pasteurized milk is expected to last 14 to 20 days, the shelf life of pasteurized milk stored at room temperature depends on the efficiency of the pasteurization process (Garedew et al., 2012, Hahn, 1996). The microbiological status of raw milk is affected by several factors including animal health status, farm management practices, environmental health, and inappropriate temperature control (Martin et al., 2018).
Yersinia is a foodborne pathogen that has at least 16 species. Three species of Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis are the most important pathogenic species of Yersinia. These bacteria are among the gram-negative, facultative anaerobic bacteria, and psychrophilic bacteria in the Enterobacteriaceae family. These bacteria are widely isolated from soil, water, and food, including milk, meat, and cause common diseases between humans and animals (Fredriksson-Ahomaa and Korkeala, 2003, Jamali et al., 2015, Rahman et al., 2011, Soltan Dallal and Salmanian, 1996).
Y. enterocolitica is the cause of several human diseases including diarrhea, appendicitis, mesenteric lymphadenitis, arthritis, and sepsis. Y. pseudotuberculosis causes tuberculosis-like symptoms in animals and causes mesenteric lymphadenitis and diarrhea in humans. Also, Yersinia species are cold-resistant agents that can grow at 4 °C and can be used as risk factors for cold chain food transfer (Huovinen et al., 2010, Rivas et al., 2021, Soltan Dallal and Moezardalan, 2004).
Pathogenic serotypes and biotypes of Y. enterocolitica serogroups O:3 (biogroup 4), O:5,27 (biogroups 2 and 3), O:8 (biogroup 1B), and O:9 (biogroup 2) are globally widespread (Soltan Dallal et al., 2017a). In Japan, O:3/3 has increased as the most important cause of Yersiniosis (Fukushima et al., 2011). In Europe, more than 90% of Yersiniosis agents are O: 3 and O: 9 serotypes. This bacterium is normally excreted from animals and is present in smaller amounts in food (Bottone, 2015, Syczyło et al., 2018). In East Asia, varieties 1b, 2a, 2b, 2c, 3, 4a, 4b, 5a, 5b were isolated from patients with systemic infection and fever, and almost all isolated strains were able to secrete YPMa super antigenic toxin (Fukushima et al., 2011, Kameyama et al., 2016). In Europe, strains 1a, 1b, and 3 were isolated from patients with gastroenteritis (Espenhain et al., 2019, Rosner et al., 2013).
One of the factors that transmit Yersinia to humans is milk. Milk in various forms including raw milk, yogurt, cheese, cream, buttermilk, ice cream, cocoa milk, skim milk powder, and low-lactose milk are in the food basket of households. The use of this valuable and high-consumption product requires careful and sensitive monitoring and observation, since this important and valuable food product can easily transmit dangerous diseases to humans. Therefore, sensitivity and accuracy require high attention (Górska-Warsewicz et al., 2019, Rahman et al., 2011).
In many countries, milk and its products make up an important part of the human diet. By subsidizing dairy products, the consumption of such products in the household nutrition basket will increase. The history of milk subsidies and school milk programs in industrialized countries is about a century old. In all European and American countries, subsidies are paid to milk producers. In Western European and North American countries, the per capita consumption of milk and its products, which are converted into liquid milk, varies between 300 and 490 kg per year, and this source provides more than 85% of the calcium needed by consumers (Raghiante et al., 2018, Soltan-Dallal et al., 2004).
According to statistics, more than 60% of the country milk passes through health channels and is supplied to the consumer market. However, a large part of the produced milk is consumed without passing through these channels, which greatly increases the risk of foodborne illnesses, especially biological factors. Some areas are used for processing under traditional conditions or in a non-pasteurized or even non-pasteurized form. Therefore, the possibility of contamination with pathogens is common, which prevents the spread of foodborne illness in the population (Al-Jawaldeh et al., 2020, Msalya, 2017, Rahimi et al., 2014). This study aimed to investigate the prevalence and characteristics of Y. enterocolitica and Y. pseudotuberculosis from raw milk supplied in Tehran.
Materials and Methods
Sampling: In this descriptive cross-sectional study, sampling was performed in cold seasons of the year, since Yersinia grows better in cold weather. Sampling was performed from autumn to winter 2018. In total, 360 samples of raw milk from farms around Tehran were prepared in closed containers under sterile conditions. They were then transferred to a microbiology laboratory in the Department of Food Microbiology at Tehran University of Medical Sciences, according to the Iranian National Standard No. 164 (milk and its products test methods, ISIRI, 2003), and the Iranian National Standard No. 3549 (milk protection conditions after milking, ISIRI, 1995). Sample transportation took place in cold chain conditions. Samples without numbering label in the open container that were not taken by cold chain method and more than twelve hours had been passed from milking were excluded. The samples were tested immediately upon arrival.
Isolation of Yersinia: The samples were tested for the presence or absence of the genus Yersinia according to the FDA method (Ahmed et al., 2019).
Enrichment: In this study, 25 ml of milk sample was mixed with 225 ml of Peptone Sorbitol Broth (PSB) in sterile glass containers. After homogenization, they were kept in the refrigerator for 10 days. After 10 days, the samples were taken out of the refrigerator and an alkaline treatment method was performed to remove milk flora (Ahmed et al., 2019). In this method, KOH-sensitive bacteria were killed. The streaking culture was performed on Cefsulodin-Irgasan-Novobiocin agar (CIN agar, Merck, Germany). The samples were stored at 25 °C for 24 to 48 h (Ahmed et al., 2019). Differentiation of Yersinia growth on CIN agar medium was done based on mannitol fermentation, staining, and production of colonies with a red center and a white border resembling a cow's eye. In this medium, the presence of sodium deoxycholate and violet crystal prevents the growth of gram-positive and many gram-negative bacteria (Figure 1). After 48 h, suspicious colonies (colonies with red center and white border) were isolated and biochemical tests of O-Nitrophenyl-β-D-galactopyranoside (ONPG), Kligler's Iron Agar (KIA, Ornithine Decarboxylase Test (ODC), and urease test (Merck, Germany) were performed. The motility test on the Sulphide Indole Motility(SIM) environment was checked. All species of Yersinia except Yersinia pestis are immobile at 37 °C and 25 °C. The API 20E kit manufactured by the French company BioMérieux was used for final identification (Soltan Dalal et al., 2009).
Also, to determine the biotypes of Yersinia enterocolitica, xylose fermentation test, trehalose fermentation test, lipase test, Bile Esculin test, Voges–Proskauer (VP) test, and Indol fermentation test were used. To determine the serotype, MAST antiserum (England) was used according to the manufacturer's instructions (Soltan Dallal et al., 2017a).
Isolation of other species of Enterobacteriaceae: To isolate other species of Enterobacteriaceae, all gram-negative bacteria were isolated during the study period on general media identified to the species level based on gram stain and biochemical tests (Ewing, 1968, Soltan Dallal et al., 2017b).
Results
In terms of livestock age, 105 (29.16%) samples aged 2-3 years, 140 (28.88%) 3-4 years, and 115 (31.94%) samples aged over 4 years, respectively. Also, in terms of the type of livestock, 247 (68.61%) samples were small and 113 (31.38%) samples were domestic.
Regarding milking type, 155 (43.05%) samples were milked by milking equipment and 205 (56.94%) samples were manually milked. Regarding the type of drinking water, 101 (28.05%) samples consumed tap water, 94 (26.11%) samples running water, and 165 (45.83%) samples consumed well water. The sampled cows included 300 (83.33%) crossbred, 41(11.38%) native cows, and 19 (5.27%) purebred cows. In this study, of 360 samples of raw milk tested, one (0.27%) sample was infected with Y. pseudotuberculosis and three (0.83%) samples were infected with Y. enterocolitica. The identification was confirmed by the API20E kit. The influential parameters in Yersinia identification are given in Table 1.
In addition, out of three samples of isolated Y. enterocolitica, 1 sample biotype 3, 1 sample biotype 1A, and 1 sample biotype 1B were identified (Table 2).
Of 360 milk samples apart from Yersinia (61.7%), other bacteria including 32 (8.9%), Klebsiella pneumonia, 50 (13.9%) Klebsiella oxytoca, 60 (16.7%) Citrobacter freundii 52 (14.4%) Serratia liquefaciens, 3 (0.83%) Serratia nematodiphilia, and 25 (6.9%) Providencia rettgeri were isolated from CIN agar medium (Figure 2).
Yersinia is a foodborne pathogen that has at least 16 species. Three species of Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis are the most important pathogenic species of Yersinia. These bacteria are among the gram-negative, facultative anaerobic bacteria, and psychrophilic bacteria in the Enterobacteriaceae family. These bacteria are widely isolated from soil, water, and food, including milk, meat, and cause common diseases between humans and animals (Fredriksson-Ahomaa and Korkeala, 2003, Jamali et al., 2015, Rahman et al., 2011, Soltan Dallal and Salmanian, 1996).
Y. enterocolitica is the cause of several human diseases including diarrhea, appendicitis, mesenteric lymphadenitis, arthritis, and sepsis. Y. pseudotuberculosis causes tuberculosis-like symptoms in animals and causes mesenteric lymphadenitis and diarrhea in humans. Also, Yersinia species are cold-resistant agents that can grow at 4 °C and can be used as risk factors for cold chain food transfer (Huovinen et al., 2010, Rivas et al., 2021, Soltan Dallal and Moezardalan, 2004).
Pathogenic serotypes and biotypes of Y. enterocolitica serogroups O:3 (biogroup 4), O:5,27 (biogroups 2 and 3), O:8 (biogroup 1B), and O:9 (biogroup 2) are globally widespread (Soltan Dallal et al., 2017a). In Japan, O:3/3 has increased as the most important cause of Yersiniosis (Fukushima et al., 2011). In Europe, more than 90% of Yersiniosis agents are O: 3 and O: 9 serotypes. This bacterium is normally excreted from animals and is present in smaller amounts in food (Bottone, 2015, Syczyło et al., 2018). In East Asia, varieties 1b, 2a, 2b, 2c, 3, 4a, 4b, 5a, 5b were isolated from patients with systemic infection and fever, and almost all isolated strains were able to secrete YPMa super antigenic toxin (Fukushima et al., 2011, Kameyama et al., 2016). In Europe, strains 1a, 1b, and 3 were isolated from patients with gastroenteritis (Espenhain et al., 2019, Rosner et al., 2013).
One of the factors that transmit Yersinia to humans is milk. Milk in various forms including raw milk, yogurt, cheese, cream, buttermilk, ice cream, cocoa milk, skim milk powder, and low-lactose milk are in the food basket of households. The use of this valuable and high-consumption product requires careful and sensitive monitoring and observation, since this important and valuable food product can easily transmit dangerous diseases to humans. Therefore, sensitivity and accuracy require high attention (Górska-Warsewicz et al., 2019, Rahman et al., 2011).
In many countries, milk and its products make up an important part of the human diet. By subsidizing dairy products, the consumption of such products in the household nutrition basket will increase. The history of milk subsidies and school milk programs in industrialized countries is about a century old. In all European and American countries, subsidies are paid to milk producers. In Western European and North American countries, the per capita consumption of milk and its products, which are converted into liquid milk, varies between 300 and 490 kg per year, and this source provides more than 85% of the calcium needed by consumers (Raghiante et al., 2018, Soltan-Dallal et al., 2004).
According to statistics, more than 60% of the country milk passes through health channels and is supplied to the consumer market. However, a large part of the produced milk is consumed without passing through these channels, which greatly increases the risk of foodborne illnesses, especially biological factors. Some areas are used for processing under traditional conditions or in a non-pasteurized or even non-pasteurized form. Therefore, the possibility of contamination with pathogens is common, which prevents the spread of foodborne illness in the population (Al-Jawaldeh et al., 2020, Msalya, 2017, Rahimi et al., 2014). This study aimed to investigate the prevalence and characteristics of Y. enterocolitica and Y. pseudotuberculosis from raw milk supplied in Tehran.
Materials and Methods
Sampling: In this descriptive cross-sectional study, sampling was performed in cold seasons of the year, since Yersinia grows better in cold weather. Sampling was performed from autumn to winter 2018. In total, 360 samples of raw milk from farms around Tehran were prepared in closed containers under sterile conditions. They were then transferred to a microbiology laboratory in the Department of Food Microbiology at Tehran University of Medical Sciences, according to the Iranian National Standard No. 164 (milk and its products test methods, ISIRI, 2003), and the Iranian National Standard No. 3549 (milk protection conditions after milking, ISIRI, 1995). Sample transportation took place in cold chain conditions. Samples without numbering label in the open container that were not taken by cold chain method and more than twelve hours had been passed from milking were excluded. The samples were tested immediately upon arrival.
Isolation of Yersinia: The samples were tested for the presence or absence of the genus Yersinia according to the FDA method (Ahmed et al., 2019).
Enrichment: In this study, 25 ml of milk sample was mixed with 225 ml of Peptone Sorbitol Broth (PSB) in sterile glass containers. After homogenization, they were kept in the refrigerator for 10 days. After 10 days, the samples were taken out of the refrigerator and an alkaline treatment method was performed to remove milk flora (Ahmed et al., 2019). In this method, KOH-sensitive bacteria were killed. The streaking culture was performed on Cefsulodin-Irgasan-Novobiocin agar (CIN agar, Merck, Germany). The samples were stored at 25 °C for 24 to 48 h (Ahmed et al., 2019). Differentiation of Yersinia growth on CIN agar medium was done based on mannitol fermentation, staining, and production of colonies with a red center and a white border resembling a cow's eye. In this medium, the presence of sodium deoxycholate and violet crystal prevents the growth of gram-positive and many gram-negative bacteria (Figure 1). After 48 h, suspicious colonies (colonies with red center and white border) were isolated and biochemical tests of O-Nitrophenyl-β-D-galactopyranoside (ONPG), Kligler's Iron Agar (KIA, Ornithine Decarboxylase Test (ODC), and urease test (Merck, Germany) were performed. The motility test on the Sulphide Indole Motility(SIM) environment was checked. All species of Yersinia except Yersinia pestis are immobile at 37 °C and 25 °C. The API 20E kit manufactured by the French company BioMérieux was used for final identification (Soltan Dalal et al., 2009).
Also, to determine the biotypes of Yersinia enterocolitica, xylose fermentation test, trehalose fermentation test, lipase test, Bile Esculin test, Voges–Proskauer (VP) test, and Indol fermentation test were used. To determine the serotype, MAST antiserum (England) was used according to the manufacturer's instructions (Soltan Dallal et al., 2017a).
Isolation of other species of Enterobacteriaceae: To isolate other species of Enterobacteriaceae, all gram-negative bacteria were isolated during the study period on general media identified to the species level based on gram stain and biochemical tests (Ewing, 1968, Soltan Dallal et al., 2017b).
Results
In terms of livestock age, 105 (29.16%) samples aged 2-3 years, 140 (28.88%) 3-4 years, and 115 (31.94%) samples aged over 4 years, respectively. Also, in terms of the type of livestock, 247 (68.61%) samples were small and 113 (31.38%) samples were domestic.
Regarding milking type, 155 (43.05%) samples were milked by milking equipment and 205 (56.94%) samples were manually milked. Regarding the type of drinking water, 101 (28.05%) samples consumed tap water, 94 (26.11%) samples running water, and 165 (45.83%) samples consumed well water. The sampled cows included 300 (83.33%) crossbred, 41(11.38%) native cows, and 19 (5.27%) purebred cows. In this study, of 360 samples of raw milk tested, one (0.27%) sample was infected with Y. pseudotuberculosis and three (0.83%) samples were infected with Y. enterocolitica. The identification was confirmed by the API20E kit. The influential parameters in Yersinia identification are given in Table 1.
In addition, out of three samples of isolated Y. enterocolitica, 1 sample biotype 3, 1 sample biotype 1A, and 1 sample biotype 1B were identified (Table 2).
Of 360 milk samples apart from Yersinia (61.7%), other bacteria including 32 (8.9%), Klebsiella pneumonia, 50 (13.9%) Klebsiella oxytoca, 60 (16.7%) Citrobacter freundii 52 (14.4%) Serratia liquefaciens, 3 (0.83%) Serratia nematodiphilia, and 25 (6.9%) Providencia rettgeri were isolated from CIN agar medium (Figure 2).
| Table 1. Impact of environmental conditions on Yersinia isolation. | ||
| Environmental condition | Yersinia entercolitica | Yersinia pseudotuberculosis |
| Water type | ||
| Tap water Running water Well water |
0 | 0 |
| 1 | 1 | |
| 2 | 0 | |
| Livestock type | ||
| Small Domestic |
1 | 1 |
| 2 | 0 | |
| Type of milk preparation | ||
| By machine Manual |
1 | 1 |
| 2 | 0 | |
| Type of cow | ||
| Hybrid Domestic Purebred |
1 | 0 |
| 2 | 1 | |
| 0 | 0 | |
| Age (Year) | ||
| 2-3 3-4 > 4 |
1 | 0 |
| 1 | 1 | |
| 1 | 0 | |
| Table 2. Biotyping tests of Y. entercolitica. | |||||||
| 1A | 1B | 2 | 3 | 4 | 5 | 6 | Biochemical test |
| + | + | - | - | - | - | - | lipase |
| +/- | - | - | - | - | - | - | Aesculin/salicin(24 h) |
| + | + | (+) | - | - | - | - | Indole |
| + | + | + | + | - | V | + | Xylose |
| + | + | + | + | + | - | + | Trehalose |
| + | - | - | - | - | - | + | Pyrazinamidase |
| + | - | - | - | - | - | - | d- glucosidase |
| + | + | + | +/-(c) | + | (+) | - | VP |
| V: Variable; VP: Voges-Proskauer; (c): Biotype of serotype O:3 found in Japan. | |||||||
Discussion
From a total of 360 raw milk samples taken during the cold seasons in 1994, 3 strains of Y. enterocolitica (0.83%) and 1 strain of Y. pseudotuberculosis (0.27%) were isolated. Y. frederiksenii, Yersinia kristensenii, Yersinia Intermedia, and Yersinia aldovae were not separated. In these experiments, the species of Serratia, Citrobacter, Klebsiella, and Providence were repeatedly isolated. In the authors’ previous study (Soltan Dallal and Moezardalan, 2004) on the contamination of raw milk with Yersinia enterocolitica in Iran, the prevalence of Y. enterocolitica was 1.6%, which was very close to the prevalence of the present study. In a study from Chaharmahal Bakhtiari, 200 samples of raw milk and 200 samples of pasteurized milk were prepared by random cluster sampling and Yersinia enterocolitica was isolated in 6 cases (3%) of raw milk and 2 cases (1%) of pasteurized milk (Sharifzadeh et al., 2004). In a study by Hanifian and Khani in Tabriz, using PCR and tracing of ail and virf genes on pasteurized milk, Yersinia enterocolitica was reported (Hanifian and Khani, 2012)). Due to the sensitivity of Yersinia to high temperatures during pasteurization, the importance of secondary contamination of milk after pasteurization is notable. The enrichment of the samples was similar to the present study and the PSBB medium was used at a ratio of 25 by 225 ml. Only one case of Yersinia enterocolitica was isolated by culture method, while 16 cases were isolated by PCR method. This explains the high sensitivity difference of PCR. Although there is no difference between live and dead bacteria in PCR, the presence of Yersinia enterocolitica DNA in pasteurized milk is notable (Hanifian and Khani, 2012). In 2015, Jamali et al. conducted a study on 446 raw milk of cows, sheep, and goats in the Varamin region. They isolated 29 strains of Yersinia, 19 strains of which were Yersinia enterocolitica with biotypes of 1A / O: 9, 1B / O: 8, and 4 / O; 3. No isolates of Yersinia pseudotuberculosis were obtained (Jamali et al., 2015). In the present study, 1 Yersinia pseudotuberculosis isolate was isolated from raw milk. Unlike Yersinia pseudotuberculosis, many studies have been performed on the isolation of Yersinia enterocolitica from raw milk. In another study by Tavassoli et al., in 2019, 200 traditional kinds of cheese were randomly collected from northeastern Iran (100 samples from Khorasan Razavi and 100 samples from Golestan) and 100 samples of raw milk from Mashhad. In total, 38 Yersinia enterocolitica were obtained from cheese and 33 isolates from raw milk. All isolates belonged to two biotypes of 1A and 1B (Tavassoli et al., 2019). In a study conducted in Egypt by Darwish et al., 27 Yersinia intermedia, 13 Yersinia kristensenii, and 3 Yersinia frederiksenii were separated. Examination of their working method shows that enrichment was similar to the current study, but their selected environments included CIN agar, McConkey, and Blood agar (Darwish et al., 2015). In 2013, Bernardino-Varo et al. studied the prevalence and diversity of Yersinia species from 1,300 samples of raw milk collected from stables in Mexico City. A total of 454 samples were declared positive; this number included 44.25% of Yersinia enterocolitica, 18.26% of Yersinia kristensenii, 13.65% of Yersinia intermedia, 14.85% of Y. frederiksenii, and Yersinia aldovae 9.14% (Bernardino-Varo et al., 2013). The high prevalence of Yersinia in this study might be due to the stable environment, which may be in the process of increasing Yersinia pollution from the workplace, especially if the stables were shared with pigs that are Yersinia reservoirs. In a study in Egypt, Yersinia enterocolitica was isolated from 10% of raw milk and dairy samples (Ahmed et al., 2019). The highest separation rates were from raw milk with 22%, followed by 12%, 4%, and 2% of fermented milk, pasteurized milk, and cheese, respectively.
Conclusion
The results of this study highlight the role of milk as an important factor in the transmission of potentially pathogenic strains of Y. enterocolitica and Y. pseudotuberculosis. The findings show that tap water is effective in preventing Yersiniacontamination. Also, the presence of purebred cattle in livestock can be effective in preventing Yersinia contamination.Furthermore, the presence of O9, O: 8, O: 5 pathogenic serotypes in milk should increase attention to Yersinia in milk tests.
Acknowledgments
This article is the result of a research project approved by Tehran University of Medical Sciences and Health Services under the contract number 34878. Thanks are owed to the Vice-Chancellor of Research in Tehran University of Medical Sciences who supported this research project.
Authors' contributions
Soltan Dallal MM designed the research; Mobassei B conducted the research; Sharifi Yazdi S analyzed the data; Sharifi Yazdi S, Mirbagheri Z wrote the manuscript; Soltan Dallal MM had primary responsibility for final content. All authors read and approved the final manuscript.
Conflict of interest
The authors declared that there is no conflict of interest.
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Fukushima H, Shimizu S & Inatsu Y 2011. Yersinia enterocolitica and Yersinia pseudotuberculosis detection in foods. Journal of pathogens. 2011.
Garedew L, Berhanu A, Mengesha D & Tsegay G 2012. Identification of gram-negative bacteria from critical control points of raw and pasteurized cow milk consumed at Gondar town and its suburbs, Ethiopia. BMC public health. 12 (1): 1-7.
Górska-Warsewicz H, Rejman K, Laskowski W & Czeczotko M 2019. Milk and dairy products and their nutritional contribution to the average polish diet. Nutrients. 11 (8): 1771.
Hahn G 1996. Pathogenic bacteria in raw milk. Situation and significance [in Germany]. International Dairy Federation.
Hanifian S & Khani S 2012. Prevalence of virulent Yersinia enterocolitica in bulk raw milk and retail cheese in northern-west of Iran. International journal of food microbiology. 155 (1-2): 89-92.
Huovinen E, et al. 2010. Symptoms and sources of Yersinia enterocolitica-infection: a case-control study. BMC infectious diseases. 10 (1): 1-9.
Jamali H, Paydar M, Radmehr B & Ismail S 2015. Prevalence, characterization, and antimicrobial resistance of Yersinia species and Yersinia enterocolitica isolated from raw milk in farm bulk tanks. Journal of dairy science. 98 (2): 798-803.
Kameyama M, Yabata J, Obane N, Otsuka H & Nomura Y 2016. Detection of pathogenic Yersinia enterocolitica in pet Djungarian hamsters in Japan. Journal of Veterinary Medical Science. 15-0654.
Martin NH, Boor KJ & Wiedmann M 2018. Symposium review: Effect of post-pasteurization contamination on fluid milk quality. Journal of dairy science. 101 (1): 861-870.
McAuley CM, McMillan K, Moore SC, Fegan N & Fox EM 2014. Prevalence and characterization of foodborne pathogens from Australian dairy farm environments. Journal of dairy science. 97 (12): 7402-7412.
Msalya G 2017. Contamination levels and identification of bacteria in milk sampled from three regions of Tanzania: evidence from literature and laboratory analyses. Veterinary Medicine International. 2017.
Muehlhoff E, Bennett A & McMahon D 2013. Milk and dairy products in human nutrition. Food and Agriculture Organization of the United Nations (FAO).
Owusu-Kwarteng J, Akabanda F, Agyei D & Jespersen L 2020. Microbial safety of milk production and fermented dairy products in Africa. Microorganisms. 8 (5): 752.
Raghiante F, dos Santos EA & Martins OA 2018. Yersinia enterocolitica in milk and dairy products: a review. Revista Brasileira de Higiene e Sanidade Animal. 12 (4): 420-427.
Rahimi E, Sepehri S, Dehkordi FS, Shaygan S & Momtaz H 2014. Prevalence of Yersinia species in traditional and commercial dairy products in Isfahan Province, Iran. Jundishapur journal of microbiology. 7 (4): e9249.
Rahman A, Bonny TS, Stonsaovapak S & Ananchaipattana C 2011. Yersinia enterocolitica: epidemiological studies and outbreaks. Journal of pathogens. 2011: 1-11.
Rivas L, Strydom H, Paine S, Wang J & Wright J 2021. Yersiniosis in New Zealand. Pathogens. 10 (2): 191.
Rosner BM, Werber D, Höhle M & Stark K 2013. Clinical aspects and self-reported symptoms of sequelae of Yersinia enterocolitica infections in a population-based study, Germany 2009–2010. BMC infectious diseases. 13 (1): 1-8.
Sharifzadeh A, Akhavan M, Zarasvandi A & Al Agha S 2004. Isolation of Yersinia entrocolitica and Listeria monocytogenes from raw and pasteurized milks supplied at dairies in Chahar mahal bakhtiary province. Iranian journal of food science and technology. 1 (1): 15-19.
Soltan-Dallal MM, Tabarraie A & MoezArdalan K 2004. Comparison of four methods for isolation of Yersinia enterocolitica from raw and pasteurized milk from northern Iran. International journal of food microbiology. 94 (1): 87-91.
Soltan Dalal MM, Akhi MT & Partou Azar AR 2009. Study of yersina enterocolitica in acute children diarrhea under frouteen years old. Medical journal of Tabriz university of medical sciences. 30 (4): 49-52.
Soltan Dallal MM & Moezardalan K 2004. Frequency of Yersinia species infection in paediatric acute diarrhoea in Tehran. Eastern Mediterranean health journal. 10 (1-2): 152-158.
Soltan Dallal MM & Salmanian H 1996. A survey on frequency of Yersinia SPP, in surface and drinking Water in Kan, Tehran. Iranian journal of infectious diseases and tropical medicine. 2: 44-49.
Soltan Dallal MM, SharifiYazdi MK & Vahedi S 2017a. The Relationship ofYersinia Isolates Bioserotypeswith Minimum Inhibitory Concentration of Ampicillin, Cefazolin, and Cefotaxime.
Soltan Dallal MM, SharifiYazdi MK & Vahedi S 2017b. The Relationship ofYersinia Isolates Bioserotypeswith Minimum Inhibitory Concentration of Ampicillin, Cefazolin, and Cefotaxime. Journal of Kerman University of medical sciences. 24 (1): 38-49.
Syczyło K, et al. 2018. The prevalence of Yersinia enterocolitica in game animals in Poland. PLoS One. 13 (3): e0195136.
Tavassoli M, Jamshidi A, Movafagh F & Afshari A 2019. Virulence Characteristics of Yersinia enterocolitica Isolated from Dairy Products in the Northeast of Iran. Journal of human, environment and health promotion. 5 (2): 72-78.
van den Brom R, de Jong A, van Engelen E, Heuvelink A & Vellema P 2020. Zoonotic risks of pathogens from sheep and their milk borne transmission. Small ruminant research. 189: 106123.
From a total of 360 raw milk samples taken during the cold seasons in 1994, 3 strains of Y. enterocolitica (0.83%) and 1 strain of Y. pseudotuberculosis (0.27%) were isolated. Y. frederiksenii, Yersinia kristensenii, Yersinia Intermedia, and Yersinia aldovae were not separated. In these experiments, the species of Serratia, Citrobacter, Klebsiella, and Providence were repeatedly isolated. In the authors’ previous study (Soltan Dallal and Moezardalan, 2004) on the contamination of raw milk with Yersinia enterocolitica in Iran, the prevalence of Y. enterocolitica was 1.6%, which was very close to the prevalence of the present study. In a study from Chaharmahal Bakhtiari, 200 samples of raw milk and 200 samples of pasteurized milk were prepared by random cluster sampling and Yersinia enterocolitica was isolated in 6 cases (3%) of raw milk and 2 cases (1%) of pasteurized milk (Sharifzadeh et al., 2004). In a study by Hanifian and Khani in Tabriz, using PCR and tracing of ail and virf genes on pasteurized milk, Yersinia enterocolitica was reported (Hanifian and Khani, 2012)). Due to the sensitivity of Yersinia to high temperatures during pasteurization, the importance of secondary contamination of milk after pasteurization is notable. The enrichment of the samples was similar to the present study and the PSBB medium was used at a ratio of 25 by 225 ml. Only one case of Yersinia enterocolitica was isolated by culture method, while 16 cases were isolated by PCR method. This explains the high sensitivity difference of PCR. Although there is no difference between live and dead bacteria in PCR, the presence of Yersinia enterocolitica DNA in pasteurized milk is notable (Hanifian and Khani, 2012). In 2015, Jamali et al. conducted a study on 446 raw milk of cows, sheep, and goats in the Varamin region. They isolated 29 strains of Yersinia, 19 strains of which were Yersinia enterocolitica with biotypes of 1A / O: 9, 1B / O: 8, and 4 / O; 3. No isolates of Yersinia pseudotuberculosis were obtained (Jamali et al., 2015). In the present study, 1 Yersinia pseudotuberculosis isolate was isolated from raw milk. Unlike Yersinia pseudotuberculosis, many studies have been performed on the isolation of Yersinia enterocolitica from raw milk. In another study by Tavassoli et al., in 2019, 200 traditional kinds of cheese were randomly collected from northeastern Iran (100 samples from Khorasan Razavi and 100 samples from Golestan) and 100 samples of raw milk from Mashhad. In total, 38 Yersinia enterocolitica were obtained from cheese and 33 isolates from raw milk. All isolates belonged to two biotypes of 1A and 1B (Tavassoli et al., 2019). In a study conducted in Egypt by Darwish et al., 27 Yersinia intermedia, 13 Yersinia kristensenii, and 3 Yersinia frederiksenii were separated. Examination of their working method shows that enrichment was similar to the current study, but their selected environments included CIN agar, McConkey, and Blood agar (Darwish et al., 2015). In 2013, Bernardino-Varo et al. studied the prevalence and diversity of Yersinia species from 1,300 samples of raw milk collected from stables in Mexico City. A total of 454 samples were declared positive; this number included 44.25% of Yersinia enterocolitica, 18.26% of Yersinia kristensenii, 13.65% of Yersinia intermedia, 14.85% of Y. frederiksenii, and Yersinia aldovae 9.14% (Bernardino-Varo et al., 2013). The high prevalence of Yersinia in this study might be due to the stable environment, which may be in the process of increasing Yersinia pollution from the workplace, especially if the stables were shared with pigs that are Yersinia reservoirs. In a study in Egypt, Yersinia enterocolitica was isolated from 10% of raw milk and dairy samples (Ahmed et al., 2019). The highest separation rates were from raw milk with 22%, followed by 12%, 4%, and 2% of fermented milk, pasteurized milk, and cheese, respectively.
Conclusion
The results of this study highlight the role of milk as an important factor in the transmission of potentially pathogenic strains of Y. enterocolitica and Y. pseudotuberculosis. The findings show that tap water is effective in preventing Yersiniacontamination. Also, the presence of purebred cattle in livestock can be effective in preventing Yersinia contamination.Furthermore, the presence of O9, O: 8, O: 5 pathogenic serotypes in milk should increase attention to Yersinia in milk tests.
Acknowledgments
This article is the result of a research project approved by Tehran University of Medical Sciences and Health Services under the contract number 34878. Thanks are owed to the Vice-Chancellor of Research in Tehran University of Medical Sciences who supported this research project.
Authors' contributions
Soltan Dallal MM designed the research; Mobassei B conducted the research; Sharifi Yazdi S analyzed the data; Sharifi Yazdi S, Mirbagheri Z wrote the manuscript; Soltan Dallal MM had primary responsibility for final content. All authors read and approved the final manuscript.
Conflict of interest
The authors declared that there is no conflict of interest.
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Garedew L, Berhanu A, Mengesha D & Tsegay G 2012. Identification of gram-negative bacteria from critical control points of raw and pasteurized cow milk consumed at Gondar town and its suburbs, Ethiopia. BMC public health. 12 (1): 1-7.
Górska-Warsewicz H, Rejman K, Laskowski W & Czeczotko M 2019. Milk and dairy products and their nutritional contribution to the average polish diet. Nutrients. 11 (8): 1771.
Hahn G 1996. Pathogenic bacteria in raw milk. Situation and significance [in Germany]. International Dairy Federation.
Hanifian S & Khani S 2012. Prevalence of virulent Yersinia enterocolitica in bulk raw milk and retail cheese in northern-west of Iran. International journal of food microbiology. 155 (1-2): 89-92.
Huovinen E, et al. 2010. Symptoms and sources of Yersinia enterocolitica-infection: a case-control study. BMC infectious diseases. 10 (1): 1-9.
Jamali H, Paydar M, Radmehr B & Ismail S 2015. Prevalence, characterization, and antimicrobial resistance of Yersinia species and Yersinia enterocolitica isolated from raw milk in farm bulk tanks. Journal of dairy science. 98 (2): 798-803.
Kameyama M, Yabata J, Obane N, Otsuka H & Nomura Y 2016. Detection of pathogenic Yersinia enterocolitica in pet Djungarian hamsters in Japan. Journal of Veterinary Medical Science. 15-0654.
Martin NH, Boor KJ & Wiedmann M 2018. Symposium review: Effect of post-pasteurization contamination on fluid milk quality. Journal of dairy science. 101 (1): 861-870.
McAuley CM, McMillan K, Moore SC, Fegan N & Fox EM 2014. Prevalence and characterization of foodborne pathogens from Australian dairy farm environments. Journal of dairy science. 97 (12): 7402-7412.
Msalya G 2017. Contamination levels and identification of bacteria in milk sampled from three regions of Tanzania: evidence from literature and laboratory analyses. Veterinary Medicine International. 2017.
Muehlhoff E, Bennett A & McMahon D 2013. Milk and dairy products in human nutrition. Food and Agriculture Organization of the United Nations (FAO).
Owusu-Kwarteng J, Akabanda F, Agyei D & Jespersen L 2020. Microbial safety of milk production and fermented dairy products in Africa. Microorganisms. 8 (5): 752.
Raghiante F, dos Santos EA & Martins OA 2018. Yersinia enterocolitica in milk and dairy products: a review. Revista Brasileira de Higiene e Sanidade Animal. 12 (4): 420-427.
Rahimi E, Sepehri S, Dehkordi FS, Shaygan S & Momtaz H 2014. Prevalence of Yersinia species in traditional and commercial dairy products in Isfahan Province, Iran. Jundishapur journal of microbiology. 7 (4): e9249.
Rahman A, Bonny TS, Stonsaovapak S & Ananchaipattana C 2011. Yersinia enterocolitica: epidemiological studies and outbreaks. Journal of pathogens. 2011: 1-11.
Rivas L, Strydom H, Paine S, Wang J & Wright J 2021. Yersiniosis in New Zealand. Pathogens. 10 (2): 191.
Rosner BM, Werber D, Höhle M & Stark K 2013. Clinical aspects and self-reported symptoms of sequelae of Yersinia enterocolitica infections in a population-based study, Germany 2009–2010. BMC infectious diseases. 13 (1): 1-8.
Sharifzadeh A, Akhavan M, Zarasvandi A & Al Agha S 2004. Isolation of Yersinia entrocolitica and Listeria monocytogenes from raw and pasteurized milks supplied at dairies in Chahar mahal bakhtiary province. Iranian journal of food science and technology. 1 (1): 15-19.
Soltan-Dallal MM, Tabarraie A & MoezArdalan K 2004. Comparison of four methods for isolation of Yersinia enterocolitica from raw and pasteurized milk from northern Iran. International journal of food microbiology. 94 (1): 87-91.
Soltan Dalal MM, Akhi MT & Partou Azar AR 2009. Study of yersina enterocolitica in acute children diarrhea under frouteen years old. Medical journal of Tabriz university of medical sciences. 30 (4): 49-52.
Soltan Dallal MM & Moezardalan K 2004. Frequency of Yersinia species infection in paediatric acute diarrhoea in Tehran. Eastern Mediterranean health journal. 10 (1-2): 152-158.
Soltan Dallal MM & Salmanian H 1996. A survey on frequency of Yersinia SPP, in surface and drinking Water in Kan, Tehran. Iranian journal of infectious diseases and tropical medicine. 2: 44-49.
Soltan Dallal MM, SharifiYazdi MK & Vahedi S 2017a. The Relationship ofYersinia Isolates Bioserotypeswith Minimum Inhibitory Concentration of Ampicillin, Cefazolin, and Cefotaxime.
Soltan Dallal MM, SharifiYazdi MK & Vahedi S 2017b. The Relationship ofYersinia Isolates Bioserotypeswith Minimum Inhibitory Concentration of Ampicillin, Cefazolin, and Cefotaxime. Journal of Kerman University of medical sciences. 24 (1): 38-49.
Syczyło K, et al. 2018. The prevalence of Yersinia enterocolitica in game animals in Poland. PLoS One. 13 (3): e0195136.
Tavassoli M, Jamshidi A, Movafagh F & Afshari A 2019. Virulence Characteristics of Yersinia enterocolitica Isolated from Dairy Products in the Northeast of Iran. Journal of human, environment and health promotion. 5 (2): 72-78.
van den Brom R, de Jong A, van Engelen E, Heuvelink A & Vellema P 2020. Zoonotic risks of pathogens from sheep and their milk borne transmission. Small ruminant research. 189: 106123.
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Received: 2021/11/27 | Published: 2023/02/21 | ePublished: 2023/02/21
Received: 2021/11/27 | Published: 2023/02/21 | ePublished: 2023/02/21
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