Listeria contamination in milk-processing chain and proficiency in Listeria monocytogenes decontamination of small-scale milk retailers

Listeria contamination in milk-processing chain and proficiency in Listeria monocytogenes decontamination of small-scale milk retailers | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Listeria contamination in milk-processing chain and proficiency in Listeria monocytogenes decontamination of small-scale milk retailers Suppada Kananub, Papavarin Lertsakkongkul, Patsara Aryatawong, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1998662/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Listeria spp. is an important-foodborne bacterium. This microorganism can be discarded from milk using high temperatures such as pasteurization. The milk processing methods of many small-scale retailers lack quality control. This study was to survey Listeria contamination at the farm and retailer levels. The retailers were to be interviewed for knowledge, attitude, and practice as well. Finally, we were to determine the heating processes employed to decontaminate microorganisms by the retailers using a reference strain of L. monocytogenes . Milk samples were collected from milk collecting centers and small-scale retailers. In clinical trial, the processing measures were proved the proficiency in L. monocytogenes decontamination. One out of 99 farms presented Listeria contamination, confirmed to L. marthii . Fifty small-scale retailers participated in the second part, including 13 males and 37 females. No Listeria spp., but Staphylococcus spp. and Bacillus spp. were identified in the processed milk. Data analyses revealed that the location of the retailer was significantly associated with the volume they routinely ordered per lot and the milk-processing time the retailers used to treat milk. Knowledge on raw milk contamination is significantly associated with the stocking or processing of the whole milk lot. Processing measures pre-sale significantly depended on the sex of the retailer. The male retailer reportedly spent less time treating milk than the female retailer. L. monocytogenes was used in the trial to evaluate the efficiency of the processes. No L. monocytogenes were after sample treatment, but S. epidermidis, S. warneri, and Escherichia coli . Although the trial implied that the retailer’s processes were effective in L. monocytogenes decontamination, the study presented the inappropriate practices of the retailer and the probability of cross-contamination. Product safety from small-scale milk retailers should be monitored incessantly. Food Science & Technology food contamination milk milk treatment foodborne bacteria Listeria spp. Figures Figure 1 Figure 2 Introduction Foodborne diseases are a major global problem. International institutes such as the World Health Organization (WHO) and the Centers for Disease Control and Prevention (CDC) are concerned about listeriosis because of the infectious and severe nature of the disease [1–2]. Listeriosis is a fatal foodborne disease that can be caused by Listeria spp., especially L. monocytogenes . However, listeriosis has a low incidence in humans and animals [3–5]. Listeria spp. most likely contaminate raw milk, cheese, seafood, and frozen foods. At the farm level, Listeria spp. are contagious bacteria. Transmission normally occurs during milking and from the environment [6–8]. The prevalence of Listeria spp. in raw milk was reported in wide range such as 3–7% in North America, 0–50% in Europe, and about 5% in Iran [9–11]. In Thailand, there was not the report of Listeria contamination in raw milk [12]. Unpasteurized or improper processed milk caused three out of 36 outbreaks of listeriosis. The illness has many forms, gastroenteritis, septicaemia, and meningitis, for example. Pregnant women and their neonates, 65 years or older people, and immunosuppressed patients were susceptible groups for the disease [1–2]. According to WHO, the risk group should become aware of consumption of fresh or improper treated-milk [3, 13–14]. Raw milk is a substantial risk to human health; therefore, milk should be heated before consumption [9]. Several small-scale retailers presently buy untreated milk directly and treat it at home with processes that might not follow the protocol or be substandard. The chance of Listeria spp. infection increases owing to inefficient processes [15]. Size of dairy farms in Thailand varies from less than 10 up to over 100 milking cows per farm. Almost all farms were crossbred with a high percentage of Holstein Friesians. The annual raw milk averaged 1,200 tons between 2017 and 2022, related to the production of 11 Kg/cow/day, approximately [16] The whole raw milk has supported the consumption within the country. Milk normally is sold as pasteurization, UHT, and sterilization products, but some are uncontrollably heated and sold in small retailers. The product from milk retailers is highly risky because the quality of milk processing is not controlled. Listeria spp. can contaminate in all processes to which the condition is proper for bacterial growth, even packaging or storage after heat treatment [7, 17]. This study was to operate into three sections. The first and second objective was to survey Listeria contamination in fresh milk from farms and milk from small-scale retailers. The retailers were additionally interviewed for knowledge; attitude; and practice. For the final part, we were to determine the heating processes employed to decontaminate microorganisms by the retailers using a reference strain of L. monocytogenes . Materials And Methods Study sample description Milk was collected from three collecting milk centers, Kamphaeng Saen Dairy Co-operative; Nakhon Pathom Dairy Co-operative; and Nong Pho Dairy Co-operative at the farm level. These three centers are in central part of Thailand and received fresh milk from 3,000 farms in total. The raw milk, which passes quality check, is to be distributed to large-scale commercial dairy manufacturers or processed by the milk collecting centers. Both the large-scale dairy manufacturer and the milk collecting center treat milk by the standard measures and sell it as commercial products. However, there is still the small-scale milk retailer, which produces and sells unpasteurized products in the local area. They usually buy raw milk from dairy farms directly and treat milk with their own measures. Study design This study was designed to explore the possibility of the contamination of L. monocytogenes in the small milk-processing line. The first part focused on the raw milk from dairy farms. The next step explored the product from the small-scale milk retailers. Additionally, the perception of milk safety was interviewed by questionnaire of knowledge, attitude, and practice. Their practices in treating milk were concluded to design the treatment groups in the trial. The last step was to confirm the retailer’s treatments to see if the methods were able to decontaminate L. monocytogenes from milk. Sample size and sampling techniques The sample number was run by ProMESA 2.3.0.2 (INTA & Massey University, Castelar, Argentina). The sample size in farm level was calculated based on the disease detection at 3% prevalence of Listeria spp. [8] and population size of 3,000 farms. The sample numbers, weighted by the total number of registered farms, of Kamphaeng Saen Dairy Co-op; Nakhon Pathom Dairy Co-op; and Nong Pho Dairy Co-op, were 33; 23; and 43 samples, respectively. A simple random sampling method was used for selecting the dairy farms. In the retailer level, the number of samples was 50 retailers, who was based on the population size of 500 shops and 5% of prevalence in pasteurized milk [18]. The retailers were selected by a purposive sampling method from five provinces, which surrounded the positive farm from the first survey. The samples were in Nakhon Pathom, Ratchaburi, and Bangkok for 11 retailers each, whereas those from Kanchanaburi and Suphanburi were nine and eight retailers, respectively. Sample collection and transportation Both farm and retailer level collected milk at least 30 mL in a sterile container. The samples were kept in a cool box during transported to the Laboratory, Faculty of Veterinary Medicine, Kasetsart University, Thailand. Milk was to be kept at 4°C and analysis of Listeria spp. within 24 h. Questionnaire survey description The retailers were asked for consent to an interview. The questionnaire was about their knowledge, attitude, and practice on milk safety (Appendix). The retailers who responded to the questionnaires included 72% women and 28% men. The qualification of approximately 80% of the respondents fell under a bachelor’s degree. The data from the questionnaire were to be used in data analysis and design of the treatment groups in the trial. The retailers treated milk without monitoring temperature and time. Their processes were completed by noticing the appearance of heated milk. The retailer’s processes were classified into; 1) using double boiling until bubbles formed, 2) using double boiling until a film layer formed, 3) using double boiling for 2 min (in 100 ℃ water), 4) using direct heating until bubbles formed, and 5) using direct heating until a film layer formed. Table 1 was the data of temperature and time of each process from the trial. Table 1 Milk temperature during process and processing time for each treatment Measures* Temperature (℃) Processing time (min) Double boiling until bubbles formed 78 19 Double boiling until a film layer formed 83 24 Double boiling for 2 min (in 100 ℃ water) 75 2 Direct heating until bubbles formed 88 14 Direct heating until a film layer formed 90 35 * Each treatment operated in 1 liters of milk Experiment description The trial consisted of five treatments, following the retailer’s processes (Table 1), and control groups as pasteurization of 63 ℃ for 30 min and 72 ℃ for 15 s. Sterile milk added L. monocytogenes (ATCC ® 51414™, American Type Culture Collection, VA, USA) was used in the experiment. Each treatment was assessed with 10 5 , 10 3 , and 10 1 colony forming units [CFU]/mL of L. monocytogenes. The sample was collected in duplicates at three time points: pre-treatment, post-treatment, and 30 min post-treatment. Bacteriological test Vidas ® LDUO (bioMérieux, Marcy-l'Étoile, France), based on an enzyme-linked fluorescent assay (validated by AFNOR/ISO16140 (BIO 12/12–07/04)), was used in qualitatively screening Listeria spp. and L. monocytogenes contamination in farm level. The positive samples from Vidas ® LDUO and the samples from retailer level were cultured on ALOA ® One Day (bioMérieux, Marcy-l'Étoile, France). ALOA ® One Day is the alternative method to detect the contamination of Listeria spp. in foods and environment samples, based on chromogenic agar. This analysis technique follows ISO 11290‐1:2017. The samples from the experiment were cultured on ALOA ® One Day and brain heart infusion Agar (BHI) for identifying and enumerating Listeria spp. and L. monocytogenes . The typical appearance of Listeria spp. and L. monocytogenes on ALOA ® one day were blue green colonies and blue green colonies with opaque, respectively. Listeria spp. grow on BHI agar as white colonies. The typical colonies from both ALOA ® and BHI agar were cultured on Trypticase Soy Agar (TSA) to prepare the colonies for VITEK ® MS (bioMerieux, Marcy l’Etoile, France), based on Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) technology. VITEK ® MS reported as genus with/without species according to their mass-to-charge (m/z) comparing the database. The confidence level presented the certainty of the result. E. coli ATCC 8739 was used as a positive control. Data analysis Milk volume per lot between provinces was compared by Kruskal-Wallis test. The associations between variables were analyzed by Chi-squared test and Fisher’s exact test using Stata 13.1 (Stata Corp LLC, College Station, TX, USA). The statistical significance was set at the significance level of 0.05. Results Farm level Ninety-nine samples of bulk tank milk were collected from three locations. All samples were negative for L. monocytogenes , but 1% positive for L. marthii from the Nong Pho milk collecting center, Ratchaburi province. The contaminating species were confirmed as L. marthii . at a confidence value of 98.7%. Retailer level Fifty milk samples from small-scale retailers were detected contamination of Listeria spp. No; however, VITEK ® MS reported Staphylococcus spp. and Bacillus spp. contamination. The questionnaire results suggested that 56% of individuals had knowledge regarding the severity of raw-milk consumption, bacterial contamination in raw milk, and the necessity for treatment before consumption. Almost all retailers identified diarrhea as the most common illness associated with untreated milk. Other consequences included vomiting, fever, headache, convulsions, and flatulence. Notably, the results showed that 4% of individuals lacked awareness on the health impacts of untreated milk. Regarding milk treatment, 90% of respondents knew that bacteria could be killed by heating milk. Less than 10% understood the pasteurization process, but 25% of respondents could explain it correctly. Unfortunately, 10% of milk retailers believed milk did not require processing before sale. The data on the practices revealed that approximately half of the responders ordered milk daily, and 6% ordered weekly or at longer intervals. Dairy farms were the primary source of raw milk for 42% of retailers, and the rest received milk from co-operations or intermediaries. The median milk volume ordered for each lot was approximately 20 kg, and Q1 and Q3 were 10 and 40 kg, respectively. Thirty-five percent of retailers processed whole milk on the day of reception. Over 50% stored the non-processed milk under cold conditions and 10% froze milk. A mere 10% of the processed milk was subjected to pasteurization at controlled temperature and time pre-sale. The remaining retailers only checked the appearance of milk whilst heating to finish the treatment; they did not follow a time-controlled heating. The data from questionnaires suggested that 62% of retailers heated milk for less than 30 min. The daily leftover-processed milk was not resold by 20% of retailers. Over 50% of the retailers kept previously heated milk under cold storage, and approximately 15% froze milk. The major health concern associated with this process was gastrointestinal illness. However, only a few respondents were aware of fever and neurological problems associated with the illness. The results of the association test were presented in Table 2. The location of retail was associated with the volume of milk ordered. Retails in Bangkok significantly stocked a larger volume (p<0.05). Table 2 Test of association using Chi-squared test and Fisher ’s exact test . Variables Explanatory variables Chi 2 P value Raw milk management Milk treatment Processed milk management Sex Province Knowledge of milk contamination Knowledge of bacterial virulence Sex Province Knowledge of milk contamination Knowledge of bacterial virulence Knowledge of pasteurization Sex Province Knowledge of milk contamination Knowledge of bacterial virulence - - 7.26 - 10.38 9.88 0.004 - - - - - - 0.32 0.07 0.007 0.70 0.001 0.04 0.95 0.15 0.10 0.13 0.39 0.73 0.09 Additionally, knowledge about contamination was significantly associated with the measures for managing milk after reception (p<0.05). The knowledge about the methods by which retailers processed whole milk in a lot instead of stocking raw milk was six-fold higher than those who lacked relevant knowledge. The sex of the retailer was not associated with the knowledge that milk treatment could reduce contamination, but the decontamination practices did. Men were 18 times less likely to heat milk for less than 30 min than women (p<0.01). The practices significantly depended on the location of the retailers (p<0.05). Retailers in Bangkok were five times more likely to treat milk improperly compared to those in other locations. Table 3 Concentration (CFU/mL) of bacteria, growing on BHI agar after treatment. L. monocytogenes added in milk (CFU/mL) Time a Processes b 1 2 3 4 5 10 10 3 10 5 T1 T2 T1 T2 T1 T2 15 0 0 0 0 0 0 10 15 0 5 0 0 0 0 5 0 0 0 0 15 5 0 0 0 10 0 75 0 5 a T1: post-treatment, T2: 30 min post-treatment b 1: using double boiling until bubbles formed, 2: using double boiling until a film layer formed, 3: using double boiling for 1–2 min, 4: using direct heating until bubbles formed, and 5: using direct heating until a film layer formed. None of the retailers sold unprocessed milk. The products were treated by double boiling or direct heating before selling. The retailers prepared milk by double boiling and direct heating without checking the temperature and processing time. The appearances i.e., bubbles or layers formed after heating were used to notice that the products were ready to sell. Trial level In the trial, the typical colony of L. monocytogenes was not shown from any processes post-treatment and 30 min post-treatment; however, the white colonies grew on BHI agar (Table 3). Neither of colonies on BHI agar confirmed the presence of L. monocytogenes contamination. Vitek ® MS showed S. epidermidis, S. warneri, and E. coli at the confidence level of 99.9%. Discussion This study reported 1% of Listeria contamination in bulk tank milk and identified as L. marthii . The first isolation of this bacterium was in 2010 from the environmental samples [19] . This microorganism was classified to be “Listeria sensu strictu” as same as L. monocytogenes . The bacteria in this group share common characteristics. L. marthii was not globally distributed; however, the members in “Listeria sensu strictu” group probably identified from healthy animals and in animal-origin food [20–21]. The presence of this microorganism might imply that the environment of dairy farms in the study area was suitable for the survival of Listeria spp. L. marthii was reported to be no risk to human and animal health . This species was just found in 2010. Its characteristic and severity should be concerned continually [20]. Listeria spp. contamination incidence in bulk-tank milk was notably low in this study, however the contamination occurrence differed depending on the location [8–9]. L. monocytogenes appearance in raw milk was reported to spread from indigenous silage. Therefore, farmers should carefully consider hygiene practices and high-quality feeds [8, 22]. Improper practices during milking and post-milking on the farm level (especially for small-size farms) could cause differences in these occurrences [8, 14, 23]. Fresh milk from farms could be the source of the health risk from Listeria spp. if people consumed raw or unpasteurized milk [13–14, 24]. Additionally, bacteria could remain in milk in cases of improper cooling, cross-contamination during handling, packaging, or storage [13, 25]. Listeria spp. can still exist in food even after refrigeration because the organism can survive at low temperatures [25]. The leukocytes in milk would degrade if the raw or improper pasteurised milk were stored at 4°C for over 3 days. As a result of this deterioration, the number of heat-resistant L. monocytogenes could increase [26]. Even though the small-scale retailers were suspected of treating milk improperly, the result in this study was not to identify Listeria spp. contamination. The current result differed from the earlier articles. [15] reported approximately 18% Listeria spp. contamination in boiled milk. Listeria prevalence, even in pasteurised milk, ranged from 5 to 40% [27–28]. Raw milk consumption or improper processing is the cause of the contamination in ready-to-drink milk [15, 29]. We cultured Staphylococcus spp. and Bacillus spp. in the retailer level. The Bacillus spp. were heat-resistant species that could be killed in a wide range of temperatures and time [30]. Bacillus spp. spores may resist heat treatment. Spores from some bacilli species can be isolated in milk, even after sterilization [30–31]. Additionally, spores are commonly found in the environment, including soil, dust, air, and surfaces [32]. Staphylococcus spp. are normal microbiota on human skin and mucous membranes [33]; however, its contamination possibly resulted in cross-contamination. Many studies have elucidated the contamination of Staphylococcus spp., especially S. aureus, and its severity on human health [17, 34–35]. Pasteurization products were identified as the source of S. aureus contamination. The incidence of S. aureus contamination in pasteurization was 4% in China, while that in South Africa was high, up to 20% [36–37]. Milk from the retailers needed to be concerned about safety, even with no Listeria spp. being shown from milk. The bacteria in milk might be the result of cross-contamination from environment and human, or improper processing steps. The sample collection was the limitation in this part. Milk from retailers was analyzed only one time in the different period of the sample collection at the farm level. The result was like the snapshot situation of Listeria contamination. The location was associated with the milk volume of each lot that the retailer ordered and their pasteurization practices. The order volume is related to the number of dairy farms in the area. Retailers in Bangkok may have a larger stock than other locations because of the transport limitations. The magnitude of milk treatment was unexpectedly related to location. People with unawareness of the risk of milk-borne pathogens were twice as likely to contract abdominal illness than those who were aware [38]. Earlier articles showed the risk of consuming raw milk as well as the products produced from unpasteurized milk on human health [4, 39–40]. With quality-uncontrolled processes, public perceptions of the product safety were certainly doubtful. Surprisingly, we had reason to say that the five milk-processing measures effectively destroyed L. monocytogenes because of no appearance of L. monocytogenes . The trial revealed that all processes reached the pasteurization condition including temperature and time; however, the temperature and processing time in this study might not be the same as the retailers because each person might consider stopping heating differently. L. monocytogenes would be reduced to greater than log 10 6.9 mL -1 after heating at 65.5°C for 15 s [26]. However, [41] reported that low temperature, long-time pasteurization at 63°C for 30 min or high temperature and short time could make a negligible log 10 2 mL -1 reduction. This variation implied the possibility of failure in milk treatment. The low pathogen load following treatment could also be attributed to the lack of detectable Listeria spp. in the culture. In the experiment, we isolated S. epidermidis, S. warneri, and E. coli, which may result from cross-contamination. Staphylococcus spp. is usually found on the skin and surfaces [32]. E. coli is a human pathogen that causes gastrointestinal illness. Typically, the source is the host intestine; however, E. coli can persist on surfaces [1, 42]. Pasteurized milk was the source of up to 9% E. coli contamination [43]. Milk processed with inadequate measures presented approximately two-fold higher E. coli numbers than adequate treatment measures [44]. According to cross-contamination suspected, the processes after heating treatment were strictly hygienic [2, 17, 43, 45]. This study collected samples from farms and retailers only one time. The incidence of Listeria contamination might be underestimated. The retailers notice the bubble or film layer to complete the milk heating process, that the temperature and time might highly deviate from person to person. The temperature and processing time in the trial might differ from the retailers used. Listeria monocytogenes in the experiment were lastly interrupted by other bacteria from cross-contamination. Conclusions In conclusion, on a farm level, which was the main source of raw milk observed 1% Listeria incidence, whereas no identification of Listeria spp . on the retailer level and post processed milk in trial. The presence of Listeria spp. at farm level warns people about the risk of raw or improper-treated milk to human health, even low incidence of Listeria. Milk should be processed following the method of pasteurization. The retailers should prevent cross-contamination during other steps after treatment as well. Finally, the responsible organizations should incessantly educate the milk retailers in adequate protocols as well as routinely check their product safety. Declarations Data availability Datasets of the current research are available at https://doi.org/10.5281/zenodo.7014148 . Conflicts of interest This study has nonfinancial competing interests. Funding statement This research was financially supported by the Start-up Research Fund: SRF, Faculty of Veterinary Medicine, Kasetsart University (SRF63_01). Acknowledgements We sincerely thank all farmers and small-scale retailers who kindly consent to sample collection. We would like to thank Editage (www.editage.com) for English language editing. Preprint is readily available from https://www.researchsquare.com/article/rs-1998662/v1 [46]. 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Janstova, and J. Lukasova, “Heat resistance of Bacillus spp. spores isolated from cow’s milk and environment,” Journal of the University of Veterinary Sciences Brno , vol. 70, no. 2, pp. 179–184, 2001. M. Heyndrickx, A. Coorevits, P. Scheldeman, L. Lebbe, P. Schumann, M. Rodríguez-Diaz, G. Forsyth, A. Dinsdale, J. Heyrman, N.A. Logan, and P.D. Vos, “Emended descriptions of Bacillus sporothermodurans and Bacillus oleronius with the inclusion of dairy farm isolates of both species,” International Journal of Systematic and Evolutionary Microbiology , vol. 62, pp. 307–314, 2012. S. Sarkar, “Microbiological considerations: pasteurized milk,” International Journal of Dairy Sciences , vol. 10, pp. 206–218, 2015. U. Ribič, A. Klančnik, and B. Jeršek, “Characterization of Staphylococcus epidermidis strains isolated from industrial cleanrooms under regular routine disinfection,” Journal of Applied Microbiolog y, vol. 122, pp. 1186–1196, 2017. R. Oliveira, E. Pinho, G. Almeida, N.F. Azevedo, and C. Almeida, “Prevalence and diversity of Staphylococcus aureus and Staphylococcal Enterotoxins in raw milk from Northern Portugal,” Frontiers in Microbiology , vol. 13, 846653, 2022. Y. Titouche, M. Akkou, K. Houali, F. Auvray, and J.A. Hennekinne, “Role of milk and milk products in the spread of methicillin-resistant Staphylococcus aureus in the dairy production chain,” Journal of Food Science , pp. 1–25, 2022. J. Dai, S. Wu1, J. Huang, Q. Wu, F. Zhang, J. Zhang, J. Wang, Y. Ding, S. Zhang, X. Yang, T. Lei, L. Xue1, and H. Wu, “Prevalence and characterization of Staphylococcus aureus isolated from pasteurized milk in China,” Frontiers in Microbiology , vol. 1, pp. 1–10, 2019. M.J. Machria, “Prevalence, susceptibility patterns and risk factors associated with Staphylococcus aureus presence in marketed milk and milk products within Nairobi city country, Kenya,” Kenyatta University, Nairobi, Kenya, 2016, Master Thesis. R. Fagnani, L.A. Nero, and C.P. Rosolem, “Why knowledge is the best way to reduce the risks associated with raw milk and raw milk products,” Journal of Dairy Research , vol. 88, no. 2, pp. 238–243, 2021. D.J. D’Amico, E. Groves, and C.W. Donnelly, “Low Incidence of Foodborne Pathogens of Concern in Raw Milk Utilized for Farmstead Cheese Production,” Journal of Food Protection , vol. 71, no. 8, pp. 1580–1589, 2008. D.R. Bangieva and V.N. Rusev, “Prevalence of Listeria monocytogenes in raw cow milk – a review,” Bulgarian Journal of Veterinary Medicine , vol. 20, no. 1, pp. 430–436, 2017. V.S. Jayamanne and U. Samarajeewa, “Evaluation of the heat resistance of pathogenic Listeria monocytogenes in milk and milk products in Sri Lanka,” Tropical Agricultural Research and Extension , vol. 13, no. 3, pp. 73–80, 2010. J. Jang, H.-G. Hur, M.J. Sadowsky, M.N. Byappanahalli, T. Yan, and S. Ishii “Environmental Escherichia coli: ecology and public health implications-a review,” Journal of Applied Microbiology , vol. 123, no. 3, pp. 570–581, 2017. M. Vahedi, M. Nasrolahei, M. Sharif, and A.M. Mirabi, “Bacteriological study of raw and unexpired pasteurized cow’s milk collected at the dairy farms and super markets in Sari city in 2011,” Journal of Preventive Medicine and Hygiene , vol. 54, 120–123, 2013. A.S. Paraffin, T. J. Zindove, and M. Chimonyo, “Effect of structural condition of milk processing facilities and food safety systems on Escherichia coli and Coliforms presence in cultured buttermilk,” Journal of Food Quality , 7365983; 10.1155/2019/7365983, 2019. 45. M. Kannaiyan, C. Uma, S. Paramasivam, and S. Kumar, “Occurrence of Listeria monocytogenes in milk and milk products,” International Journal of Current Research in Life Sciences , vol. 7, pp. 1572–1574, 2018. 46. S. Kananub, P. Lertsakkongkul, P. Aryatawong, W. Horhirunkhajohn, N. Pinniam, P. Krajanglikit, K. Sonthong, and S. Kasemsuwan “Listeria species contamination in the milk-processing chain and decontamination proficiency of small-scale milk retailers,” Research Square . Preprint [Version 1]. September 1, 2022. https://www.researchsquare.com/article/rs-1998662/v1. Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1998662","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":133498181,"identity":"b5d7fbbe-6820-4b66-b1cb-9875a07e40b9","order_by":0,"name":"Suppada 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15:59:10","currentVersionCode":2,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-1998662/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-1998662/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49483796,"identity":"70bc67e2-9ef3-4321-bc0f-86f2600467c6","added_by":"auto","created_at":"2024-01-11 15:48:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":439017,"visible":true,"origin":"","legend":"\u003cp\u003eColonies on ALOA® agar (a) and brain heart infusion (BHI) agar (b).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-1998662/v2/e65d6c59e8ac411352b6545f.png"},{"id":49483798,"identity":"1099b28e-ed58-4b3e-b49c-afba2bfc82ba","added_by":"auto","created_at":"2024-01-11 15:48:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":752586,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of retailers that colour intensity representing the number of dairy farms.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-1998662/v2/1355a0ffd4f55a33bead0e83.png"},{"id":49483973,"identity":"e1f78cc8-4132-4533-8f2f-a15dbc8e1307","added_by":"auto","created_at":"2024-01-11 15:56:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1322927,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1998662/v2/68342f46-6666-48a3-af25-81363f0b5999.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eListeria \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003econtamination in milk-processing chain and proficiency in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eListeria monocytogenes \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003edecontamination of small-scale milk retailers\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFoodborne diseases are a major global problem. International institutes such as the World Health Organization (WHO) and the Centers for Disease Control and Prevention (CDC) are concerned about listeriosis because of the infectious and severe nature of the disease [1\u0026ndash;2]. Listeriosis is a fatal foodborne disease that can be caused by \u003cem\u003eListeria\u003c/em\u003e spp., especially \u003cem\u003eL. monocytogenes\u003c/em\u003e. However, listeriosis has a low incidence in humans and animals [3\u0026ndash;5].\u003cem\u003e\u0026nbsp;Listeria\u003c/em\u003e spp. most likely contaminate raw milk, cheese, seafood, and frozen foods. At the farm level, \u003cem\u003eListeria\u003c/em\u003e spp. are contagious bacteria. Transmission normally occurs during milking and from the environment [6\u0026ndash;8]. The prevalence of \u003cem\u003eListeria\u0026nbsp;\u003c/em\u003espp. in raw milk was reported in wide range such as 3\u0026ndash;7% in North America, 0\u0026ndash;50% in Europe, and about 5% in Iran [9\u0026ndash;11]. In Thailand, there was not the report of \u003cem\u003eListeria\u003c/em\u003e contamination in raw milk [12]. Unpasteurized or improper processed milk caused three out of 36 outbreaks of listeriosis. The illness has many forms, gastroenteritis, septicaemia, and meningitis, for example. Pregnant women and their neonates, 65 years or older people, and immunosuppressed patients were susceptible groups for the disease [1\u0026ndash;2]. According to WHO, the risk group should become aware of consumption of fresh or improper treated-milk [3, 13\u0026ndash;14].\u003c/p\u003e\n\u003cp\u003eRaw milk is a substantial risk to human health; therefore, milk should be heated before consumption [9]. Several small-scale retailers presently buy untreated milk directly and treat it at home with processes that might not follow the protocol or be substandard. The chance of \u003cem\u003eListeria\u003c/em\u003e spp. infection increases owing to inefficient processes [15].\u003c/p\u003e\n\u003cp\u003eSize of dairy farms in Thailand varies from less than 10 up to over 100 milking cows per farm. Almost all farms were crossbred with a high percentage of Holstein Friesians. The annual raw milk averaged 1,200 tons between 2017 and 2022, related to the production of 11 Kg/cow/day, approximately [16] The whole raw milk has supported the consumption within the country. Milk normally is sold as pasteurization, UHT, and sterilization products, but some are uncontrollably heated and sold in small retailers. The product from milk retailers is highly risky because the quality of milk processing is not controlled. \u003cem\u003eListeria\u003c/em\u003e spp. can contaminate in all processes to which the condition is proper for bacterial growth, even packaging or storage after heat treatment [7, 17].\u003c/p\u003e\n\u003cp\u003eThis study was to operate into three sections. The first and second objective was to survey \u003cem\u003eListeria\u003c/em\u003e contamination\u0026nbsp;in\u0026nbsp;fresh milk from farms and milk from small-scale retailers. The retailers were additionally interviewed for knowledge; attitude; and practice.\u0026nbsp;For the final part, we were to determine the heating processes employed to decontaminate microorganisms by the retailers using a reference strain of \u003cem\u003eL. monocytogenes\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cem\u003eStudy sample description\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMilk was collected from three collecting milk centers, Kamphaeng Saen Dairy Co-operative; Nakhon Pathom Dairy Co-operative; and Nong Pho Dairy Co-operative at the farm level. These three centers are in central part of Thailand and received fresh milk from 3,000 farms in total. The raw milk, which passes quality check, is to be distributed to large-scale commercial dairy manufacturers or processed by the milk collecting centers. Both\u0026nbsp;the\u0026nbsp;large-scale dairy manufacturer and the milk collecting center treat milk by the standard measures and sell it as commercial products. However, there is still the small-scale milk retailer, which produces and sells unpasteurized products in the local area. They usually buy raw milk from dairy farms directly and treat milk with their own measures.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStudy design\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis study was designed to explore the possibility of the contamination of \u003cem\u003eL. monocytogenes\u003c/em\u003e in the small milk-processing line. The first part focused on the raw milk from dairy farms. The next step explored the product from the small-scale milk retailers. Additionally, the perception of milk safety was interviewed by questionnaire of knowledge, attitude, and practice. Their practices in treating milk were concluded to design the treatment groups in the trial. The last step was to confirm the retailer\u0026rsquo;s treatments to see if the methods were able to decontaminate \u003cem\u003eL. monocytogenes\u003c/em\u003e from milk.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSample size and sampling techniques\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe sample number was run by ProMESA 2.3.0.2 (INTA \u0026amp; Massey University, Castelar, Argentina).\u0026nbsp;The sample size in\u0026nbsp;farm level was calculated based on the disease detection at 3% prevalence of \u003cem\u003eListeria\u003c/em\u003e spp. [8] and\u0026nbsp;population\u0026nbsp;size of 3,000 farms. The sample numbers, weighted by the\u0026nbsp;total\u0026nbsp;number of registered farms, of Kamphaeng Saen Dairy Co-op; Nakhon Pathom Dairy Co-op; and Nong Pho Dairy Co-op, were 33; 23; and 43 samples, respectively. A simple random sampling method was used for selecting the dairy farms.\u003c/p\u003e\n\u003cp\u003eIn the retailer level, the number of samples was 50 retailers, who was based on the population size of 500 shops and 5% of prevalence in pasteurized milk [18]. The retailers were selected by a purposive sampling method from five provinces, which surrounded the positive farm from the first survey. The samples were in Nakhon Pathom, Ratchaburi, and Bangkok for 11 retailers each, whereas those from Kanchanaburi and Suphanburi were nine and eight retailers, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSample collection and transportation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBoth farm and retailer level collected milk at least 30 mL in a sterile container. The samples were kept in a cool box during transported to the Laboratory, Faculty of Veterinary Medicine, Kasetsart University, Thailand. Milk was to be kept at 4\u0026deg;C and analysis of \u003cem\u003eListeria\u003c/em\u003e spp. within 24 h.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eQuestionnaire survey description\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe retailers were asked for consent to an interview. The questionnaire was about their knowledge, attitude, and practice on milk safety (Appendix). The retailers who responded to the questionnaires included 72% women and 28% men. The qualification of approximately 80% of the respondents fell under a bachelor\u0026rsquo;s degree. The data from the questionnaire were to be used in data analysis and\u0026nbsp;design of the treatment groups in the trial.\u003c/p\u003e\n\u003cp\u003eThe retailers treated milk without monitoring temperature and time. Their processes were completed by noticing the appearance of heated milk. The retailer\u0026rsquo;s processes were classified into; 1) using double boiling until bubbles formed, 2) using double boiling until a film layer formed, 3) using double boiling for 2 min (in 100 ℃ water), 4) using direct heating until bubbles formed, and 5) using direct heating until a film layer formed. Table 1 was the data of temperature and time of each process from the trial.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eTable 1 \u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eMilk temperature during process and processing time for each treatment\u003c/span\u003e\u003c/p\u003e\n\u003cdiv align=\"center\" style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\n \u003ctable style=\"border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:219.75pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eMeasures*\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:106.35pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eTemperature (℃)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:127.35pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eProcessing time (min)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:219.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eDouble boiling until bubbles formed\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:106.35pt;border:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e78\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:127.35pt;border:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e19\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:219.75pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eDouble boiling until a film layer formed\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:106.35pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e83\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:127.35pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e24\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:219.75pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eDouble boiling for 2 min\u0026nbsp;\u003c/span\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e(in 100\u0026nbsp;\u003c/span\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e℃\u003c/span\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;water)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:106.35pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e75\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:127.35pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:219.75pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eDirect heating until bubbles formed\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:106.35pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e88\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:127.35pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e14\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:219.75pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eDirect heating until a film layer formed\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:106.35pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e90\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:127.35pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e35\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e*\u0026nbsp;\u003c/span\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eEach treatment operated in 1 liters of milk\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eExperiment description\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe trial consisted of five treatments, following\u0026nbsp;the retailer\u0026rsquo;s processes (Table 1), and control groups as pasteurization of 63 ℃ for 30 min and 72 ℃ for 15 s. Sterile milk added\u0026nbsp;\u003cem\u003eL. monocytogenes\u003c/em\u003e (ATCC\u003csup\u003e\u0026reg;\u003c/sup\u003e 51414\u0026trade;, American Type Culture Collection, VA, USA) was used in the experiment. Each treatment was assessed\u0026nbsp;with\u0026nbsp;10\u003csup\u003e5\u003c/sup\u003e, 10\u003csup\u003e3\u003c/sup\u003e,\u003csup\u003e\u0026nbsp;\u003c/sup\u003eand 10\u003csup\u003e1\u003c/sup\u003e colony forming units [CFU]/mL of \u003cem\u003eL. monocytogenes.\u0026nbsp;\u003c/em\u003eThe sample was collected in duplicates at three time points: pre-treatment, post-treatment, and 30 min post-treatment.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBacteriological test\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eVidas\u003csup\u003e\u0026reg;\u003c/sup\u003e LDUO (bioM\u0026eacute;rieux, Marcy-l\u0026apos;\u0026Eacute;toile, France), based on an enzyme-linked fluorescent assay (validated by AFNOR/ISO16140 (BIO 12/12\u0026ndash;07/04)), was used in qualitatively screening \u003cem\u003eListeria\u0026nbsp;\u003c/em\u003espp. and \u003cem\u003eL. monocytogenes\u003c/em\u003e contamination in farm level. The positive samples from Vidas\u003csup\u003e\u0026reg;\u003c/sup\u003e LDUO and the samples from retailer level were cultured on ALOA\u003csup\u003e\u0026reg;\u003c/sup\u003e One Day (bioM\u0026eacute;rieux, Marcy-l\u0026apos;\u0026Eacute;toile, France). ALOA\u003csup\u003e\u0026reg;\u003c/sup\u003e One Day is the alternative method to detect the contamination of \u003cem\u003eListeria\u003c/em\u003e spp. in foods and environment samples, based on chromogenic agar. This analysis technique follows ISO 11290‐1:2017.\u003c/p\u003e\n\u003cp\u003eThe samples from the experiment were cultured on ALOA\u003csup\u003e\u0026reg;\u003c/sup\u003e One Day and brain heart infusion Agar (BHI) for identifying and enumerating \u003cem\u003eListeria\u0026nbsp;\u003c/em\u003espp. and \u003cem\u003eL. monocytogenes\u003c/em\u003e. The typical appearance of \u003cem\u003eListeria\u0026nbsp;\u003c/em\u003espp. and \u003cem\u003eL. monocytogenes\u003c/em\u003e on ALOA\u003csup\u003e\u0026reg;\u003c/sup\u003e one day were\u003cem\u003e\u0026nbsp;\u003c/em\u003eblue green colonies and blue green colonies with opaque, respectively. \u003cem\u003eListeria\u0026nbsp;\u003c/em\u003espp. grow on BHI agar as white colonies.\u003c/p\u003e\n\u003cp\u003eThe typical colonies from both ALOA\u003csup\u003e\u0026reg;\u0026nbsp;\u003c/sup\u003eand\u0026nbsp;BHI agar were cultured on Trypticase Soy Agar (TSA) to prepare the colonies for VITEK\u003csup\u003e\u0026reg;\u003c/sup\u003e MS (bioMerieux, Marcy l\u0026rsquo;Etoile, France), based on Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) technology. VITEK\u003csup\u003e\u0026reg;\u003c/sup\u003e MS reported as genus with/without species according to their mass-to-charge (m/z) comparing the database. The confidence level presented the certainty of the result. \u0026nbsp;\u003cem\u003eE. coli\u003c/em\u003e ATCC 8739 was used as a positive control.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eData analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMilk volume per lot between provinces was compared by Kruskal-Wallis test. The associations between variables were analyzed by Chi-squared test and Fisher\u0026rsquo;s exact test using Stata 13.1 (Stata Corp LLC, College Station, TX, USA). The statistical significance was set at the significance level of 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eFarm level\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNinety-nine samples of bulk tank milk were collected from three locations. All samples were negative for \u003cem\u003eL. monocytogenes\u003c/em\u003e, but 1% positive for \u003cem\u003eL. marthii\u003c/em\u003e from the Nong Pho milk collecting center, Ratchaburi province. The contaminating species were confirmed as \u003cem\u003eL. marthii\u003c/em\u003e. at a confidence value of 98.7%.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eRetailer level\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFifty milk samples from small-scale retailers were detected contamination of \u003cem\u003eListeria\u0026nbsp;\u003c/em\u003espp. No; however, VITEK\u003csup\u003e\u0026reg;\u003c/sup\u003e MS reported \u003cem\u003eStaphylococcus\u003c/em\u003e spp. and \u003cem\u003eBacillus\u0026nbsp;\u003c/em\u003espp. contamination.\u003c/p\u003e\n\u003cp\u003eThe questionnaire results suggested that 56% of individuals had knowledge regarding the severity of raw-milk consumption, bacterial contamination in raw milk, and the necessity for treatment before consumption. Almost all retailers identified diarrhea as the most common illness associated with untreated milk. Other consequences included vomiting, fever, headache, convulsions, and flatulence. Notably, the results showed that 4% of individuals lacked awareness on the health impacts of untreated milk.\u003c/p\u003e\n\u003cp\u003eRegarding milk treatment, 90% of respondents knew that bacteria could be killed by heating milk. Less than 10% understood the pasteurization process, but 25% of respondents could explain it correctly. Unfortunately, 10% of milk retailers believed milk did not require processing before sale.\u003c/p\u003e\n\u003cp\u003eThe data on the practices revealed that approximately half of the responders ordered milk daily, and 6% ordered weekly or at longer intervals. Dairy farms were the primary source of raw milk for 42% of retailers, and the rest received milk from co-operations or intermediaries. The median milk volume ordered for each lot was approximately 20 kg, and Q1 and Q3 were 10 and 40 kg, respectively. Thirty-five percent of retailers processed whole milk on the day of reception. Over 50% stored the non-processed milk under cold conditions and 10% froze milk. A mere 10% of the processed milk was subjected to pasteurization at controlled temperature and time pre-sale. The remaining retailers only checked the appearance of milk whilst heating to finish the treatment; they did not follow a time-controlled heating.\u003c/p\u003e\n\u003cp\u003eThe data from questionnaires suggested that 62% of retailers heated\u0026nbsp;milk\u0026nbsp;for less than 30 min. The daily leftover-processed milk was not resold by 20% of retailers. Over 50% of the retailers kept previously heated milk under cold storage, and approximately 15% froze milk. The major health concern associated with this process was gastrointestinal illness. However, only a few respondents were aware of fever and neurological problems associated with the illness.\u003c/p\u003e\n\u003cp\u003eThe results of the association test were presented in Table 2. The location of retail was associated with the volume of milk ordered. Retails in Bangkok significantly stocked a larger volume (p\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eTable 2\u003c/span\u003e\u003c/strong\u003e\u003cem\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u0026nbsp;\u003c/span\u003e\u003c/em\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eTest of association using Chi-squared \u003cem\u003etest\u0026nbsp;\u003c/em\u003eand Fisher\u003cem\u003e\u0026rsquo;s\u003c/em\u003e exact test\u003cem\u003e.\u003c/em\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cdiv align=\"center\" style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\n \u003ctable style=\"width:6.3in;border-collapse:collapse;border:none;\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 155.95pt;border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5.4pt;height: 30.6pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height: 150%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height: 150%;font-family:\"Times New Roman\",serif;'\u003eVariables\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 184.25pt;border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5.4pt;height: 30.6pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height: 150%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height: 150%;font-family:\"Times New Roman\",serif;'\u003eExplanatory variables\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5.4pt;height: 30.6pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height: 150%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height: 150%;font-family:\"Times New Roman\",serif;'\u003eChi\u003csup\u003e2\u003c/sup\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5.4pt;height: 30.6pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height: 150%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height: 150%;font-family:\"Times New Roman\",serif;'\u003eP value\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 155.95pt;border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 0in 5.4pt;height: 49.95pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eRaw milk management\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eMilk treatment\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eProcessed milk management\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 184.25pt;border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 0in 5.4pt;height: 49.95pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eSex\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eProvince\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-indent:1.45pt;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eKnowledge of milk contamination\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-indent:1.45pt;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eKnowledge of bacterial virulence\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eSex\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eProvince\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-indent:1.45pt;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eKnowledge of milk contamination\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-indent:1.45pt;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eKnowledge of bacterial virulence\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eKnowledge of pasteurization\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eSex\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eProvince\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-indent:1.45pt;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eKnowledge of milk contamination\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-indent:1.45pt;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eKnowledge of bacterial virulence\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 0in 5.4pt;height: 49.95pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e-\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e-\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e7.26\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e-\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e10.38\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e9.88\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e0.004\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e-\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e-\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e-\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e-\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e-\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e-\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border-width: medium medium 1pt;border-style: none none solid;border-color: currentcolor currentcolor windowtext;border-image: none;padding: 0in 5.4pt;height: 49.95pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e0.32\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e0.07\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e0.007\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e0.70\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e0.001\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e0.04\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e0.95\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e0.15\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e0.10\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e0.13\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e0.39\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e0.73\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e0.09\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:200%;'\u003e\u003cspan style=\"line-height:200%;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eAdditionally, knowledge about contamination was significantly associated with the measures for managing milk after reception (p\u0026lt;0.05). The knowledge about the methods by which retailers processed whole milk in a lot instead of stocking raw milk was six-fold higher than those who lacked relevant knowledge. The sex of the retailer was not associated with the knowledge that milk treatment could reduce contamination, but the decontamination practices did. Men were 18 times less likely to heat milk for less than 30 min than women (p\u0026lt;0.01). The practices significantly depended on the location of the retailers (p\u0026lt;0.05). Retailers in Bangkok were five times more likely to treat milk improperly compared to those in other locations.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:200%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eTable 3 \u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eConcentration\u0026nbsp;\u003c/span\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e(CFU/mL)\u003c/span\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eof bacteria, growing on BHI agar after treatment.\u003c/span\u003e\u003c/p\u003e\n\u003cdiv align=\"center\" style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\n \u003ctable style=\"width:425.25pt;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width:155.95pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cem\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eL. monocytogenes\u003c/span\u003e\u003c/em\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eadded in milk (CFU/mL)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:71.45pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eTime \u003csup\u003ea\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" style=\"width:197.85pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eProcesses \u003csup\u003eb\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:41.95pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.45pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.5pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.45pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.5pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:155.95pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e10\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New 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style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:200%;'\u003e\u003csup\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003ea\u0026nbsp;\u003c/span\u003e\u003c/sup\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eT1: post-treatment, T2: 30 min post-treatment\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:200%;'\u003e\u003csup\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eb\u003c/span\u003e\u003c/sup\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;1: using double boiling until bubbles formed, 2: using double boiling until a film layer formed, \u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp; 3: using double boiling for 1\u0026ndash;2 min, 4: using direct heating until bubbles formed, and 5: using\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:200%;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp; direct heating until a film layer formed.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eNone of the retailers sold unprocessed milk. The products were treated by double boiling or direct heating before selling. The retailers prepared milk by double boiling and direct heating without checking the temperature and processing time. The appearances i.e., bubbles or layers formed after heating were used to notice that the products were ready to sell.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTrial level\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn the trial, the typical colony of \u003cem\u003eL. monocytogenes\u003c/em\u003e was not shown from any processes post-treatment and 30 min post-treatment; however, the white colonies grew on BHI agar (Table 3). Neither of colonies on BHI agar confirmed the presence of \u003cem\u003eL.\u003c/em\u003e \u003cem\u003emonocytogenes\u0026nbsp;\u003c/em\u003econtamination. Vitek\u003csup\u003e\u0026reg;\u003c/sup\u003e MS showed \u003cem\u003eS. epidermidis, S. warneri,\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;E. coli\u0026nbsp;\u003c/em\u003eat the confidence level of 99.9%.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study reported 1% of \u003cem\u003eListeria\u003c/em\u003e contamination in bulk tank milk and identified as\u003cem\u003e\u0026nbsp;L. marthii\u003c/em\u003e. The first isolation of this bacterium was in 2010 from the environmental samples [19]\u003cstrong\u003e.\u003c/strong\u003e This microorganism was classified to be \u0026ldquo;Listeria sensu strictu\u0026rdquo; as same as \u003cem\u003eL. monocytogenes\u003c/em\u003e. The bacteria in this group share common characteristics. \u003cem\u003eL. marthii\u0026nbsp;\u003c/em\u003ewas not globally distributed; however, the members in \u0026ldquo;Listeria sensu strictu\u0026rdquo; group probably identified from healthy animals and in animal-origin food [20\u0026ndash;21]. The presence of this microorganism might imply that the environment of dairy farms in the study area was suitable for the survival of \u003cem\u003eListeria\u0026nbsp;\u003c/em\u003espp. \u003cem\u003eL. marthii\u0026nbsp;\u003c/em\u003ewas reported to be no risk to human and animal health\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eThis species was just found in 2010. Its characteristic and severity should be concerned continually [20].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eListeria\u003c/em\u003e spp. contamination incidence in bulk-tank milk was notably low in this study, however the contamination occurrence differed depending on the location [8\u0026ndash;9]. \u003cem\u003eL. monocytogenes\u003c/em\u003e appearance in raw milk was reported to spread from indigenous silage. Therefore, farmers should carefully consider hygiene practices and high-quality feeds [8, 22]. Improper practices during milking and post-milking on the farm level (especially for small-size farms) could cause differences in these occurrences [8, 14, 23]. Fresh milk from farms could be the source of the health risk from \u003cem\u003eListeria\u003c/em\u003e spp. if people consumed raw or unpasteurized milk [13\u0026ndash;14, 24].\u003c/p\u003e\n\u003cp\u003eAdditionally, bacteria could remain in milk in cases of improper cooling, cross-contamination during handling, packaging, or storage [13, 25]. \u003cem\u003eListeria\u003c/em\u003e spp. can still exist in food even after refrigeration because the organism can survive at low temperatures [25].\u003c/p\u003e\n\u003cp\u003eThe leukocytes in milk would degrade if the raw or improper pasteurised milk were stored at 4\u0026deg;C for over 3 days. As a result of this deterioration, the number of heat-resistant \u003cem\u003eL. monocytogenes\u0026nbsp;\u003c/em\u003ecould increase [26]. Even though the small-scale retailers were suspected of treating milk improperly, the result in this study was not to identify \u003cem\u003eListeria\u003c/em\u003e spp. contamination. The current result differed from the earlier articles. [15] reported approximately 18% \u003cem\u003eListeria\u003c/em\u003e spp. contamination in boiled milk. \u003cem\u003eListeria\u003c/em\u003e prevalence, even in pasteurised milk, ranged from 5 to 40% [27\u0026ndash;28]. Raw milk consumption or improper processing is the cause of the contamination in ready-to-drink milk [15, 29].\u003c/p\u003e\n\u003cp\u003eWe cultured \u003cem\u003eStaphylococcus\u003c/em\u003e spp. and \u003cem\u003eBacillus\u0026nbsp;\u003c/em\u003espp. in the retailer level. The \u003cem\u003eBacillus\u003c/em\u003e spp. were heat-resistant species that could be killed in a wide range of temperatures and time [30]. \u003cem\u003eBacillus\u003c/em\u003e spp. spores may resist heat treatment. Spores from some bacilli species can be isolated in milk, even after sterilization [30\u0026ndash;31]. Additionally, spores are commonly found in the environment, including soil, dust, air, and surfaces [32].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStaphylococcus\u003c/em\u003e spp. are normal microbiota on human skin and mucous membranes [33]; however, its contamination possibly resulted in cross-contamination. Many studies have elucidated the contamination of \u003cem\u003eStaphylococcus\u0026nbsp;\u003c/em\u003espp., especially \u003cem\u003eS. aureus,\u0026nbsp;\u003c/em\u003eand its severity on human health [17, 34\u0026ndash;35]. Pasteurization products were identified as the source of \u003cem\u003eS. aureus\u003c/em\u003e contamination. The incidence of \u003cem\u003eS. aureus\u003c/em\u003e contamination in pasteurization was 4% in China, while that in South Africa was high, up to 20% [36\u0026ndash;37].\u003c/p\u003e\n\u003cp\u003eMilk from the retailers needed to be concerned about safety,\u0026nbsp;even with no \u003cem\u003eListeria\u0026nbsp;\u003c/em\u003espp. being shown from milk. The bacteria in milk might be the result of cross-contamination from environment and human, or improper processing steps. The sample collection was the limitation in this part. Milk from retailers was analyzed only one time in the different period of the sample collection at the farm level. The result was like the snapshot situation of \u003cem\u003eListeria\u003c/em\u003e contamination.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe location was associated with the milk volume of each lot that the retailer ordered and their pasteurization practices. The order volume is related to the number of dairy farms in the area. Retailers in Bangkok may have a larger stock than other locations because of the transport limitations. The magnitude of milk treatment was unexpectedly related to location. People with unawareness of the risk of milk-borne pathogens were twice as likely to contract abdominal illness than those who were aware [38].\u003c/p\u003e\n\u003cp\u003eEarlier articles showed the risk of consuming raw milk as well as the products produced from unpasteurized milk on human health [4, 39\u0026ndash;40]. With quality-uncontrolled processes, public perceptions of the product safety were certainly doubtful. Surprisingly, we had reason to say that the five milk-processing measures effectively destroyed \u003cem\u003eL. monocytogenes\u003c/em\u003e because of no appearance of \u003cem\u003eL. monocytogenes\u003c/em\u003e. The trial revealed that all processes reached the pasteurization condition including temperature and time; however, the temperature and processing time in this study might not be the same as the retailers because each person might consider stopping heating differently.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eL. monocytogenes\u003c/em\u003e would be reduced to greater than log\u003csub\u003e10\u0026nbsp;\u003c/sub\u003e6.9 mL\u003csup\u003e-1\u003c/sup\u003e after heating at 65.5\u0026deg;C for 15 s [26]. However, [41] reported that low temperature, long-time pasteurization at 63\u0026deg;C for 30 min or high temperature and short time could make a negligible log\u003csub\u003e10\u0026nbsp;\u003c/sub\u003e2 mL\u003csup\u003e-1\u003c/sup\u003e reduction. This variation implied the possibility of failure in milk treatment. The low pathogen load following treatment could also be attributed to the lack of detectable \u003cem\u003eListeria\u003c/em\u003e spp. in the culture.\u003c/p\u003e\n\u003cp\u003eIn the experiment, we isolated \u003cem\u003eS. epidermidis, S. warneri,\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;E. coli,\u003c/em\u003e which may result from cross-contamination. \u003cem\u003eStaphylococcus\u003c/em\u003e spp. is usually found on the skin and surfaces [32]. \u003cem\u003eE. coli\u003c/em\u003e is a human pathogen that causes gastrointestinal illness. Typically, the source is the host intestine; however, \u003cem\u003eE. coli\u003c/em\u003e can persist on surfaces [1, 42]. Pasteurized milk was the source of up to 9% \u003cem\u003eE. coli\u003c/em\u003e contamination [43]. Milk processed with inadequate measures presented approximately two-fold higher \u003cem\u003eE. coli\u003c/em\u003e numbers than adequate treatment measures [44]. According to cross-contamination suspected, the processes after heating treatment were strictly hygienic [2, 17, 43, 45].\u003c/p\u003e\n\u003cp\u003eThis study collected samples from farms and retailers only one time. The incidence of Listeria contamination might be underestimated. The retailers notice the bubble or film layer to complete the milk heating process, that the temperature and time might highly deviate from person to person. The temperature and processing time in the trial might differ from the retailers used. \u003cem\u003eListeria monocytogenes\u003c/em\u003e in the experiment were lastly interrupted by other bacteria from cross-contamination.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, on a farm level, which was the main source of raw milk observed 1% \u003cem\u003eListeria\u003c/em\u003e incidence,\u0026nbsp;whereas no identification of \u003cem\u003eListeria\u0026nbsp;\u003c/em\u003espp\u003cem\u003e.\u0026nbsp;\u003c/em\u003eon the retailer level and post processed milk in trial. The presence of \u003cem\u003eListeria\u0026nbsp;\u003c/em\u003espp. at farm level warns people about the risk of raw or improper-treated milk to human health, even low incidence of Listeria. Milk should be processed following the method of pasteurization. The retailers should prevent cross-contamination during other steps after treatment as well. Finally, the responsible organizations should incessantly educate the milk retailers in adequate protocols as well as routinely check their product safety.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDatasets of the current research are available at\u0026nbsp;\u003ca href=\"https://doi.org/10.5281/zenodo.7014148\"\u003ehttps://doi.org/10.5281/zenodo.7014148\u003c/a\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study has nonfinancial competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was financially supported by the Start-up Research Fund: SRF, Faculty of Veterinary Medicine, Kasetsart University (SRF63_01).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely thank all farmers and small-scale retailers who kindly consent to sample collection. We would like to thank Editage (www.editage.com) for English language editing. Preprint is readily available from\u0026nbsp;\u003ca href=\"https://www.researchsquare.com/article/rs-1998662/v1\"\u003ehttps://www.researchsquare.com/article/rs-1998662/v1\u003c/a\u003e [46].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuestionnaire in the topic: \u0026ldquo;\u003cem\u003eListeria\u003c/em\u003e contamination in milk-processing chain and proficiency in \u003cem\u003eListeria monocytogenes\u003c/em\u003e decontamination of small-scale milk retailers\u0026rdquo; was presented as supplementary data.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eCenters for Disease Control and Prevention (CDC). Surveillance for foodborne disease outbreaks United States, 2017: Annual Report, 2017.\u003c/li\u003e\n \u003cli\u003eWorld Health Organization (WHO). 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Chimonyo, \u0026ldquo;Effect of structural condition of milk processing facilities and food safety systems on \u003cem\u003eEscherichia coli\u003c/em\u003e and Coliforms presence in cultured buttermilk,\u0026rdquo; \u003cem\u003eJournal of Food Quality\u003c/em\u003e, 7365983; 10.1155/2019/7365983, 2019.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e45. M. Kannaiyan, C. Uma, S. Paramasivam, and S. Kumar, \u0026ldquo;Occurrence of Listeria monocytogenes in milk and milk products,\u0026rdquo; \u003cem\u003eInternational Journal of Current Research in Life Sciences\u003c/em\u003e, vol. 7, pp. 1572\u0026ndash;1574, 2018.\u003c/p\u003e\n\u003cp\u003e46. S. Kananub, P. Lertsakkongkul, P. Aryatawong, W. Horhirunkhajohn, N. Pinniam, P. Krajanglikit, K. Sonthong, and S. Kasemsuwan \u0026ldquo;Listeria species contamination in the milk-processing chain and decontamination proficiency of small-scale milk retailers,\u0026rdquo; \u003cem\u003eResearch Square\u003c/em\u003e. Preprint [Version 1]. September 1, 2022. https://www.researchsquare.com/article/rs-1998662/v1.\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"food contamination, milk, milk treatment, foodborne bacteria, Listeria spp.","lastPublishedDoi":"10.21203/rs.3.rs-1998662/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1998662/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eListeria \u003c/em\u003espp. is an important-foodborne bacterium. This microorganism can be discarded from milk using high temperatures such as pasteurization. The milk processing methods of many small-scale retailers lack quality control. This study was to survey \u003cem\u003eListeria\u003c/em\u003e contamination at the farm and retailer levels. The retailers were to be interviewed for knowledge, attitude, and practice as well. Finally, we were to determine the heating processes employed to decontaminate microorganisms by the retailers using a reference strain of \u003cem\u003eL. monocytogenes\u003c/em\u003e. Milk samples were collected from milk collecting centers and small-scale retailers. In clinical trial, the processing measures were proved the proficiency in \u003cem\u003eL. monocytogenes\u003c/em\u003e decontamination.\u003cem\u003e \u003c/em\u003eOne out of 99 farms presented \u003cem\u003eListeria\u003c/em\u003e contamination, confirmed to \u003cem\u003eL. marthii\u003c/em\u003e. Fifty small-scale retailers participated in the second part, including 13 males and 37 females. No \u003cem\u003eListeria \u003c/em\u003espp., but \u003cem\u003eStaphylococcus\u003c/em\u003e spp. and \u003cem\u003eBacillus \u003c/em\u003espp. were identified in the processed milk. Data analyses revealed that the location of the retailer was significantly associated with the volume they routinely ordered per lot and the milk-processing time the retailers used to treat milk. Knowledge on raw milk contamination is significantly associated with the stocking or processing of the whole milk lot. Processing measures pre-sale significantly depended on the sex of the retailer. The male retailer reportedly spent less time treating milk than the female retailer. \u003cem\u003eL. monocytogenes\u003c/em\u003e was used in the trial to evaluate the efficiency of the processes. No \u003cem\u003eL. monocytogenes\u003c/em\u003e were after sample treatment, but \u003cem\u003eS. epidermidis, S. warneri, \u003c/em\u003eand\u003cem\u003e Escherichia coli\u003c/em\u003e. Although the trial implied that the retailer’s processes were effective in \u003cem\u003eL. monocytogenes\u003c/em\u003e decontamination, the study presented the inappropriate practices of the retailer and the probability of cross-contamination. Product safety from small-scale milk retailers should be monitored incessantly.\u003c/p\u003e","manuscriptTitle":"Listeria contamination in milk-processing chain and proficiency in Listeria monocytogenes decontamination of small-scale milk retailers","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2024-01-11 15:48:46","doi":"10.21203/rs.3.rs-1998662/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2022-09-01 19:37:16","doi":"10.21203/rs.3.rs-1998662/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"665527f1-b2c6-4f0f-84b0-e10317a3c929","owner":[],"postedDate":"January 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":28089968,"name":"Food Science \u0026 Technology"}],"tags":[],"updatedAt":"2022-10-04T03:59:21+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-11 15:48:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-1998662","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1998662","identity":"rs-1998662","version":["v2"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}