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Monitoring of foodborne pathogenic bacteria in vending machine raw milk in Piedmont, Italy
Food Control 32 (2013) 435e439
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Food Control journal homepage: www.elsevier.com/locate/foodcont
Short communication
Monitoring of foodborne pathogenic bacteria in vending machine raw milk in Piedmont, Italy Daniela Manila Bianchi a, b, *, Antonio Barbaro a, c, Silvia Gallina a, b, Nicoletta Vitale a, c, Laura Chiavacci a, c, Maria Caramelli a, Lucia Decastelli a, b a b c
Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, 10154 Turin, Italy Struttura Complessa Controllo Alimenti e Igiene delle Produzioni, Italy Struttura Complessa Epidemiologia e Osservatorio Epidemiologico, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 July 2012 Received in revised form 22 December 2012 Accepted 5 January 2013
Raw milk consumption in Italy has increased over the past three years following the enactment of a national law that allows the sale of unpacked and unpasteurized cows’ milk via vending machines on the farm and at markets. From 2009 to 2011, a three-part monitoring survey of raw milk sold though vending machines was carried out to investigate for the occurrence of Salmonella spp., Escherichia coli O:157, Campylobacter spp. and Listeria monocytogenes. A total of 618 raw milk samples were collected from 112 dairy herds supplying 131 raw milk vending machines. Of the samples tested, 0.3% were positive for Salmonella spp., 0.2% for E. coli O:157, 1.5% for Campylobacter spp., and 1.6% for Listeria monocytogens. Multivariate analysis showed no effect of seasonality, average daily temperature, herd size, sample collection point or distance between herd and vending machine; however, there was a statistically significant correlation between a previous finding of pathogens and recurrence of contamination. The monitoring survey results confirm that unpasteurized milk can be a vehicle of a variety of microorganisms and an important source of foodborne illness outbreaks. Ó 2013 Published by Elsevier Ltd.
Keywords: Raw milk Foodborne pathogenic bacteria
1. Introduction Milk and dairy products are basic components of human diet and provide a dietary source of proteins, vitamins and other minerals (Huth, DiRienzo, & Miller, 2006). A vast variety of dairy products are available, including liquid milk, soft and hard cheese, yogurt, as well as specialty foods such as ricotta cheese and mascarpone. Almost all are made from pasteurized milk. Alongside this established market is a growing consumer demand for raw milk. However, raw milk consumption is accompanied by the risk of ingesting pathogenic bacteria that can pose an elevated health hazard (Latorre et al., 2009). And although zoonotic diseases like brucellosis and tuberculosis have been or are nearly completely eradicated in European Union countries (EFSA, 2010), the milk of asymptomatic animals may harbor other infectious agents: milk pasteurization was, and remains, key to preventing milk-borne zoonosis. European regulations and Regulation EC 853/2004 (Regulation CE 853/2004, 2004) in particular, allow the sale of raw milk,
* Corresponding author. Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, 10154 Turin, Italy. E-mail addresses: [email protected], [email protected] (D.M. Bianchi). 0956-7135/$ e see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.foodcont.2013.01.004
defining it as “the secretion of the mammary gland of farmed animals that has not been heated to more than 40 C or undergone any treatment that has an equivalent effect”. This regulation sets forth the guidelines for raw milk sale and distribution on the market. In Italy, the sale of raw milk via automatic self-service vending machines is regulated by an agreement between the state and the regions “Intesa Stato Regioni 2007 [ISR 2007]” (Intesa Stato Regioni, 2007a). Under the provisions of the ISR agreement, dairy farms, milk intended for raw consumption, and vending machines must comply with specific norms: biosafety measures and own-check for producers, microbiological and chemical criteria for milk, and vending machine installation and management specifications. In 2007 the Italian Ministry of Health authorized the sale and distribution of unpacked raw milk via automatic self-service vending machines. The milk dispensers boost sales revenues for farmers, as the milk is sold directly on the farm; consumers appreciate this new distribution channel as they can purchase a product they perceive as wholesome at a convenient price. The ISR agreement also details vending machine set up and management. Raw milk dispensers must be located in a welldefined separate area, far away from any source of waste or dirtiness. They must be equipped with a temperature thermometer
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with an external display and a thermostat that ensures a constant milk temperature between 0 C and 4 C. The machines must also have a dispenser valve that will interrupt the sale of raw milk if the milk temperature exceeds 4 C. The milk supply valve must be not exposed to contamination and it must allow easy removal for cleaning and disinfection. A vending machine can contain the raw milk from only one dairy herd. The front of the vending machine must display the following information: identification of the farm where the milk is produced, including the address of the farm, the date of milking, the date the machine was restocked, the expiry date, and instructions for home conservation at a temperature between 0 C and 4 C. Since December 2008, vending machines must also display a notice to customers that the raw milk must be boiled before consumption (Ordinance by the Ministry of Health, 2008). This warning to customers was deemed necessary owing to increased outbreaks of the hemolytic uremic syndrome (HUS) (Italian Registry of Familial and Recurrent HUS/TTP) and evidence from scientific studies that confirmed that unpasteurized milk poses a health hazard, especially for children (Scavia, Escher, Baldinelli, Pecoraro, & Caprioli, 2009; Weber, Behne, & Fruth, 2007). Under the provisions of ISR 2007 (Intesa Stato Regioni, 2007a), the Regional Competent Authority is responsible for devising and implementing monitoring plans for raw milk safety and hygiene and for the continuous control of microbiological criteria to ensure adequate consumer protection. In Italy, raw milk vending machines are supplied by 1032 dairy herds (Integrated National Annual Report of Italian Ministry of Health (Italian Ministry of Health, 2010)). Each herd can supply one or more vending machines. Of a national total of 1411 authorized vending machines, 170 (12%) supplied by 112 dairy herds are authorized for operation in Piedmont. Since 2008, monitoring surveys have been conducted in Piedmont to check that the raw milk sold via vending machines complies with the criteria set forth in the national regulation. During a pilot monitoring survey carried out from November 2008 to July 2009, Listeria monocytogenes and Campylobacter spp. were isolated from some samples of unpacked raw milk. The Regional Veterinary Authority subsequently conducted three annual mandatory monitoring surveys to inspect all dairy herds and at least one of the vending machines they supplied. This study reports the results of the three monitoring surveys conducted from November 2009 to June 2011. The microbiological safety of raw milk is discussed, focusing on commonly isolated pathogenic bacteria and potential risk factors that can increase the probability of contamination by pathogens in raw milk. 2. Materials and methods 2.1. Sampling plans Between November 2009 and July 2011, three surveys were carried out: the first from November 2009 to March 2010, the second from July to December 2010, and the last from January to July 2011. During each survey, all herds supplying raw milk for a vending machine and at least one vending machine for each herd were inspected. Sampling was performed by the local veterinary service. The samples were collected into sterile containers and brought refrigerated to the Food Control Laboratory of the Istituto Zooprofilattico Sperimentale del Piemonte Liguria e Valle d’Aosta in Turin. Sampling at the vending machine and the bulk milk tank was done without advance notification of the farmers or the vending machine operators. However, because of the product’s short shelflife, the milk producers and food business operators were informed about the date of testing so that they or a legal representative could
be present in the laboratory during analysis. In compliance with ISO 7218:2007 (Microbiology of food and animal feeding stuffs, 2007b), the analyses were started within 24 h of sampling. In accordance with ISR 2007 guidelines, each milk sample was composed of five units: each unit contained at least 200 mL of raw milk. Microbiological analyses were performed on all five units forming the sample. 2.2. Laboratory testing In accordance with national legal requirements (Intesa Stato Regioni, 2007a) for food safety criteria, the milk samples were tested for the presence of: Salmonella spp., L. monocytogenes, Escherichia coli O:157 and Campylobacter spp. Each test was performed on 25 mL of raw milk by means of qualitative methods; the laboratory and all test procedures are accredited according to ISO 17025:2005 (General requirements for the competence of testing and calibration laboratories, 2005) at the ACCREDIA Italian accreditation body. A biomolecular method (real-time PCR) (AFNOR, 2004) was performed to detect Salmonella spp. Samples testing positive for Salmonella spp. DNA underwent a second round for confirmation with a microbiological method according to ISO 6579 (Microbiology of food and animal feeding stuffs, 2002). Viable colonies suspected of belonging to the genus Salmonella were biochemically confirmed by API20EÒ (BioMérieux). Serotyping of Salmonella spp. strains was performed according to the KauffmanneWhite protocol. A biomolecular method (real-time PCR; Biorad AFNOR BRD 07/ 10-04/05) was also performed to detect L. monocytogenes DNA. Samples testing positive were retested under a microbiological protocol according to ISO 11290 (Microbiology of food and animal feeding stuffs, 1996b). Viable colonies suspected to be L. monocytogenes were biochemically confirmed by GP-VITEKÒ (BioMérieux). Confirmed L. monocytogenes strains were then analyzed to identify the serotype (serum provided by Denka Seiken Co). An enzyme-linked-fluorescence assay (AFNOR, 2000) was applied to detect E. coli O:157. Positive samples were then retested for microbiological isolation and identification according to ISO 16654 (Microbiology of food and animal feeding stuffs, 1996a). E. coli O:157 strains were investigated for the presence of shiga-like toxinproducing genes according to ISO TS 13136 (Microbiology of food and animal feeding stuffs, 2011). The microbiological method described in ISO 10272 (Microbiology of food and animal feeding stuffs, 2006) was used to detect Campylobacter spp. Colonies suspected of belonging to the genus Campylobacter were biochemically confirmed by API (BioMérieuxÒ). A unit of the milk sample was considered incompliant with food safety norms if one pathogenic bacterium was isolated in 25 mL; the whole milk sample was considered incompliant if one or more units were not compliant. 2.3. Statistical analyses Data were analyzed using SASÒ software ver. 9.2 (SAS Institute Inc., Cary, NC, USA). Descriptive analysis was performed to determine the occurrence of pathogens. The c2 test was applied to test for associations between the occurrence of pathogens and the following variables: distance between dairy herds and vending machine; herd size; previous occurrence of pathogens; daily average temperature; months and seasons. Categorical variables were reduced to as few categories as possible, ideally two. The univariate odds ratios were calculated. Linear associations were confirmed for significant continuous variables by dividing them into four groups according to quartiles and assessing the linearity in the odds ratios
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consumption. Nonetheless, many consumers still drink raw milk for several reasons: the milk dispensed at vending machines is less expensive than pasteurized milk sold at a market; consuming raw milk is viewed as customary practice; raw milk is believed to be more wholesome and nutritious than pasteurized milk. Whatever the consumer convictions about raw milk, epidemiological data have highlighted the growing involvement of Campylobacter spp. in foodborne zoonosis outbreaks. The Community Zoonoses Reports of the European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (ECDC) in their Community Zoonoses Reports have in the last 5 years cited campylobacteriosis as the most commonly reported zoonosis in the European Union (EFSA, 2007) (EFSA, 2010). In the present study, Campylobacter was found in 1.5% of samples. Previous studies reported the prevalence of Campylobacter spp. in raw milk samples ranging from 0.3% (Cavirani, 2008), to 2% (Jayarao et al., 2006), to 4.6% (Wysok, Wiszniewska-qaszczych, ski, & Szteyn, 2011) and 9.2% (Jayarao & Henning, 2001). Uradzin Similarly, in a study that included 99 raw milk samples in Northern Italy, thermotolerant Campylobacter were isolated or PCR-detected in 2.02% of samples (Giacometti et al., 2012). All isolated strains identified in this study belonged to the species C. jejuni: this finding is supported by an epidemiological survey on bovine fecal culture which reported an infection rate from 5 to 50%, with C. jejuni ranking highest, followed by Campylobacter coli and Campylobacter upsaliensis (Wesley et al., 2000), confirming that milk contamination during or immediately after milking is most probably of stool origin. About 155,000 confirmed cases of human salmonellosis are reported annually in European Europe (EFSA, 2009). According to the Regional Center for Foodborne Disease, 1571 people were affected by salmonellosis in Piedmont in 2009 (MTA, 2009). According to a previous study and official control data, very few Salmonella findings are reported from cows’ milk. In Italy Salmonella spp. was isolated from 0.7% of the milk samples tested (EFSA, 2009) and recently Giacometti et al. (2012) found one milk sample positive out of 99 analyzed. Here, two milk samples out of 618 (0.3%) were positive for Salmonella enterica and S. Anatum and S. Ndolo were identified. Although Typhimurium and Dublin serovars are considered to be the primary serotypes isolated from the bovine reservoir, other serovars have been detected (Murphy, Buckley, O’Connor, Gilroy, & Fanning, 2008) and S. Anatum was isolated from bovine bulk milk (Van Kessel, Karns, & Gorski, 2004). The prevalence of L. monocytogenes in bulk tank milk is reported to range from 1 to 12% (Oliver, Jayarao, & Almeida, 2005) and a previous Italian survey reports 1% of milk sample positive for Salmonella spp. (Giacometti et al., 2012). In the present study, L. monocytogenes was isolated in ten milk samples (1.6%). This species is widespread in nature and lives naturally in plants and soil environments; its high adaptability enables it to grow also in refrigerated raw milk. Among the twelve known serotypes, the only two identified in this study were 1/2a and 4b/4e. This result is in line with previous findings (Jayarao et al., 2006; Oliver et al., 2005).
over groups. A generalized linear model (GLM) was performed to evaluate factors affecting the occurrence of pathogens. A logit link function for binomial distribution was used. The significance of the predictors in the model was set at a ¼ 0.05 and tested using Wald’s chi square. 3. Results In all, 618 raw milk samples were collected from 112 dairy herds and 131 raw milk vending machines; 12,980 tests, including confirmation and serotyping, were performed. Tables 1 and 2 present the results of the three monitoring surveys; Table 3 reports the occurrence of the bacterial pathogens isolated from the raw milk samples. During the survey periods, two milk samples tested positive for Salmonella spp. Salmonella Ndolo and Salmonella Anatum were identified, respectively, from the milk samples collected at the herd and a few weeks later at the vending machine supplied by this herd. E. coli O:157 was isolated from a milk sample collected at a vending machine: the strain was non verocytotoxigenic. All nine Campylobacter spp. strains were identified as Campylobacter jejuni; six were isolated from milk samples collected from four different herds; in two cases, the herd was found positive for C. jejuni twice during the survey periods. The three other Campylobacter strains were isolated from milk samples collected at three vending machines: two of which were supplied from two positive herds. During the first monitoring survey, a milk sample from a vending machine tested positive both for C. jejuni and Salmonella Ndolo. During the three surveys, ten L. monocytogenes strains were identified: five were isolated from milk samples from four different herds (one bulk milk tank tested positive at two successive monitoring surveys): the five other L. monocytogenes strains were isolated from milk samples collected at five different vending machines, two of which were supplied by two positive herds. The most commonly identified serotypes were 1/2a (n ¼ 6) and 4b/4e (n ¼ 4). Only previous occurrence correlated with renewed detection of a pathogen and was statistically significant at bivariate analysis (Table 4). The odds ratio to be noncompliant in relation to a previous occurrence of pathogens as estimated by the GLM model was 23.26 (95% confidence interval [CI], 5.3e101.9). No other factors (sampling site, vending machine site, average daily temperature, herd size, season) were associated with the presence of pathogens (Listeria, Salmonella, Campylobacter). 4. Discussion The sale of raw milk from automatic self-service vending machines was authorized in Italy in 2004. Since then, the demand for raw milk has grown enormously. In 2008, the Italian Ministry of Health made it compulsory that all raw milk vending machines carry a warning that the milk should be boiled at home before
Table 1 Total number of raw milk samples collected at dairy herds and tested between 2009 and 2011. Period
Dairy herds
Noncompliant herds (%)
Samples
Nov. 2009eMarch 2010 July 2010eDec. 2010 Jan. 2011eJuly 2011 Total
95 89 93 277
4a (4.2) 0 5b (5.4) 9 (3.2)
107 92 99 298
a b
2 Pathogens were isolated in 3 samples. 1 Pathogen was isolated in 2 samples.
Noncompliant samples (%) 6 (5.6) 0 6 (6.1) 12 (4)
Unit-forming samples
Tests
535 460 495 1490
2238 1889 2115 6242
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Table 2 Total number of raw milk samples collected at vending machines (VM) and tested between 2009 and 2011. Period
VM
Noncompliant VM (%)
Samples
Noncompliant samples (%)
Unit-forming samples
Tests
Nov. 2009eMarch 2010 July 2010eDec. 2010 Jan. 2011eJuly 2011 Total
100 102 94 296
3 2 4 9
113 103 320
2 (1.8) 4 (3.9) 9 (2.8)
565 515 1600
2409 2169 6738
(3.0) (2.0) (4.3) (3.0)
In the reported survey, both biomolecular and microbiological traditional methods based on bacteria isolation were used at the laboratory level: samples were considered not compliant when target bacteria were isolated on cultural media indicated in ISO standards methods. In certain case, the limit of detection of microbiological methods can lead to an underestimation of real positive samples, official control strategies are still bound to the isolation of viable and cultivable micro-organisms, such as indicated in the ISO methods used for the present study. Bivariate analysis showed no seasonality effect. As expected, three pathogens (Listeria spp., Salmonella, and Campylobacter) were isolated from samples taken in all four seasons (Franciosi, Pecile, CAvazza, & Poznasnski, 2009). Nor was there an association between the sampling site (herd or vending machine) and the occurrence of pathogens. Pathogens can be found indifferently in dairy herds and in vending machines. In spite of restrictive measures adopted across the years, no substantial difference in the occurrence of pathogens was observed: the number is low but noncompliance with regulations appears irreducible. The vending machine installation site (farm or market) had no effect on the occurrence of pathogen, as the distribution of noncompliant samples was the same for the farm as the
market. In this study, the only factor affecting the probability to be incompliant was a previous instance of noncompliance. This suggests the need for more attention in herd management and the implementation of biosafety procedures. This survey allowed checking all 112 herds supplying vending machines in Piedmont and 131 vending machines out of 170 present in the study area. Due to sample size the real number of positive samples could be higher than the one observed: in particular the real percentage of target microorganism it is expected to be higher than the one detected. In conclusion, this monitoring survey confirms that unpasteurized milk can be vehicle of a variety of microorganisms and can be an important source of foodborne illness outbreaks, especially if consumers are young, elderly or ill. Pathogenic bacteria can be transmitted to milk through contact with contaminated sources on the farm and feces from infected animals. Pasteurization is the most effective method for ensuring the microbiological safety of milk. Furthermore, this study underlines the importance of educational training on food safety measures and the application of good practices for farmers selling raw milk. Also, these results underscore the importance of a consumer protection approach and confirm that the advice of Italian Ministry
Table 3 Number and percent (%) of pathogens isolated from raw milk samples collected from herds and vending machines (VM). Period
Sampling site
No. of samples
Salmonella spp.
E. coli O:157
Campylobacter spp.
Nov. 2009 e March 2010
HERD VM HERD VM HERD VM
107 104 92 113 99 103 618
1 (0.9) 1 (1) 0 0 0 0 2 (0.3)
0 0 0 1 (0.9) 0 0 1 (0.2)
3 2 0 0 3 1 9
July 2010 - Dec. 2010 Jan. 2011 e July 2010 Total
L. monocytogenes
(2.8) (1.9)
2 (1.9) 1 (1) 0 1 3 3 10
(3) (1) (1.5)
(0.9) (3) (2.9) (1.6)
Table 4 Bivariate analysis of factors influencing the occurrence of pathogens in raw milk samples. Factor
Category
No. of samples
Percentage noncompliant
Season
Winter Spring Autumn Summer 4.1 C 4.1 e10 C 10.1 e18 C >18C 68 69e127 128e196 >196 Herd Vending machine First Second Third Farm Market Yes No
125 203 180 110 148 150 171 149 155 167 143 153 298 320 211 205 202 455 163 14 604
2.40 5.42 1.67 3.64 2.70 4.00 2.92 4.03 3.23 3.59 4.90 1.96 4.03 2.81 4.27 0.98 4.95 3.30 3.68 28.57 2.81
Average daily temperature
Herd size
Sampling site Survey
Vending machine site Previous ccurrence of pathogens
OR denotes odds ratio; CI confidence interval.
c2
P value
4.56
0.21
0.68
0.8
1.97
0.57
0.01
0.41
5.63
0.06
0.05
0.81
27.60
0.0001
OR
95% CI
11.83
3.9e33.3
D.M. Bianchi et al. / Food Control 32 (2013) 435e439
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Food Control journal homepage: www.elsevier.com/locate/foodcont
Short communication
Monitoring of foodborne pathogenic bacteria in vending machine raw milk in Piedmont, Italy Daniela Manila Bianchi a, b, *, Antonio Barbaro a, c, Silvia Gallina a, b, Nicoletta Vitale a, c, Laura Chiavacci a, c, Maria Caramelli a, Lucia Decastelli a, b a b c
Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, 10154 Turin, Italy Struttura Complessa Controllo Alimenti e Igiene delle Produzioni, Italy Struttura Complessa Epidemiologia e Osservatorio Epidemiologico, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 July 2012 Received in revised form 22 December 2012 Accepted 5 January 2013
Raw milk consumption in Italy has increased over the past three years following the enactment of a national law that allows the sale of unpacked and unpasteurized cows’ milk via vending machines on the farm and at markets. From 2009 to 2011, a three-part monitoring survey of raw milk sold though vending machines was carried out to investigate for the occurrence of Salmonella spp., Escherichia coli O:157, Campylobacter spp. and Listeria monocytogenes. A total of 618 raw milk samples were collected from 112 dairy herds supplying 131 raw milk vending machines. Of the samples tested, 0.3% were positive for Salmonella spp., 0.2% for E. coli O:157, 1.5% for Campylobacter spp., and 1.6% for Listeria monocytogens. Multivariate analysis showed no effect of seasonality, average daily temperature, herd size, sample collection point or distance between herd and vending machine; however, there was a statistically significant correlation between a previous finding of pathogens and recurrence of contamination. The monitoring survey results confirm that unpasteurized milk can be a vehicle of a variety of microorganisms and an important source of foodborne illness outbreaks. Ó 2013 Published by Elsevier Ltd.
Keywords: Raw milk Foodborne pathogenic bacteria
1. Introduction Milk and dairy products are basic components of human diet and provide a dietary source of proteins, vitamins and other minerals (Huth, DiRienzo, & Miller, 2006). A vast variety of dairy products are available, including liquid milk, soft and hard cheese, yogurt, as well as specialty foods such as ricotta cheese and mascarpone. Almost all are made from pasteurized milk. Alongside this established market is a growing consumer demand for raw milk. However, raw milk consumption is accompanied by the risk of ingesting pathogenic bacteria that can pose an elevated health hazard (Latorre et al., 2009). And although zoonotic diseases like brucellosis and tuberculosis have been or are nearly completely eradicated in European Union countries (EFSA, 2010), the milk of asymptomatic animals may harbor other infectious agents: milk pasteurization was, and remains, key to preventing milk-borne zoonosis. European regulations and Regulation EC 853/2004 (Regulation CE 853/2004, 2004) in particular, allow the sale of raw milk,
* Corresponding author. Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, 10154 Turin, Italy. E-mail addresses: [email protected], [email protected] (D.M. Bianchi). 0956-7135/$ e see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.foodcont.2013.01.004
defining it as “the secretion of the mammary gland of farmed animals that has not been heated to more than 40 C or undergone any treatment that has an equivalent effect”. This regulation sets forth the guidelines for raw milk sale and distribution on the market. In Italy, the sale of raw milk via automatic self-service vending machines is regulated by an agreement between the state and the regions “Intesa Stato Regioni 2007 [ISR 2007]” (Intesa Stato Regioni, 2007a). Under the provisions of the ISR agreement, dairy farms, milk intended for raw consumption, and vending machines must comply with specific norms: biosafety measures and own-check for producers, microbiological and chemical criteria for milk, and vending machine installation and management specifications. In 2007 the Italian Ministry of Health authorized the sale and distribution of unpacked raw milk via automatic self-service vending machines. The milk dispensers boost sales revenues for farmers, as the milk is sold directly on the farm; consumers appreciate this new distribution channel as they can purchase a product they perceive as wholesome at a convenient price. The ISR agreement also details vending machine set up and management. Raw milk dispensers must be located in a welldefined separate area, far away from any source of waste or dirtiness. They must be equipped with a temperature thermometer
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with an external display and a thermostat that ensures a constant milk temperature between 0 C and 4 C. The machines must also have a dispenser valve that will interrupt the sale of raw milk if the milk temperature exceeds 4 C. The milk supply valve must be not exposed to contamination and it must allow easy removal for cleaning and disinfection. A vending machine can contain the raw milk from only one dairy herd. The front of the vending machine must display the following information: identification of the farm where the milk is produced, including the address of the farm, the date of milking, the date the machine was restocked, the expiry date, and instructions for home conservation at a temperature between 0 C and 4 C. Since December 2008, vending machines must also display a notice to customers that the raw milk must be boiled before consumption (Ordinance by the Ministry of Health, 2008). This warning to customers was deemed necessary owing to increased outbreaks of the hemolytic uremic syndrome (HUS) (Italian Registry of Familial and Recurrent HUS/TTP) and evidence from scientific studies that confirmed that unpasteurized milk poses a health hazard, especially for children (Scavia, Escher, Baldinelli, Pecoraro, & Caprioli, 2009; Weber, Behne, & Fruth, 2007). Under the provisions of ISR 2007 (Intesa Stato Regioni, 2007a), the Regional Competent Authority is responsible for devising and implementing monitoring plans for raw milk safety and hygiene and for the continuous control of microbiological criteria to ensure adequate consumer protection. In Italy, raw milk vending machines are supplied by 1032 dairy herds (Integrated National Annual Report of Italian Ministry of Health (Italian Ministry of Health, 2010)). Each herd can supply one or more vending machines. Of a national total of 1411 authorized vending machines, 170 (12%) supplied by 112 dairy herds are authorized for operation in Piedmont. Since 2008, monitoring surveys have been conducted in Piedmont to check that the raw milk sold via vending machines complies with the criteria set forth in the national regulation. During a pilot monitoring survey carried out from November 2008 to July 2009, Listeria monocytogenes and Campylobacter spp. were isolated from some samples of unpacked raw milk. The Regional Veterinary Authority subsequently conducted three annual mandatory monitoring surveys to inspect all dairy herds and at least one of the vending machines they supplied. This study reports the results of the three monitoring surveys conducted from November 2009 to June 2011. The microbiological safety of raw milk is discussed, focusing on commonly isolated pathogenic bacteria and potential risk factors that can increase the probability of contamination by pathogens in raw milk. 2. Materials and methods 2.1. Sampling plans Between November 2009 and July 2011, three surveys were carried out: the first from November 2009 to March 2010, the second from July to December 2010, and the last from January to July 2011. During each survey, all herds supplying raw milk for a vending machine and at least one vending machine for each herd were inspected. Sampling was performed by the local veterinary service. The samples were collected into sterile containers and brought refrigerated to the Food Control Laboratory of the Istituto Zooprofilattico Sperimentale del Piemonte Liguria e Valle d’Aosta in Turin. Sampling at the vending machine and the bulk milk tank was done without advance notification of the farmers or the vending machine operators. However, because of the product’s short shelflife, the milk producers and food business operators were informed about the date of testing so that they or a legal representative could
be present in the laboratory during analysis. In compliance with ISO 7218:2007 (Microbiology of food and animal feeding stuffs, 2007b), the analyses were started within 24 h of sampling. In accordance with ISR 2007 guidelines, each milk sample was composed of five units: each unit contained at least 200 mL of raw milk. Microbiological analyses were performed on all five units forming the sample. 2.2. Laboratory testing In accordance with national legal requirements (Intesa Stato Regioni, 2007a) for food safety criteria, the milk samples were tested for the presence of: Salmonella spp., L. monocytogenes, Escherichia coli O:157 and Campylobacter spp. Each test was performed on 25 mL of raw milk by means of qualitative methods; the laboratory and all test procedures are accredited according to ISO 17025:2005 (General requirements for the competence of testing and calibration laboratories, 2005) at the ACCREDIA Italian accreditation body. A biomolecular method (real-time PCR) (AFNOR, 2004) was performed to detect Salmonella spp. Samples testing positive for Salmonella spp. DNA underwent a second round for confirmation with a microbiological method according to ISO 6579 (Microbiology of food and animal feeding stuffs, 2002). Viable colonies suspected of belonging to the genus Salmonella were biochemically confirmed by API20EÒ (BioMérieux). Serotyping of Salmonella spp. strains was performed according to the KauffmanneWhite protocol. A biomolecular method (real-time PCR; Biorad AFNOR BRD 07/ 10-04/05) was also performed to detect L. monocytogenes DNA. Samples testing positive were retested under a microbiological protocol according to ISO 11290 (Microbiology of food and animal feeding stuffs, 1996b). Viable colonies suspected to be L. monocytogenes were biochemically confirmed by GP-VITEKÒ (BioMérieux). Confirmed L. monocytogenes strains were then analyzed to identify the serotype (serum provided by Denka Seiken Co). An enzyme-linked-fluorescence assay (AFNOR, 2000) was applied to detect E. coli O:157. Positive samples were then retested for microbiological isolation and identification according to ISO 16654 (Microbiology of food and animal feeding stuffs, 1996a). E. coli O:157 strains were investigated for the presence of shiga-like toxinproducing genes according to ISO TS 13136 (Microbiology of food and animal feeding stuffs, 2011). The microbiological method described in ISO 10272 (Microbiology of food and animal feeding stuffs, 2006) was used to detect Campylobacter spp. Colonies suspected of belonging to the genus Campylobacter were biochemically confirmed by API (BioMérieuxÒ). A unit of the milk sample was considered incompliant with food safety norms if one pathogenic bacterium was isolated in 25 mL; the whole milk sample was considered incompliant if one or more units were not compliant. 2.3. Statistical analyses Data were analyzed using SASÒ software ver. 9.2 (SAS Institute Inc., Cary, NC, USA). Descriptive analysis was performed to determine the occurrence of pathogens. The c2 test was applied to test for associations between the occurrence of pathogens and the following variables: distance between dairy herds and vending machine; herd size; previous occurrence of pathogens; daily average temperature; months and seasons. Categorical variables were reduced to as few categories as possible, ideally two. The univariate odds ratios were calculated. Linear associations were confirmed for significant continuous variables by dividing them into four groups according to quartiles and assessing the linearity in the odds ratios
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consumption. Nonetheless, many consumers still drink raw milk for several reasons: the milk dispensed at vending machines is less expensive than pasteurized milk sold at a market; consuming raw milk is viewed as customary practice; raw milk is believed to be more wholesome and nutritious than pasteurized milk. Whatever the consumer convictions about raw milk, epidemiological data have highlighted the growing involvement of Campylobacter spp. in foodborne zoonosis outbreaks. The Community Zoonoses Reports of the European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (ECDC) in their Community Zoonoses Reports have in the last 5 years cited campylobacteriosis as the most commonly reported zoonosis in the European Union (EFSA, 2007) (EFSA, 2010). In the present study, Campylobacter was found in 1.5% of samples. Previous studies reported the prevalence of Campylobacter spp. in raw milk samples ranging from 0.3% (Cavirani, 2008), to 2% (Jayarao et al., 2006), to 4.6% (Wysok, Wiszniewska-qaszczych, ski, & Szteyn, 2011) and 9.2% (Jayarao & Henning, 2001). Uradzin Similarly, in a study that included 99 raw milk samples in Northern Italy, thermotolerant Campylobacter were isolated or PCR-detected in 2.02% of samples (Giacometti et al., 2012). All isolated strains identified in this study belonged to the species C. jejuni: this finding is supported by an epidemiological survey on bovine fecal culture which reported an infection rate from 5 to 50%, with C. jejuni ranking highest, followed by Campylobacter coli and Campylobacter upsaliensis (Wesley et al., 2000), confirming that milk contamination during or immediately after milking is most probably of stool origin. About 155,000 confirmed cases of human salmonellosis are reported annually in European Europe (EFSA, 2009). According to the Regional Center for Foodborne Disease, 1571 people were affected by salmonellosis in Piedmont in 2009 (MTA, 2009). According to a previous study and official control data, very few Salmonella findings are reported from cows’ milk. In Italy Salmonella spp. was isolated from 0.7% of the milk samples tested (EFSA, 2009) and recently Giacometti et al. (2012) found one milk sample positive out of 99 analyzed. Here, two milk samples out of 618 (0.3%) were positive for Salmonella enterica and S. Anatum and S. Ndolo were identified. Although Typhimurium and Dublin serovars are considered to be the primary serotypes isolated from the bovine reservoir, other serovars have been detected (Murphy, Buckley, O’Connor, Gilroy, & Fanning, 2008) and S. Anatum was isolated from bovine bulk milk (Van Kessel, Karns, & Gorski, 2004). The prevalence of L. monocytogenes in bulk tank milk is reported to range from 1 to 12% (Oliver, Jayarao, & Almeida, 2005) and a previous Italian survey reports 1% of milk sample positive for Salmonella spp. (Giacometti et al., 2012). In the present study, L. monocytogenes was isolated in ten milk samples (1.6%). This species is widespread in nature and lives naturally in plants and soil environments; its high adaptability enables it to grow also in refrigerated raw milk. Among the twelve known serotypes, the only two identified in this study were 1/2a and 4b/4e. This result is in line with previous findings (Jayarao et al., 2006; Oliver et al., 2005).
over groups. A generalized linear model (GLM) was performed to evaluate factors affecting the occurrence of pathogens. A logit link function for binomial distribution was used. The significance of the predictors in the model was set at a ¼ 0.05 and tested using Wald’s chi square. 3. Results In all, 618 raw milk samples were collected from 112 dairy herds and 131 raw milk vending machines; 12,980 tests, including confirmation and serotyping, were performed. Tables 1 and 2 present the results of the three monitoring surveys; Table 3 reports the occurrence of the bacterial pathogens isolated from the raw milk samples. During the survey periods, two milk samples tested positive for Salmonella spp. Salmonella Ndolo and Salmonella Anatum were identified, respectively, from the milk samples collected at the herd and a few weeks later at the vending machine supplied by this herd. E. coli O:157 was isolated from a milk sample collected at a vending machine: the strain was non verocytotoxigenic. All nine Campylobacter spp. strains were identified as Campylobacter jejuni; six were isolated from milk samples collected from four different herds; in two cases, the herd was found positive for C. jejuni twice during the survey periods. The three other Campylobacter strains were isolated from milk samples collected at three vending machines: two of which were supplied from two positive herds. During the first monitoring survey, a milk sample from a vending machine tested positive both for C. jejuni and Salmonella Ndolo. During the three surveys, ten L. monocytogenes strains were identified: five were isolated from milk samples from four different herds (one bulk milk tank tested positive at two successive monitoring surveys): the five other L. monocytogenes strains were isolated from milk samples collected at five different vending machines, two of which were supplied by two positive herds. The most commonly identified serotypes were 1/2a (n ¼ 6) and 4b/4e (n ¼ 4). Only previous occurrence correlated with renewed detection of a pathogen and was statistically significant at bivariate analysis (Table 4). The odds ratio to be noncompliant in relation to a previous occurrence of pathogens as estimated by the GLM model was 23.26 (95% confidence interval [CI], 5.3e101.9). No other factors (sampling site, vending machine site, average daily temperature, herd size, season) were associated with the presence of pathogens (Listeria, Salmonella, Campylobacter). 4. Discussion The sale of raw milk from automatic self-service vending machines was authorized in Italy in 2004. Since then, the demand for raw milk has grown enormously. In 2008, the Italian Ministry of Health made it compulsory that all raw milk vending machines carry a warning that the milk should be boiled at home before
Table 1 Total number of raw milk samples collected at dairy herds and tested between 2009 and 2011. Period
Dairy herds
Noncompliant herds (%)
Samples
Nov. 2009eMarch 2010 July 2010eDec. 2010 Jan. 2011eJuly 2011 Total
95 89 93 277
4a (4.2) 0 5b (5.4) 9 (3.2)
107 92 99 298
a b
2 Pathogens were isolated in 3 samples. 1 Pathogen was isolated in 2 samples.
Noncompliant samples (%) 6 (5.6) 0 6 (6.1) 12 (4)
Unit-forming samples
Tests
535 460 495 1490
2238 1889 2115 6242
438
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Table 2 Total number of raw milk samples collected at vending machines (VM) and tested between 2009 and 2011. Period
VM
Noncompliant VM (%)
Samples
Noncompliant samples (%)
Unit-forming samples
Tests
Nov. 2009eMarch 2010 July 2010eDec. 2010 Jan. 2011eJuly 2011 Total
100 102 94 296
3 2 4 9
113 103 320
2 (1.8) 4 (3.9) 9 (2.8)
565 515 1600
2409 2169 6738
(3.0) (2.0) (4.3) (3.0)
In the reported survey, both biomolecular and microbiological traditional methods based on bacteria isolation were used at the laboratory level: samples were considered not compliant when target bacteria were isolated on cultural media indicated in ISO standards methods. In certain case, the limit of detection of microbiological methods can lead to an underestimation of real positive samples, official control strategies are still bound to the isolation of viable and cultivable micro-organisms, such as indicated in the ISO methods used for the present study. Bivariate analysis showed no seasonality effect. As expected, three pathogens (Listeria spp., Salmonella, and Campylobacter) were isolated from samples taken in all four seasons (Franciosi, Pecile, CAvazza, & Poznasnski, 2009). Nor was there an association between the sampling site (herd or vending machine) and the occurrence of pathogens. Pathogens can be found indifferently in dairy herds and in vending machines. In spite of restrictive measures adopted across the years, no substantial difference in the occurrence of pathogens was observed: the number is low but noncompliance with regulations appears irreducible. The vending machine installation site (farm or market) had no effect on the occurrence of pathogen, as the distribution of noncompliant samples was the same for the farm as the
market. In this study, the only factor affecting the probability to be incompliant was a previous instance of noncompliance. This suggests the need for more attention in herd management and the implementation of biosafety procedures. This survey allowed checking all 112 herds supplying vending machines in Piedmont and 131 vending machines out of 170 present in the study area. Due to sample size the real number of positive samples could be higher than the one observed: in particular the real percentage of target microorganism it is expected to be higher than the one detected. In conclusion, this monitoring survey confirms that unpasteurized milk can be vehicle of a variety of microorganisms and can be an important source of foodborne illness outbreaks, especially if consumers are young, elderly or ill. Pathogenic bacteria can be transmitted to milk through contact with contaminated sources on the farm and feces from infected animals. Pasteurization is the most effective method for ensuring the microbiological safety of milk. Furthermore, this study underlines the importance of educational training on food safety measures and the application of good practices for farmers selling raw milk. Also, these results underscore the importance of a consumer protection approach and confirm that the advice of Italian Ministry
Table 3 Number and percent (%) of pathogens isolated from raw milk samples collected from herds and vending machines (VM). Period
Sampling site
No. of samples
Salmonella spp.
E. coli O:157
Campylobacter spp.
Nov. 2009 e March 2010
HERD VM HERD VM HERD VM
107 104 92 113 99 103 618
1 (0.9) 1 (1) 0 0 0 0 2 (0.3)
0 0 0 1 (0.9) 0 0 1 (0.2)
3 2 0 0 3 1 9
July 2010 - Dec. 2010 Jan. 2011 e July 2010 Total
L. monocytogenes
(2.8) (1.9)
2 (1.9) 1 (1) 0 1 3 3 10
(3) (1) (1.5)
(0.9) (3) (2.9) (1.6)
Table 4 Bivariate analysis of factors influencing the occurrence of pathogens in raw milk samples. Factor
Category
No. of samples
Percentage noncompliant
Season
Winter Spring Autumn Summer 4.1 C 4.1 e10 C 10.1 e18 C >18C 68 69e127 128e196 >196 Herd Vending machine First Second Third Farm Market Yes No
125 203 180 110 148 150 171 149 155 167 143 153 298 320 211 205 202 455 163 14 604
2.40 5.42 1.67 3.64 2.70 4.00 2.92 4.03 3.23 3.59 4.90 1.96 4.03 2.81 4.27 0.98 4.95 3.30 3.68 28.57 2.81
Average daily temperature
Herd size
Sampling site Survey
Vending machine site Previous ccurrence of pathogens
OR denotes odds ratio; CI confidence interval.
c2
P value
4.56
0.21
0.68
0.8
1.97
0.57
0.01
0.41
5.63
0.06
0.05
0.81
27.60
0.0001
OR
95% CI
11.83
3.9e33.3
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