Microbiology of raw milk in New Zealand

International Journal of Food Microbiology 157 (2012) 305–308

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Microbiology of raw milk in New Zealand Bruce Hill ⁎, Betty Smythe, Denise Lindsay, Joanna Shepherd Fonterra Research Centre, Fitzherbert Science Centres, Dairy Farm Road, Palmerston North, New Zealand

a r t i c l e

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Article history: Received 4 December 2011 Received in revised form 26 March 2012 Accepted 27 March 2012 Available online 9 April 2012 Keywords: New Zealand Raw milk Microbiology

a b s t r a c t The results of this study demonstrate the occurrence of the non-spore-forming pathogens, Staphylococcus aureus, Escherichia coli (total count and O157:H7), Listeria, Campylobacter and Salmonella, in New Zealand's raw milk supply. Samples of raw milk were collected monthly within five major dairying regions over one year. Each month, samples from five randomly selected farm vats in each region were collected for analysis (297 samples in total). Methods based on plate count techniques were used to enumerate S. aureus and E. coli. Enrichment methods in combination with a modified most probable number detection method were used to monitor samples for the presence of E. coli O157:H7, Listeria, Campylobacter and Salmonella. Salmonella was not detected in this study, and Campylobacter was isolated once (0.34%). E. coli was present at b 100 cfu/ml in 99% of samples and exceeded 103 cfu/ml in 0.7% of samples. E. coli O157:H7 was not detected whereas non-pathogenic E. coli O157 strains (i.e. lacking genes for stx1, stx2, eae and Hly A) were detected in 1% of samples. S. aureus was not detected (b1 cfu/ml) in 21% of samples; levels were >1 but b 100 cfu/ml in 60% of samples and on one occasion (0.34%) S. aureus exceeded 104 cfu/ml. L. monocytogenes was isolated from 0.68% of samples and L. innocua was present in 4% of samples. The results demonstrate that raw milk sampled from farm vats in New Zealand, as in other countries, inevitably contains recognised pathogens and, hence, control by pasteurisation or an equivalent treatment of raw milk remains paramount. Even so, the prevalence of most of these pathogens was lower than those reported in many of the studies performed in other countries. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Bovine milk is highly nutritious — it contains lipids, proteins (casein, whey), carbohydrates (lactose), amino acids, vitamins and minerals (calcium), essential for the nutritional requirements of the growing calf (Haug et al., 2007). However, because of its nutritional properties, milk is also a good growth matrix for a variety of spoilage and potentially pathogenic microorganisms. A considerable amount of information about pathogens in raw milk has already been published internationally (Coia et al., 2001; Desmasures et al., 1997; Heuvelink et al., 1998; Jayarao et al., 2006; Normanno et al., 2005; Rea et al., 1992; Steele et al., 1997; Waak et al., 2002). Unfortunately, none of the more comprehensive publications has addressed the occurrence of food borne pathogens in New Zealand's raw milk supplies. A preliminary study by Stone (1987) investigated the prevalences of Campylobacter jejuni, Listeria and Yersinia enterocolitica in a small number of New Zealand milk samples. Although studies performed in other countries might be relevant to New Zealand, in reality these studies have generally been performed in countries in which the milking practices are very different from those in New Zealand. For example, dairy herds in New Zealand are much larger (average herd size — 376 in 2009/10) than those generally seen in the EU (herd

⁎ Corresponding author. Tel.: + 64 6 350 4649; fax: + 64 6 356 1476. E-mail address: [email protected] (B. Hill). 0168-1605/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2012.03.031

sizes range from approximately 40 to 100 cows), and larger volumes of milk (total of 13 billion litres in 2010) are processed. In addition, New Zealand dairy herds are generally not housed and are predominantly grass fed for a large part of the year. All these factors have been suggested to affect the prevalence of foodborne pathogens, in particular Listeria monocytogenes, in raw milk (Husu et al., 1990; O'Donnell, 1995; Rea et al., 1992; Sanaa et al., 1993; Waak et al., 2002). As a result, previously published global studies may not provide a reliable basis for predicting the pathogen status of raw milk within New Zealand. Thus, the aim of this study was to produce a snapshot of the occurrence of selected pathogens in the New Zealand raw milk supply. The information will be used by the New Zealand Food Safety Authority in the development of dairy risk assessments relating to the risk posed by the consumption of raw milk products. 2. Materials and methods 2.1. Sampling Raw milk samples were collected each month from farm vats in five of the main milk collection regions of New Zealand (Northland, Waikato, Taranaki/Manawatu, Canterbury and Southland). The aim was to test 300 samples overall and approximately equal numbers from each region. Sampling began in April 2007 (end of the 2006/07 dairying season) and ceased in May 2008 (end of the 2007/08 dairying season). Each month, individual samples (250–400 ml) were collected aseptically

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from five randomly selected farm vats within each region. An effort was made to avoid testing any supply more than once during the study. The outcome was that 297 samples were actually collected during the study: Northland — 54 samples from 53 suppliers; Waikato — 54 samples from 48 suppliers; Taranaki/Manawatu — 60 samples from 60 suppliers; Canterbury — 64 samples from 64 suppliers; Southland — 65 samples from 65 suppliers. Only seven farm vats were sampled more than once during the entire study. 2.2. Microbiological analyses The samples were analysed for Staphylococcus aureus and Escherichia coli using agar enumeration methods, and for the presence of E. coli O157:H7, Listeria, Campylobacter and Salmonella according to standard enrichment methods described in Table 1. In order to estimate concentrations of E. coli O157:H7, Listeria, Campylobacter and Salmonella, a modified most probable number (MPN) method was developed (Table 2) and used to analyse samples enriched using standard procedures. In this approach, 25, 10 and 1 ml portions of each milk sample were enriched using the appropriate enrichment methods (Table 1). The application of the modified MPN approach allowed a large number of samples to be screened to detect low numbers of pathogens. To minimise cost, all three subsamples (25, 10, 1 ml) were enriched; however, initially, only the 25 ml sample was screened with the appropriate rapid method (Table 1). If the 25 ml sample returned a positive result, the 10 and 1 ml enriched samples were also screened immediately. However, if the 25 ml sample returned a negative result, the 10 and 1 ml enrichment samples were discarded. For the pathogens enumerated using this modified MPN technique, a MPN result could be calculated from the 25, 10 and 1 ml results using Table 2. 3. Results and discussion Fig. 1 presents the enumeration results for S. aureus (n = 293) and E. coli (n = 297) as percentages of samples that contained colony concentrations ranging from b1 to >10 3 cfu/ml in 50 cfu/ml increments.

In the case of S. aureus, the results show that 60% of the raw milk samples contained b10 2 cfu/ml and 30% contained between 10 2 and 10 3 cfu/ml. On only one occasion did a sample result exceed 10 4 cfu/ml. It is difficult to compare the results from this study with those conducted in other countries, because most studies express the results qualitatively, e.g. the prevalence of S. aureus in raw milk from studies conducted in other countries has ranged widely from 13 to 100% (Adesiyun et al., 1998; Chye et al., 2004; Jørgensen et al., 2005). S. aureus is one of the causative agents of mastitis in dairy herds (Barkema et al., 2006). This disease involves inflammation of the mammary glands and a resultant sporadic shedding of S. aureus cells into the raw milk (Barkema et al., 2006). Therefore, the presence of large concentrations of S. aureus is indicative of mastitis in a dairy herd. From a food safety perspective, it is recognised that S. aureus is an enterotoxin-producing pathogen but that the concentration needs to exceed 10 5 cfu/ml for sufficient toxin to be produced to cause human illness (Hill, 1981; Jay, 2000). None of the raw milk samples in this study contained numbers of S. aureus that were close to this. In the case of E. coli, 99% of samples tested had counts b10 2 cfu/ml and only 0.7% were >10 3 cfu/ml (Fig. 1). Pathogenic E. coli O157:H7 was not detected in any samples tested in this study (n = 296). In other studies published internationally, this pathogen has also been found to be at low prevalences in raw milk (0–0.2% of samples in most trials) with the exception being an American study (Jayarao et al., 2006) in which 2.4% of samples were found to contain E. coli O157:H7. Crump et al. (2001) have credited New Zealand's system of pastoral agriculture and grass-fed herds for the delay of the appearance of this pathogen in local herds. Previous research has shown that healthy cattle are known reservoirs of such E. coli strains in New Zealand (Cookson et al., 2006). Non-pathogenic E. coli O157 (i.e. non-H7, lacking Shiga toxins 1 and 2, eae and Hyl A genes) was detected in 1% of samples, but did not exceed 1 cfu/4 ml (Table 3). Salmonella (n = 294) was not detected in any sample tested during this study, and Campylobacter (n = 296) was detected in only a single sample (0.34%). This isolate was not identified further. The

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

Reference method

Type of assay

Procedure

S. aureus count

ISO 6888-1 (1999)

Plate count identification

E. coli count

ISO 16649-2 (2001)

Plate count

E. coli O157 detection

AOAC 996.09 (1999) MIRINZ (2000c)

Detection and MPN estimate Identification

Baird–Parker agar plus egg yolk tellurite (0.5% w/v) or rabbit plasma fibrinogen agar. Presumptive colonies confirmed using a coagulase test. ß-Glucuronidase-positive E. coli colonies counted on tryptone bile X-glucuronide agar at 37 and 44 °C. TECRA O157 visual immunoassay (VIA™).

Paton and Paton (1998)

L. monocytogenes detection

Salmonella spp. detection

Fields et al. (1997) Wang et al. (2002) Speirs et al. (1977) Konowalchuk et al. (1977) Karmali (1989) AOAC 2002.09 (2003) Detection and MPN estimate FDA (2000) Identification AOAC 998.09 (2001)

Campylobacter spp. detection MIRINZ (2000a) MIRINZ (2000b) Baylis et al. (2000)

Detection and MPN estimate Identification Detection and MPN estimate

Isolation using immuno magnetic separation as per manufacturer's instructions (Dynabeads, Invitrogen USA) and plating on to MacConkey agar with sorbitol, cefixime and tellurite (CT-SMAC). Sorbitol-negative colonies with typical reactions on triple sugar iron agar and lysine iron agar were screened using an E. coli O157 latex test kit (Oxoid, UK). For latex-positive colonies, multiplex PCR was carried out to confirm the presence of the toxin gene(s) stx1 and/or stx2, and eae and Hly A genes. PCR was carried out to test for the presence of H7 as described previously. Toxin production was confirmed using a Vero Cell Assay as previously described.

TECRA Listeria Visual Immunoassay (VIA™). Presumptive isolates were identified using phenotypical and biochemical characteristics as described in the FDA bacteriological analytical manual, chapter 10. TECRA Salmonella Visual Immunoassay (VIA™). Presumptive isolates were identified using slide agglutination with Salmonella polyvalent O (A-S) and polyvalent H (phases 1 and 2) antisera (Remel Europe Limited). Based on primary enrichment in Bolton broth followed by isolation on modified Campylobacter blood-free selective agar.

B. Hill et al. / International Journal of Food Microbiology 157 (2012) 305–308 Table 2 Conversion of possible MPN results into MPN/ml or equivalent 1 cfu/x ml. Possible MPN results 1 × 25 ml

1 × 10 ml

1 × 1 ml

−

−

−

−

−

+

−

+

−

+

−

−

−

+

+

+

−

+

+

+

−

+

+

+

MPN/ml or equivalent 1 cfu/x ml b0.028 (≡b 1 cfu/35.7 ml) 0.028a (≡1 cfu/35.7 ml) 0.033a (≡1 cfu/30.3 ml) 0.047 (≡1 cfu/21.3 ml) 0.067a (≡1 cfu/14.9 ml) 0.11 (≡1 cfu/9.1 ml) 0.24 (≡1 cfu/4.2 ml) >0.24 (≡>1 cfu/4.2 ml)

Confidence intervals Lower 95%

Upper 95%

–

–

0.003

0.27

0.0038

0.28

0.0069

0.33

0.011

0.41

0.19

0.66

0.03

2.00

–

–

a Unlikely MPN outcomes because the 10 ml and 1 ml enrichments were only tested when a positive rapid screening result was obtained for the 25 ml enrichments.

prevalence of Salmonella in raw milk has ranged from ‘not detected’ to 8.9% in several studies published internationally (D'Amico et al., 2008; Jayarao and Henning, 2001; Jayarao et al., 2006; Murinda et al., 2002; Rohrbach et al., 1992; Steele et al., 1997). It is reasonable to suppose that the presence and the concentration of Salmonella in bulk tank milk on farms are dependent on various factors including geographical region, herd size and subclinical shedding, farm management practices and its presence in the environment (Ruzante et al., 2010). For example, subclinical shedding of Salmonella is reportedly common in Ohio, USA, where the prevalence of infected herds ranged from 1 to 97% (Huston et al., 2002). In the case of New Zealand, outbreaks of Salmonella infections within dairy herds seem to occur sporadically, and the organism is often not detected in herd animals or their faeces (Grinberg et al., 2005; Moriarty et al., 2008; Vermunt and Parkinson, 2000). In addition, epidemiological evidence has linked raw milk with very few outbreaks of nontyphoid salmonellosis in New Zealand (King et al., 2011). When Campylobacter is detected in raw milk, it is thought to originate from various environmental sources including indoor cow housing (Ellis-Iversen et al., 2009). In New Zealand, dairy cattle are predominantly grass fed in open fields year round. Therefore, the low prevalence of Campylobacter in raw milk in this study is not 80 70

% of samples

60 50 40 30 20

307

Table 3 Estimates of numbers of bacterial pathogens contained within positive samples according to a modified MPN method and Table 2. Bacteria

Campylobacter Non-pathogenic E. coli O157:H7 Listeria spp.b L. innocua L. monocytogenes

Number of positive (%) samples 1 (0.34%) 1 1 (1.01%) 1 2 (0.68%) 8 (4.07%) 4 2 (0.68%)

Volume (ml) in which 1 cfu was contained (according to MPN and Table 2) 21 30a 21 4 21 21 4 4

a

In relation to Table 2, this is an unusual MPN outcome. It is attributable to an initial positive rapid screening result (performed on a 25 ml enrichment) subsequently being confirmed as a negative result, while the 10 ml sample was tested and resulted in a positive. b Not identified to the species level.

unexpected and is in agreement with another publication which indicated that Campylobacter is usually detected in only a small percentage of raw milk samples in New Zealand (Hudson et al., 1999). Of the 16 raw milk samples found to contain Listeria in this study, L. innocua was detected in 4% and L. monocytogenes was detected in 0.68% of samples (n = 295). In the case of L. monocytogenes, the quantification achieved using the MPN technique indicated that the concentration in the two positive samples was 1 cfu/4 ml (Table 3). Various published studies indicate that some of the contributors to Listeria contamination of milk include the housing of cattle indoors all well as poorly made silage and poor on-farm hygiene (Husu et al., 1990; Sanaa et al., 1993). The previously mentioned absence of the practice of indoor housing of dairy cattle in New Zealand may also explain in part the lower rates of detection of L. monocytogenes in this study compared with those found in many of the studies performed in other countries (Fox et al., 2011; Hayes et al., 1986; Jayarao et al., 2006; Rohrbach et al., 1992; van Kessel et al., 2004). 4. Conclusions As with other studies conducted internationally, our results indicate that raw milk sampled from farm vats in New Zealand contained recognised pathogens. Even so, the prevalence and concentration of the pathogens included in the study were relatively low. Detection rates for Salmonella, E. coli O157:H7, Campylobacter and Listeria were generally lower than those found in many of the studies from other countries. This finding is perhaps not surprising, as the presence of these pathogens in raw milk is believed to be influenced by environmental factors, such as indoor housing of cattle and poor quality feed, such as silage, which are less likely to occur on New Zealand dairy farms where the animals are predominantly pasture fed. Notwithstanding, the inescapable presence of such pathogens in raw milk, albeit at low levels, highlights the continued need for correct pasteurisation or other equivalent destructive technique to be practised routinely to ensure the production of safe dairy products for consumption. Without the maintenance of pasteurisation or other effective controls, these pathogens have the potential to cause illnesses in consumers of raw milk or products made from raw milk.

10

Acknowledgements <1 1 -4 5 9 10 0 -9 0 9 15 - 1 0 49 20 - 19 0 9 25 -2 0 49 30 - 2 0 9 35 - 3 9 0 4 40 - 3 9 0 99 45 - 4 0 4 50 - 4 9 0 99 55 - 5 0 4 60 -5 9 0 99 65 - 6 0 4 70 - 6 9 0 99 75 - 7 0 49 80 - 79 0 9 85 -84 0 9 90 -8 0 9 95 - 9 9 0 49 -1 0 >1 00 00 0

0

cfu/ml Fig. 1. Counts of S. aureus (n = 293; black bars) and E. coli (n = 297; white bars) in New Zealand raw milk, as percentages of samples that contained colony numbers ranging from b 1 to >1000 cfu/ml, in 50 cfu/ml increments.

We would like to thank Dr. Andrew Hudson (Christchurch Science Centre) for his significant contributions to the planning of this study and for his editorial input, Dr. Rob Crawford (Fonterra) for the development of the modified MPN method used in this study and also the contributions made to the design and execution of the study by Dr. Steve Hathaway, Dr. Roger Cook and Dr. Lisa Oakley (NZFSA),

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Dr. Fiona Thompson-Carter (Environmental Science and Research), Dr. Nigel French (Massey University), Warwick Aspin (AsureQuality) and Sally Miller (Fonterra Research Centre). We also recognise the important contribution of the Fonterra milk collection staff who ensured that the required samples were collected, chilled and couriered to the analytical laboratory throughout the study. References Adesiyun, A.A., Webb, L.A., Romain, H.T., 1998. Prevalence and characteristics of Staphylococcus aureus strains isolated from bulk and composite milk and cattle handlers. Journal of Food Protection 61, 629–632. AOAC, 1999. Official Method 996.09: Escherichia coli O157:H7 in Selected Foods (EHEC Visual Immunoprecipitate Assay (VIP)). AOAC International, Gaithersburg, Maryland. AOAC, 2001. Official Method 998.09: Salmonella in Foods (TECRA Salmonella Visual Immunoassay). AOAC International, Gaithersburg, Maryland. AOAC, 2003. 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