Quality of raw milk intended for direct consumption in Estonia

Food Control 51 (2015) 135e139

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Quality of raw milk intended for direct consumption in Estonia €e b, Arvo Viltrop c Piret Kalmus a, *, Toomas Kramarenko b, d, Mati Roasto b, Kadrin Merema a

Department of Clinical Veterinary Medicine, Institute of Veterinary Medicine and Animal Sciences, Estonian University of Life Sciences, Kreutzwaldi 62, 51014, Tartu, Estonia b Department of Food Hygiene, Institute of Veterinary Medicine and Animal Sciences, Estonian University of Life Sciences, Kreutzwaldi 56/3, 51014, Tartu, Estonia c Department of Basic Veterinary Sciences and Population Medicine, Institute of Veterinary Medicine and Animal Sciences, Estonian University of Life Sciences, Kreutzwaldi 62, 51014, Tartu, Estonia d Estonian Veterinary and Food Laboratory, Kreutzwaldi 30, 51006, Tartu, Estonia

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 August 2014 Received in revised form 4 November 2014 Accepted 11 November 2014 Available online 20 November 2014

The main aim of the present study was to estimate the occurrence of zoonotic bacteria in raw milk intended for sale directly to consumers in Estonia. In-line milk filters, bulk milk samples and milk samples from selling points were collected from a total of 14 dairy farms and respective retail selling points. The somatic cell counts, total bacterial counts and the presence of Salmonella spp and Listeria monocytogenes were studied from bulk milk samples. Campylobacter spp., Salmonella spp., L. monoscytogenes and Shiga-toxin producing Escherichia coli (STEC) were studied in farms in-line milk filters. The total bacterial counts exceeded 100,000 cfu/ml in three (21.4%) bulk milk samples and in 10 samples (71.4%) collected at the retail level. STEC genes were detected in 64.3% of the in-line milk filter samples. More than one STEC serogroup-specific gene was detected in four dairy farms. L. monocytogenes was found in 36% of the in-line milk filters. Neither Salmonella spp. nor Campylobacter spp. were found in any samples. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Raw milk Direct selling Safety and quality

1. Introduction The sale of raw milk in large amounts directly to consumers through vending machines or via other means has increased in recent years in many countries (Bianchi et al., 2013; Serraino et al., 2013; Wysok, Wiszniewska-Laszczych, Uradzinski, & Szteyn, 2011). The raw milk-related health benefits that are propagated by the raw milk proponents through media channels and improved marketing possibilities have increased the general interest in raw milk consumption also in Estonia. The sale of raw milk via automatic vending machines at grocery stores began in 2012, but sales in farmers' markets have been allowed in Estonia for many years. It is a quite common practice for farmers near the cities to bring or send raw milk directly to consumers' homes. Additionally, many dairy farms sell raw milk in small amounts from the farm.

* Corresponding author. Tel.: þ372 7313219; fax: þ372 7313706. E-mail addresses: [email protected] (P. Kalmus), toomas.kramarenko@ vetlab.ee (T. Kramarenko), [email protected] (M. Roasto), kadrin.meremae@ €e), [email protected] (A. Viltrop). emu.ee (K. Merema http://dx.doi.org/10.1016/j.foodcont.2014.11.018 0956-7135/© 2014 Elsevier Ltd. All rights reserved.

Studies have shown, that customers tend to prefer raw milk due to better taste and believe in better nutritional value and several health benefits compared to heat treated milk (Claeys, Cardoen, & Herman, 2013; Haug, Høstmark, & Harstad, 2007). Among health benefits improved immunity, less milk allergy, lactose intolerance and diabetes among raw milk consumers have been advocated, however without convincing scientific evidence supporting such claims (Claeys et al., 2013). However, in a recent study consumption of raw milk in early ages and living in the farm environment have been identified as an exposure that might contribute to the pro ska, Pearce, tective effect on asthma and allergies in children (Sozan Dudek, & Cullinan, 2013; Von Mutius & Vercelli, 2010). Nevertheless, due to its nutritional properties, raw milk also creates good growth conditions for a variety of spoilage and potentially pathogenic microorganisms, such as Shiga-toxin producing Escherichia coli (STEC), Listeria (L.) monocytogenes, Salmonella enterica, Campylobacter spp., Yersinia spp. and others (Amagliani et al., 2012; Hill, Smythe, Lindsay, & Shepherd, 2012). Moreover, raw milk can contain mastitis-causing agents, such as Staphylococcus (S.) aureus and Streptococcus (Str.) agalactiae, which are potentially hazardous to humans (Haguenoer et al., 2011). Additionally, zoonotic pathogens, such as Coxiella burnetii, Mycobacterium bovis, Brucella spp.,

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samples were taken by official veterinarians during the one month period from June to July 2013 without previous notification to the farmers. The disposable in-line milk filters and bulk tank milk samples (200 ml) were aseptically collected on the farms and were sent immediately to the Veterinary and Food Laboratory under refrigerated conditions. The same batches of milk were followed to the selling point at which additional milk samples (200 ml) were aseptically taken. The samples were collected in sterile containers and sent to the Veterinary and Food Laboratory under refrigerated conditions.

Toxoplasma gondii, Leptospira hardjo and tick-borne encephalitis virus, can be excreted with milk (Dehkordi, Borujeni, Rahimi, & Abdizadeh, 2013; Hudopisk et al., 2012; Thiermann, 1982). According to the Estonian Veterinary and Food Board information from April 2013, there were 32 dairy farms with veterinary certificates permitting the sale of raw milk in large amounts through retail; e.g., from vending machines, markets, grocery storesand other selling points. The requirements and criteria of the European Commission (EC) regulation 853/2004 laying down specific hygiene rules for food of animal origin (section IX, raw milk and dairy products) are the basis of the Estonian national health requirements for the disposed raw milk and for the hygiene requirements of milk production holdings. Additionally, Regulation No. 71 of the Estonian Ministry of Agriculture determines some hygiene requirements for raw milk disposal. For example, Staphylococcus aureus quantification has to be performed once every two months. Less than 500 cfu per ml of S. aureus are allowed in raw milk meant for direct marketing. Regulations also establish that raw milk for direct sale requires a veterinary certificate provided by the veterinary authority that is valid for six months. These criteria are not applied to raw milk or the handling of raw milk that is intended for direct sale to customers in small amounts (up to 100 kg cow milk per day and 700 kg per week) from the farm. The most frequent food-borne infections in Estonia were salmonellosis and campylobacteriosis in 2011e2013. (Estonian Health Board reports, 2011e2013) The incidence of campylobacteriosis per 100,000 inhabitants has increased from 16 in year 2011 to 28,7 cases in 2013. In total, 3.1% and 3.3% of cases have been associated with the consumption of unpasteurised milk in 2012 and 2013, respectively. No association has been found between salmonellosis (16.7e24.3 cases per 100,000 inhabitants in 2011e2013) and consumption of unpasteurised milk in Estonia (Estonian Health Board report, 2013). The incidence of E. coli infections per 100,000 inhabitants has been increased from 0.7 in 2011 to 0.9 in 2013, where 8 cases out of 12 were STEC in year 2013. The main aim of the study was to estimate the prevalence of selected zoonotic pathogens in raw milk produced in dairy farms that are authorised to produce and sell unpasteurised milk outside of the farm directly to consumers.

2.2. Laboratory analyses All analyses of the samples collected from the farms began within 24 h after sampling. The samples obtained from the selling points were analysed on the day of expiry date. Laboratory analyses were performed according to ISO-standards in the Estonian Veterinary and Food Laboratory and described in Table 1. Total bacterial count (TBC) were analysed in milk samples collected from the farm bulk and selling points using horizontal method for the enumeration of microorganisms according to ISO 4833 standard procedure (Anonymous, 2003a). The microscopic method (ISO 13366-1) was used to determine total somatic cell count (SCC) in the bulk milk samples (Anonymous, 2008a). Mastitis-causing pathogens were analysed by the Estonian Milk Recording Laboratory using a commercial real-time PCR test kit (PathoProof Mastitis PCR Assay, Thermo Fisher Scientific, Espoo, Finland) described by Koskinen et al. (2009). 2.3. Statistical analyses The Stata 11.0 statistical software was used for the statistical analyses. Statistical significance was assumed at p  0.05. Multivariable linear regression analysis was used to identify the associations of bulk milk SCC and TBC with herd size and farm type (organic or conventional). Prior to analysis, the bulk milk SCC and TBC were transformed logarithmically. A logistic regression model was used to identify the associations of the presence of STEC in the milk filters depending on farm type and herd size. 3. Results and discussion

2. Materials and methods The average daily milk yield was 19.9 kg in the conventional herds and 15.3 kg in the organic herds. The total amount of produced milk on the sampling day was 24,635 kg, and 17.8% (4267 kg) of this milk was sold directly. Individual udder clothes for the cleaning of the udders before milking, the wearing of gloves during the milking process, post-milking teat disinfection and dry cow treatment were employed by 12 of the 14 farms.

2.1. Sample collection In total, 14 farms were randomly selected out of the 35 farms that sell raw milk directly to consumers in Estonia. Of these 14 farms, six (42.8%) produced milk organically. Among the selected herds, tie-stall and free-stall systems were equally used. All

Table 1 Culture media and procedures used for the microbiological testing of collected samples. Bacteria

Sample

Reference method

Type of assay

Main procedure

S. aureus

Bulk milk

ISO 6888-1/A1(2003b)

Enumeration

E. coli (STEC); serogroups O157, O111, O26, O103 and O145 L. monocytogenes

Milk filter

ISO/TS 13136 (2012)

PCR

BairdeParker agar was used and presence of coagulase was detected by using rabbit plasma stx1 and stx2 genes together with serogroup specific genes were detected by PCR after enrichment step

Bulk milk; milk filter Milk from selling points Milk filter Milk filter

ISO 11290-1/A1(2004a)

Detection

ALOA and Palcam agars were used as isolation media

ISO 11290-1/A1(2004b)

Enumeration

ALOA agar was used

ISO 10272 (2006) ISO 6579(2007)

Detection Detection

Bolton enrichment following mCCD and Skirrow agars were used XLD and BG agars were used as isolation media

L. monocytogenes Campylobacter spp. Salmonella spp.

P. Kalmus et al. / Food Control 51 (2015) 135e139

3.1. Somatic cell counts and total bacterial counts in the bulk tank milk samples and the samples from the selling points The bulk tank milk somatic cell counts (BMSCCs) varied from 143,000 to 777,000 cells/ml (median 265,000 cells/ml; Table 2). The BMSCCs exceeded 400,000 cells/ml in the milk samples from three of the farms. The medians BMSCCs were 309,000/ml in the organic farms and 255,000/ml in the conventional farms. Neither BMSCC nor TBC were associated with herd size or type (p > 0.05). The TBCs in the bulk milk samples ranged from 1500 to 16,000,000 cfu/ml (median 26,000 cfu/ml). The TBCs in the raw milk samples at the selling sites ranged from 5000 cfu/ml to 20,000,000 cfu/ml (median 540,000 cfu/ml) (Table 2). The TBCs exceeded 100,000 cfu/ml in three (21.4%) of the bulk milk samples and 10 of the samples (71.4%) collected at the retail level and analysed on the day of the expiry date. In Estonia, the official criterion for SCC is 400,000/ml based on a moving geometric average over a three-month period with at least one sample per month. The official criterion for the total bacterial count (TBC) is 100,000 cfu/ml based on a moving geometric average over a twomonth period with at least two samples per month. In the present study, only one sample was collected per location; therefore the results cannot directly be compared with the official criteria stated in Regulation 853/2004 (EU). However, the milk quality from many of the farms was not satisfactory on the sampling day. The elevated TBCs of the milk might have been caused by unsatisfactory cleaning and disinfection of the equipment or by improper storage of raw milk in the markets or vending machines. Milking hygiene and the cleaning and disinfection of milking and storage equipment should be controlled more frequently at both farms and at selling points. 3.2. Detection of mastitis pathogens in the bulk milk samples All bulk tank milk samples from the selected dairy farms contained two or more mastitis pathogens. The E. coli was detected in three bulk tank milk samples. The proportion of Str. agalactiae, Str. dysgalactiae and Str. uberis positive samples were 7.1%, 57.1% and 50%, respectively. Str. agalactiae is a potentially zoonotic pathogen and might cause significant morbidity and mortality in both infant and adult humans (Bingen et al., 1992). S. aureus was detected in bulk tank milk samples of eight (57.1%) dairy farms. According to Table 2 Quality and safety indicators of the bulk tank milks at the farm level and of the raw milk at the retail level. Farm

A B C D E F G H I J K L M N

Herd size (number of dairy cows)

Bulk tank milk at farm level

Raw milk at point of sale

Somatic cell count (cells/ml)a

Total bacterial count (cfu/ml)b

Total bacterial count (cfu/ml)b

278 178 95 573 274 95 81 178 46 339 392 148 33 134

263.000 504.000 313.000 162.000 156.000 384.000 231.000 251.000 267.000 247.000 143.000 632.000 777.000 306.000

18.000 34.000 1.500 130.000 9000 75.000 12.000 45.000 35.000 580.000 8.000 16.000.000 12.000 5.000

710.000 700.000 22.000 290.000 13.000 76.000 2.700.000 180.000 3.200.000 10.000.000 380.000 20.000.000 2.100.000 5.000

a According to (EC) No 853/2004, criterion 400,000; rolling geometric average over a three-month period with at least one sample per month. b According to (EC) No 853/2004, criterion 100,000; rolling geometric average over a two-month period with at least two samples per month.

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guen (1997), the prevalence of S. aureus Desmasures, Bazin, and Gue in raw milk in France is 62% followed by 47.3% in Norway (Jakobsen, Heggebø, Sunde, & Skjervheim, 2011), 32.5% in Poland (KorpysaDzirba & Osek, 2011) and 17% in the Netherlands (Howard, 2006). A high SCC (over 200,000 cells/ml) might indicate the presence of contagious mastitis pathogens (S. aureus and/or Str. agalactiae) in the herd (Edmondson & Bradley, 2004). Therefore, decreasing the SCC limit of bulk milk intended for direct sale to consumers might exclude farms with problematic udder health status. Dairy farms that sell raw milk directly to consumers should be Str. agalactiae free. 3.3. Determination of L. monocytogenes The in-line milk filters of five (36%) of the dairy farms were positive for Listeria monocytogenes. Bacteria were simultaneously detected in the in-line milk filters and bulk tank milk samples of four (28.5%) of the dairy farms. Previously, Kramarenko et al. (2013) reported that 18.1% of Estonian bulk tank milk samples were positive for L. monocytogenes. For comparison, the prevalences of L. monocytogenes in other studies, such as those of Ruusunen et al. n, Die guez, and Rodríguez(2013) from Finland, Vilar, Yus, Sanjua Otero (2007) from Spain, De Reu, Grijspeerdt, and Herman (2004) from Belgium and Desmasures et al. (1997) from France, range between 5.5 and 6.5%. Application of the L. monocytogenes enumeration method to all of the samples collected from the selling points (i.e., at the retail level) revealed that the counts of bacteria were below 1 cfu per ml. Nevertheless, a total of 28.6% of the 25 ml bulk tank milk samples contained L. monocytogenes, which might represent a potential public health risk if the milk is consumed without prior heat treatment. Within the framework of the Estonian state surveillance program, raw milk samples from direct sellers are studied once a year for the presence of L. monocytogenes in the bulk tank milk (absence in 25 ml; n ¼ 1), and L. monocytogenes counts are determined for milk samples that are taken at selling points. The L. monocytogenes counts should not exceed 100 cfu/ml. We believe that the sampling plan that prescribes only one 25 ml sample per year per farm is not sufficiently sensitive to detect pathogens in raw milk. Moreover, this sampling frequency does not allow for proper food safety decisions to be made. 3.4. Detection of Salmonella spp. and Campylobacter spp. Neither Salmonella spp. nor Campylobacter spp. were detected in the in-line milk filters of the dairy farms examined in the present study. In contrast, Campylobacter spp. has been detected in raw milk in many other countries, such as Northern Italy (Giacometti et al., 2012), Poland (Wysok et al., 2011), Great Britain (De Louvois & Rampling, 1998) and France (Desmasures et al., 1997), at prevalences that range from 1.4 to 4.6%. No Campylobacter-positive raw milk samples were detected in studies from Finland (Ruusunen et al., 2013), Norway (Mørk, Bergsjø, Sviland, & Kvitle, 2003) and Switzerland (Stephan & Bühler, 2002). One possible explanation for the Campylobacter-negative results of the present study is that, during the sample transportation, transportation media were not used, and campylobacteria is known to be highly sensitive to drying. Indeed, Serraino et al. (2013) placed fresh milk filters in sterile plastic bags with enough bulk tank milk to completely cover the sock filter while transporting the samples. Similar to our results, Salmonella spp. was not detected in raw milk in Finnish (Ruusunen et al., 2013), Belgian (De Reu et al., 2004) and Swiss (Stephan & Bühler, 2002) studies, but a total of 1% of the samples taken from raw milk vending machines were Salmonellapositive in Northern Italy (Giacometti et al., 2012).

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3.5. Detection of Shiga-toxin producing E. coli

4. Conclusions

PCR analyses of the in-line milk filter samples revealed that STEC virulence genes were present in nine dairy farms out of the total of 14. The presence of STEC genes was not associated with management type or herd size (p > 0.05). In a recent study from Northern Italy, 32 of 255 bulk milk samples and 68 of 225 milk filters were positive for stx genes (Trevisani et al., 2014). The patterns of the identified STEC toxin genes in the samples are presented in Table 3. The toxin gene vtx2 was found in eight (57%) milk filters, but the presence of vtx1 was detected in only one farm. Additionally, the intimin-coding gene eae was found to be present in the majority of the samples. Four important STEC serogroup genes (i.e., O103, O145, O26 and O157) were detected in our study, but O111 was not. More than one STEC serogroup-specific gene was detected in four dairy farms. Primarily, genes specific to serogroups O103 and O145 were found followed by O157 and O26. Cattle are the main natural reservoir for STEC, which is arguably the most important E. coli pathotype for humans in developed countries (Wong, Pearson, & Hartland, 2011). Generally, STEC enteritis and haemolytic uraemic syndrome (HUS) are associated with significant mortality and morbidity, particularly among patients with severe renal and neurological disorders (Borgatta, KmetLunacek, & Rello, 2012), and raw milk has been described as a risk factor for HUS (Scavia, Escher, Baldinelli, Pecoraro, & Caprioli, 2009). The presence of STEC-specific genes in milk filters demonstrate the presence of the infection in our dairy cattle. This creates a constant risk for the raw milk to be contaminated with dangerous pathogens as the in-line milk filters are not bacteria proof. Risks and benefits of the consumption of raw or heated cow milk have been comprehensively addressed in review of Claeys et al. (2013). The general conclusion of this review is that pasteurisation is not expected to affect presumed milk health benefits significantly. At present in Estonia, there is no legislative requirement to inform consumers that a product is unpasteurised and might therefore contain organisms that are harmful to health. Nevertheless, it is essential to inform customers about raw milk consumption-related risks and to provide clear recommendation to heat milk before consumption. The food safety criteria for raw milk that is intended for direct sale in large amounts should be reviewed while accounting for herd health status and the presence of certain zoonotic pathogens.

The milk quality of one-third of the dairy herds from which raw milk was directly sold to customers was not satisfactory. Presence of zoonotic pathogens (particularly STEC and L. monocytogenes) in cows and farm environment poses a permanent risk for contamination of produced milk. Therefore, raw milk that is intended for direct consumption cannot be considered microbiologically safe without heat treatment. The current risk management methods that are applied in Estonia are not sufficient to ensure raw milk safety for customers. The official criteria for raw milk that is intended for direct consumption should be reviewed and improved.

Table 3 Characteristics of the STEC detected in the in-line milk filters of the studied dairy farms based on real-time PCR. Dairy farm STEC genes

A B C D E F G H I J K L M N Totala

Serogroups

vtx1

vtx2

eae

O26

O103

O111 O145

O157

e e e e e e þ e e e e e e e 1 (7.1)

þ e þ þ e þ e þ þ e e e þ þ 8 (57.1)

þ e e þ e þ þ þ þ e e e þ þ 8 (57.1)

e e e þ e e e e e e e e e e 1 (7.1)

e e þ þ e þ þ þ e e e e þ e 6 (42.9)

e e e e e e e e e e e e e e 0 (0)

e e e þ e e e þ e e e e þ þ 4 (28.6)

þ e e þ e e e þ þ e e e þ þ 6 (42.9)

þ, detected. e, not detected. a Number and percentage of farms with positive detection results.

Acknowledgements This study was supported by the Estonian Ministry of Agriculture, project 8-2/T13091VLTO “Raw milk quality pilot study in Estonia”. We thank the Estonian Veterinary and Food Board for constructive collaboration during the planning of this study and for providing samples.

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