Pathogenic Bacteria in Finnish Bulk Tank Milk

Abstract

While the quality of raw cow milk in Finland is known for its high hygienic standard, with the national average total bacterial count being below 10⁴ CFU/mL annually, the prevalence of pathogenic bacteria in Finnish raw milk is underreported. The aim of this study was to determine the occurrence of Listeria monocytogenes, thermophilic Campylobacter spp., Salmonella spp., stx-positive Escherichia coli (STEC), coagulase-positive staphylococci, Yersinia spp., and Bacillus cereus group in raw cow milk samples collected from bulk tanks at 183 Finnish farms. Additionally, the hygienic quality of the milk was studied by determining the total bacterial and E. coli counts. L. monocytogenes was detected in 5.5% of the milk samples, with concentrations varying from <1 to 30 CFU/mL. Thermophilic Campylobacter spp. or Salmonella spp. were not detected in any of the samples. STEC with Shiga toxin–encoding stx2 was detected in 2.7% of the samples. Yersinia enterocolitica was detected in 7.7% of the samples; however, all isolates were negative for ail, suggesting that they were non-pathogenic. Coagulase-positive staphylococci were detected in 34.4% of the samples, with an average concentration of 25 CFU/mL in the positive samples. Members of the B. cereus group were detected in 20.8% of the samples, with an average concentration of 1 CFU/mL in the positive samples. No relationship was detected between E. coli or the total bacterial count and the presence of pathogenic bacteria, which suggests that pathogens can be present also in farms with excellent production hygiene. Although the concentration of pathogenic bacteria in fresh raw milk was mainly relatively low, it should be borne in mind that some of the pathogenic bacteria can survive and multiply at refrigeration temperatures and may cause a disease with a very low infectious dose. Thus, consumption of raw milk and related products poses a potential risk for food poisoning.

Introduction

I ncreasing demand for unpasteurized milk has brought the safety and quality of raw milk into the focus of research and general debate. The increased popularity of raw milk consumption has been explained by taste, convenience, cost, high nutritional quality (Kaylegian et al., 2008), and assumed protective role against allergies (Braun-Fahrländer and von Mutius, 2011). However, raw milk is an ideal growth medium for bacteria, and it can contain a large variety of pathogenic bacteria (Oliver et al., 2009). The problem may be exacerbated, as the use of automatic milking techniques has been shown to increase the total bacterial count and spore contamination of milk (Rasmussen et al., 2002).

While pasteurization destroys most vegetative pathogens in milk, consumption of raw milk or related products has been associated with large food poisoning outbreaks (Oliver et al., 2009), and the number of outbreaks appears to be increasing (Newkirk et al., 2011). From 2000 to 2011, raw milk or related products were associated with 45 outbreaks of foodborne illness in North America. A total of 686 people were affected, with eight deaths (BC Centre for Disease Control, 2012). Raw milk–related outbreaks are also common in Europe, and in the 1990s, approximately 100 milk-borne outbreaks were reported in the United Kingdom and France (De Buyser et al., 2001; Gillespie et al., 2003). While less than 1% of dairy products are consumed unpasteurized (Langer et al., 2012), 50–60% of all dairy-related outbreaks in the United States and Europe have been associated with consumption of unpasteurized products (Gillespie et al., 2003; Langer et al., 2012). The actual number of outbreaks is most likely markedly higher since not all cases are recognized or reported. Thermophilic Campylobacter spp., shigatoxic Escherichia coli (STEC), Salmonella spp., and Staphylococcus aureus were the most commonly detected pathogens in raw milk-borne outbreaks in the United States and Europe (De Buyser et al., 2001; Gillespie et al., 2003; Newkirk et al., 2011; Langer et al., 2012). Moreover, dairy products caused almost half of the food poisoning outbreaks caused by Listeria monocytogenes in Europe (Lundén et al., 2004). The occurrence of pathogens in raw milk produced in Europe and in the United States has suggested considerable variation (Oliver et al., 2005, 2009). This may be partly explained by different detection methods (Amagliani et al., 2012).

Member states of the European Union have implemented common regulations in relation to production of raw milk, which can be complemented by national rules (European Commission 853/2004). In Finland, the maximum amount of raw milk allowed for direct sale from a farm to consumers is 2,500 L per year. Sale of larger volumes requires approval of the farm facility as a food processing establishment (Food Act 23/2006). While Finnish farmers and consumers are actively demanding the release of the raw milk sales, food control authorities call for scientific data on the health risks related to raw milk consumption. The Finnish cow milk is considered to be of very high hygienic quality, with the national average total bacterial count being below 10⁴ CFU/mL annually (Finnish Association for Milk Hygiene, 2012). While an average of 3.4 L of unpasteurized milk was consumed per capita each year from 2000 to 2010 (Agricultural Statistics, 2012a), the number is prone to be significantly higher among established consumers. Consumption of raw milk in the past was associated mostly with farming families and employees, but interest among the urban population is increasing.

There are limited studies on the prevalence of pathogenic bacteria in raw milk in Scandinavian countries. The aim of this study was to determine the occurrence of L. monocytogenes, thermophilic Campylobacter spp., Salmonella spp., sorbitol-negative stx-positive E. coli (STEC), coagulase-positive staphylococci, Yersinia spp., and Bacillus cereus group in Finnish raw cow milk. Additionally, the hygienic quality of the milk was studied by determining the total bacterial count and the number of E. coli. The results provide insight into the microbiological risks associated with milk production and raw milk consumption in Finland.

Materials and Methods

Sampling and bacteriological methods

In this cross-sectional study, raw cow milk samples were collected in June–August 2011 from bulk tanks in 183 dairy farms around Southern and Central Finland representing 1.7% of all the 10 597 dairy farms in Finland in 2011 (Agricultural Statistics, 2012b). Sampling was organized by dairies in cooperation with farmers and dairy truck drivers (Thrusfield, 2007). All samples were refrigerated (<4°C) and analyzed within 2 days from sampling. The milk had been in the bulk tanks for a maximum of 2 days before sampling. Methods used for bacteriological analyses are described in Table 1.

Table 1.

Methods Used for Bacteriological Analyses

Organism	Protocol	Quantitative method (sample size)	Qualitative method (sample size)	Broths and agars	Incubation conditions	Verification of colonies
Listeria monocytogenes	Modified ISO 11290-1:1996 standard protocol	Direct plating (1 mL)	NA	ALOA^a	37°C, 48 h	Catalase test, Gram staining, API Listeria test kit^d
		NA	Selective enrichment (25 mL)	Half-Fraser broth and Fraser broth^b; followed by ALOA	30°C, 24 h (half-Fraser); 37°C, 48 h (Fraser)
		NA	Non-selective enrichment (25 mL)	TSB; followed by ALOA	30°C, 18–20 h
Thermophilic Campylobacter spp.	Modified ISO 10272-1:2006 standard protocol	NA	Enrichment (25 mL)	Bolton broth^b; followed by mCCDA^b	37°C, 48 h, microaerophilic (Bolton broth); 42°C, 48 h, microaerophilic (mCCDA)	Gram staining, hippurate hydrolysis, multiplex-PCR (Denis et al., 1999)
Salmonella spp.	Modified ISO 6579:2002 standard protocol	NA	Enrichment (25 mL)	TSB; followed by Rappaport Vassiliadis broth^b; followed by XLD agar^b	30°C, 18–20 h (TSB); 42°C, 48 h (Rappaport Vassiliadis broth); 37°C, 24 h (XLD)	Real-time PCR targeted to ttr (Malorny et al., 2004)
Sorbitol-negative stx-positive Escherichia coli	Modified ISO 16654:2001 standard protocol	NA	Enrichment (25 mL)	TSB; followed by SMAC agar^c	30°C, 18–20 h (TSB); 42°C, 24 h (SMAC)	Real-time PCR targeted to stx1 and stx2 (Sharma and Dean-Nystrom, 2003)
Coagulase-positive staphylococci	Modified ISO 6888-1:1999 standard protocol	Plating from serially diluted samples (10⁰-10⁻³ mL)	NA	Baird-Parker agar^b	37°C, 24 h	Coagulase test
Yersinia spp.	Korte et al., 2004 with modifications	Direct plating (1 mL)	NA	CIN agar^b	30°C, 24 h	Urea hydrolysis, API 20E test kit^d, PCR targeted to ail (Nakajima et al., 1992)
		NA	Overnight enrichment (25 mL)	TSB; followed by CIN	30°C, 18–20 h
		NA	Cold enrichment (25 mL)	PMB broth; followed by CIN	4°C, 1, 2, and 3 weeks
Bacillus cereus group	Modified ISO 7932:2004 standard protocol	Plating from serially diluted samples (10⁰ to 10⁻³ mL)	NA	MYP agar^b	30°C, 48 h	Catalase test, Gram staining
Escherichia coli	Modified NMKL 125:2005 standard protocol	Plating from serially diluted samples (10⁰ to 10⁻³ mL)	NA	VRB agar^b	44°C, 24 h	Oxidase test, gas formation, indole test
Total bacterial count	ISO 4833:2003	Plating from serially diluted samples (10⁻¹ to 10⁻⁴ mL)	NA	MPCA^a	30°C, 72 h

Lab M Limited, Bury, Lancashire, UK.

Oxoid, Basingstoke, Hampshire, UK.

Merck, Darmstadt, Germany.

BioMérieux, Marcy l'Etoile, France.

NA, not applicable; ALOA, Agar Listeria Ottavani & Agosti; TSB, tryptic soy broth; mCCDA, modified charcoal cephoperazone desoxycholate agar; XLD, xylose lysine deoxycholate; SMAC, sorbitol MacConkey; CIN, cefsulodin-irgasan-novobiocin; PMB, peptone mannitol bile salt; MYP, mannitol-egg yolk-polymyxin; VRB, violet red bile; MPCA, milk plate count agar; PCR, polymerase chain reaction.

Statistical analysis

A scatter plot analysis was used with PASW Statistics 18 software to detect relationships between the total bacterial count or number of E. coli and pathogenic bacteria. Confidence intervals (CI) were calculated using the equation for the exact binomial CI (Agresti and Coull, 1998).

Results and Discussion

L. monocytogenes was detected in 10 raw milk samples (5.5% [95% CI 3.0–9.8%]), with concentrations of 5 and 30 CFU/mL in the two samples testing positive with the direct plating method, and with concentrations below 1 CFU/mL in eight samples that tested positive after enrichment (Table 2). The occurrence of L. monocytogenes in Finnish raw milk samples was in agreement with the 1.0–7.0% reported in Sweden (Waak et al., 2002), Italy (Giacometti et al., 2012), France (Desmasures et al., 1997), Spain (Vilar et al., 2007), and the United States (Jayarao and Henning, 2001; Muraoka et al., 2003; Van Kessel et al., 2004; D'Amico et al., 2008). Farms with a small or large total bacterial count were equally contaminated, showing that L. monocytogenes contamination in raw milk cannot be totally avoided by good hygienic practices. Despite the relatively high occurrence of L. monocytogenes, the observed number of bacteria in raw milk was low. While L. monocytogenes levels below 100 CFU/g of food have generally been considered safe for healthy adults (European Commission 2073/2005), the infectious dose is poorly understood and may be significantly lower for risk groups. This is supported by a hospital outbreak of listeriosis among immunocompromised persons, traced back to consumption of pasteurized butter containing L. monocytogenes at levels of 5–60 CFU/g (Lyytikäinen et al., 2000). Prolonged exposure to low levels of L. monocytogenes may further increase the risk (Maijala et al., 2001). In addition, L. monocytogenes strains have been shown to survive and grow in raw milk at 4°C (Gaya et al., 1991), thus long storage of contaminated milk even at refrigeration temperatures can cause a health hazard.

Table 2.

Occurrence and Counts of Pathogenic Bacteria in Finnish Raw Milk Samples

Organism (number of samples analyzed)	Number of positive samples (percentage [95% CI])	Count in positive samples (CFU/mL [average])	Total bacterial count range in samples positive for each pathogen (CFU/mL [average])	Escherichia coli count range in samples positive for each pathogen (CFU/mL [average])
Listeria monocytogenes (n=183)	10 (5.5% [3.0–9.8%])	<1–30^a (1)	2.1×10³ to 2.3×10⁶ (2.0×10⁴)	0–6.3×10² (3.1×10¹)
Thermophilic Campylobacter spp. (n=177)	0 (0% [0–2.1%])^b	ND	NA	NA
Salmonella spp. (n=183)	0 (0% [0–2.1%])^b	ND	NA	NA
Sorbitol-negative stx-positive	5 (2.7% [1.2–6.2%])^c	ND	8.8×10³ to 1.0×10⁶ (5.6×10⁴)	0–5.2×10¹ (6.1×10⁰)
Escherichia coli (n=183)
Coagulase-positive staphylococci (n=183)	63 (34.4% [27.9–41.6%])	1–1500^a (25)	2.6×10² to 3.0×10⁶ (1.2×10⁴)	0–8.9×10² (7.6×10⁰)
Yersinia spp. (n=183)	27 (14.8% [10.3–20.6%])^c	ND	2.0×10² to 2.3×10⁶ (1.1×10⁴)	0–6.3×10² (1.7×10¹)
Yersinia enterocolitica (n=183)	14 (7.7% [4.6–12.4%])^c,d	ND	2.1×10³ to 2.3×10⁶ (1.1×10⁴)	0–6.3×10² (8.9×10⁰)
Yersinia pseudotuberculosis (n=183)	0 (0% [0–2.1%])^b	ND	NA	NA
Bacillus cereus group (n=183)	38 (20.8% [15.5–27.2%])	1–8^a (1)	2.6×10² to 3.0×10⁶ (1.6×10⁴)	0–1.7×10² (2.4×10⁰)

Detection limit of 1 CFU/mL.

Not detected in 25 mL of milk.

Detected in 25 mL of milk.

All isolates ail-negative.

CI, confidence interval; CFU, colony-forming unit; ND, not determined; NA, not applicable.

Consumption of raw milk or related products has been linked to several listeriosis outbreaks in Europe (Lundén et al., 2004). Listeriosis can manifest itself either as febrile gastroenteritis or more severe invasive forms (Allerberger and Wagner, 2010). L. monocytogenes has been shown to survive during cheese manufacture and ripening (Bachmann and Spahr, 1995; Linton et al., 2008; Schvartzman et al., 2011), so cheeses made from unpasteurized milk can pose a serious risk as demonstrated by an outbreak in France (Goulet et al., 1995). L. monocytogenes strains adhere to food contact surfaces resulting in persistent plant contamination that may last for several years (Miettinen et al., 1999; Lundén et al., 2000). Biofilm formation in milking equipment (Latorre et al., 2010) may also lead to persistent contaminations at farms. Long-term monitoring of the farms testing positive for L. monocytogenes is therefore needed to evaluate the degree of persistency of contamination in dairy production and to establish targeted sanitation.

Thermophilic Campylobacter spp. were not detected in any of the samples [95% CI 0–2.1%], which was in agreement with a previous study where no campylobacteria were detected in tank milk samples taken on five subsequent occasions at 2–4-month intervals from three Finnish dairy cattle herds (Hakkinen and Hänninen, 2009). However, C. jejuni and C. coli are very common cause of foodborne outbreaks and gastroenteritis, often also associated with raw milk-borne outbreaks (Newkirk et al., 2011). The risk was recently re-established by the large raw milk outbreak affecting nearly 100 persons in the eastern United States (Food Safety News, 2012). An outbreak of campylobacteriosis associated with the consumption of raw milk was also reported in a Finnish farmer family (Schildt et al., 2006). These epidemiological findings and detection of campylobacteria in 31% of the fecal samples from Finnish cattle (Hakkinen et al., 2007) suggest that Campylobacter can be present also in the milk chain and may thus pose a health risk for raw milk consumers also in Finland. The negative result in our data may be partly explained by poor survival of Campylobacter cells at low temperature in raw milk (Doyle and Roman, 1982).

Salmonella spp. was not detected in any of the samples [95% CI 0–2.1%]. Very low result was expected because of the Finnish Salmonella Control Programme targeted to keep the prevalence of salmonella in all production animals and foodstuffs below the level of 1%. Since 2000, ≤0.4% of Finnish meat, lymph node, or surface samples of cattle carcasses tested positive for salmonella each year. Moreover, only 7–15 farms yielded salmonella positive fecal samples in annual sampling (Finnish Zoonosis Centre, 2012). These surveillance data together with our results suggest that the risk of salmonellosis through consumption of raw milk in Finland appears relatively low. Nevertheless, several international (Mazurek et al., 2004; Dominguez et al., 2009; Van Duynhoven et al., 2009) and even Finnish (Epinorth, 2005) salmonellosis outbreaks related to consumption of raw milk or related products, and the presence of salmonella in 2.2–11.8% of raw milk in the United States (Jayarao and Henning, 2001; Murinda et al., 2002; Van Kessel et al., 2004; Karns et al., 2005; Jayarao et al., 2006) remind us of the potential of salmonella as a major milk pathogen.

STEC with Shiga toxin–encoding stx2 was detected with PCR analysis in sorbitol-negative colonies from five samples (2.7% [95% CI 1.2–6.2%]), while stx1 was not detected. Stx2 has been more frequently associated with the development of the life-threatening hemolytic uremic syndrome than Stx1 (Nataro and Kaper, 1998). The occurrence of sorbitol-negative STEC in our study was consistent with up to 3.8% reported in the United States (Jayarao and Henning, 2001; Murinda et al., 2004; Jayarao et al., 2006). The infectious dose of STEC can be low (Karmali, 2004) and serious foodborne infections through raw milk (Denny et al., 2008; Guh et al., 2010) or raw milk cheeses (Vernozy-Rozand et al., 2005; Stephan et al., 2008) have been reported, recently also in Finland (ProMED-mail, 2012). No relationship was detected between a high concentration of E. coli and the presence of STEC, suggesting the concentration of E. coli is not a reliable indicator for the occurrence of STEC in milk.

Coagulase-positive staphylococci were detected in 63 samples (34.4% [95% CI 27.9–41.6%]), with concentrations varying from 1 to 1,500 CFU/mL in the positive samples, and the average concentration being 25 CFU/mL. The occurrence of coagulase-positive staphylococci in our study was in agreement with the 32.5–47.2% reported in Poland (Korpysa-Dzirba and Osek, 2011), Italy (Normanno et al., 2005), and Norway (Jakobsen et al., 2011). However, higher occurrences, from 62% to 83%, have been detected in Norway (Jørgensen et al., 2005a), France (Desmasures et al., 1997), and Italy (Bartolomeoli et al., 2009). The most significant coagulase-positive staphylococcal species in food poisoning outbreaks is S. aureus, which is part of the normal microbial population on the skin and mucous membranes of humans and animals (Hatakka et al., 2000) and thus a common finding in raw milk. S. aureus is also a notorious cause of bovine mastitis. Several S. aureus strains isolated from milk have been shown to produce enterotoxins (Jørgensen et al., 2005a), which can cause gastrointestinal illness in humans. However, growth to numbers high enough to support toxigenesis in milk is not likely under refrigeration. Nevertheless, raw milk-related food poisonings have often been reported (De Buyser et al., 2001; Jørgensen et al. 2005b; Ostyn et al., 2010), which suggests temperature fluctuation during production or storage.

Y. enterocolitica was detected in 14 samples (7.7% [95% CI 4.6–12.4%]). Two of these samples were positive with the direct plating method, eight were positive after overnight enrichment, and 12 were positive after cold enrichment. Y. pseudotuberculosis was not detected in any of the samples (95% CI 0–2.1%). Other Yersinia spp., such as Y. frederiksenii, Y. intermedia, and Y. kristensenii, were detected in 15 samples (8.2%). Only a few studies on the prevalence of Y. enterocolitica in raw milk have been reported. The occurrence of Y. enterocolitica in our study was in agreement with 1.2–6.1% reported in the United States (Jayarao and Henning, 2001; Jayarao et al., 2006). However, all our Y. enterocolitica isolates tested negative for ail, a gene shown to correlate with pathogenicity. In previous studies all the isolates from raw milk were considered to be pathogenic based on an autoagglutination test (Jayarao and Henning, 2001; Jayarao et al., 2006). Non-pathogenic strains detected in our study indicate the survival of Y. enterocolitica in milk; thus, the presence of pathogenic strains cannot be excluded. Several yersiniosis outbreaks have been associated with the consumption of pasteurized milk (Ackers et al., 2000; Food Safety News, 2011), but the importance of cattle as a reservoir for Y. enterocolitica remains unknown. The major reservoirs for pathogenic Y. enterocolitica strains are evidently pigs (Fredriksson-Ahomaa et al., 2006).

Members of the B. cereus group were detected in 38 samples (20.8% [95% CI 15.5–27.2%]), with concentrations varying from 1 to 8 CFU/mL in the positive samples, and the average concentration being 1 CFU/mL. Thus, while B. cereus is a relatively common contaminant in milk, concentrations in fresh milk are low. Therefore the immediate risk for food poisoning appears to be low. This is supported by scarce reports on B. cereus food poisonings due to consumption of dairy products in general (De Buyser et al., 2001; Gillespie et al., 2003; Langer et al., 2012). However, relatively high number of B. cereus group strains isolated from milk are positive for the enterotoxin genes (Bartoszewicz et al., 2008), which suggests toxigenic potential in the dairy chain. For reliable assessment of B. cereus risk in raw milk it would be essential to identify the toxic potential of the isolated strains and link their genetic background to strains isolated from food poisoning cases.

E. coli was detected in 83 samples (45.4% [95% CI 38.3–52.6%]), with concentrations varying from 1 to 885 CFU/mL in the positive samples, and the average concentration being 5 CFU/mL. No relationship was detected between the number of E. coli and the total bacterial count, or the number of E. coli and the presence of pathogenic bacteria. E. coli is part of the normal microbial population of the lower intestine in humans and animals and is therefore commonly used as a hygiene indicator for fecal contamination. A high number of E. coli in milk may thus indicate contamination of the milk with other enteric bacteria, such as Yersinia spp., Salmonella spp., or Campylobacter spp. The high number of E. coli and its presence in samples with a very low total bacterial count suggest that fecal contamination of milk is very common and cannot be totally avoided, even on farms with excellent production hygiene.

The total bacterial count varied from 1.2×10² to 3.0×10⁶ CFU/mL, and the average total bacterial count for all the milk samples was 1.3×10⁴ CFU/mL. The average total bacterial count was slightly higher than the national average, 5.6×10³ CFU/mL, based on the bimonthly reports collected by the Finnish Association for Milk Hygiene (2012). This might be partly explained by storage temperature and storage time of the raw milk samples before analysis, or by seasonal variation in the total bacterial count, with higher counts reported in the summertime (Elmoslemany et al., 2010). The total bacterial counts showed variation between farms, which may reflect the prevailing weather and environmental conditions at the sampling time or different practices in farm hygiene and milking technique. No relationship was detected between the total bacterial count and the occurrence of pathogenic bacteria, with pathogens being detected in milk samples with both very low and high total bacterial counts. This confirms that the total bacterial count should not be relied upon as an indicator for the presence of pathogens.

At least one of the potentially pathogenic bacteria, L. monocytogenes, STEC, coagulase-positive staphylococci, or B. cereus, was detected in 96 samples (52.5%). Either L. monocytogenes or STEC was detected in 15 samples (8.2%).

Conclusion

The results demonstrate that despite a high hygienic standard, the presence of pathogenic bacteria in raw milk cannot be avoided. Although the average concentration of pathogenic bacteria in fresh raw milk is mainly relatively low, it should be borne in mind that some pathogenic species can survive and multiply at refrigeration temperatures. Thus, it is essential for the producer and the consumer to pay attention to the raw milk storage temperature and storage time. The high occurrence of Y. enterocolitica, L. monocytogenes, and E. coli in milk suggests fecal and/or environmental contamination. Strict hygiene measures, identification of persons at risk, and heating the raw milk before consumption are important in controlling the health risks related to raw milk consumption. In addition, routine monitoring plan of pathogenic bacteria for the farms selling raw milk directly to consumers is advisable.

Footnotes

Acknowledgments

We thank Hanna Laitinen, Timo Latomäki, and Anu Surakka, Valio Ltd.; Hanna Salonen, Arla Ingman Ltd.; and the Finnish milk truck drivers and dairy farmers for their help with sampling and sharing the raw milk samples. Anneli Luoti is thanked for technical assistance. This work was funded by the Finnish Ministry of Agriculture and Forestry (1294/311/2011).

Disclosure Statement

No competing financial interests exist.

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