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

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International Journal of Food Microbiology

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Short communication

Microbiology of raw milk in New Zealand

Bruce Hill ⁎, Betty Smythe, Denise Lindsay, Joanna ShepherdFonterra Research Centre, Fitzherbert Science Centres, Dairy Farm Road, Palmerston North, New Zealand

⁎ Corresponding author. Tel.: +64 6 350 4649; fax: +E-mail address: bruce.hill@fonterra.com (B. Hill).

0168-1605/$ – see front matter © 2012 Elsevier B.V. Alldoi:10.1016/j.ijfoodmicro.2012.03.031

a b s t r a c t

a r t i c l e i n f o

Article history:Received 4 December 2011Received in revised form 26 March 2012Accepted 27 March 2012Available online 9 April 2012

Keywords:New ZealandRaw milkMicrobiology

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 milksupply. Samples of raw milk were collected monthly within five major dairying regions over one year. Eachmonth, samples from five randomly selected farm vats in each region were collected for analysis (297 samplesin total). Methods based on plate count techniques were used to enumerate S. aureus and E. coli. Enrichmentmethods in combination with a modified most probable number detection method were used to monitor sam-ples for the presence of E. coli O157:H7, Listeria, Campylobacter and Salmonella. Salmonella was not detected inthis study, and Campylobacter was isolated once (0.34%). E. coli was present at b100 cfu/ml in 99% of samplesand exceeded 103 cfu/ml in 0.7% of samples. E. coli O157:H7 was not detected whereas non-pathogenic E. coliO157 strains (i.e. lacking genes for stx1, stx2, eae and Hly A) were detected in 1% of samples. S. aureus was notdetected (b1 cfu/ml) in 21% of samples; levels were >1 but b100 cfu/ml in 60% of samples and on one occasion(0.34%) S. aureus exceeded 104 cfu/ml. L. monocytogeneswas isolated from 0.68% of samples and L. innocuawaspresent in 4% of samples. The results demonstrate that raw milk sampled from farm vats in New Zealand, as inother countries, inevitably contains recognised pathogens and, hence, control by pasteurisation or an equivalenttreatment of raw milk remains paramount. Even so, the prevalence of most of these pathogens was lower thanthose 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 andminerals (calcium), essential for the nutritional requirements of thegrowing calf (Haug et al., 2007). However, because of its nutritionalproperties, milk is also a good growth matrix for a variety of spoilageand potentially pathogenic microorganisms.

A considerable amount of information about pathogens in rawmilkhas 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 publicationshas addressed the occurrence of food borne pathogens in NewZealand'srawmilk supplies. A preliminary study by Stone (1987) investigated theprevalences of Campylobacter jejuni, Listeria and Yersinia enterocoliticain a small number of New Zealand milk samples. Although studiesperformed in other countriesmight be relevant to New Zealand, in real-ity these studies have generally been performed in countries in whichthe milking practices are very different from those in New Zealand.For example, dairy herds in New Zealand are much larger (averageherd size — 376 in 2009/10) than those generally seen in the EU (herd

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sizes range from approximately 40 to 100 cows), and larger volumesof milk (total of 13 billion litres in 2010) are processed. In addition,New Zealand dairy herds are generally not housed and are predomi-nantly grass fed for a large part of the year. All these factors have beensuggested to affect the prevalence of foodborne pathogens, in particularListeria monocytogenes, in rawmilk (Husu et al., 1990; O'Donnell, 1995;Rea et al., 1992; Sanaa et al., 1993; Waak et al., 2002). As a result, previ-ously published global studies may not provide a reliable basis for pre-dicting the pathogen status of raw milk within New Zealand. Thus, theaim of this study was to produce a snapshot of the occurrence of select-ed pathogens in the New Zealand rawmilk supply. The informationwillbe used by the New Zealand Food Safety Authority in the developmentof dairy risk assessments relating to the risk posed by the consumptionof raw milk products.

2. Materials and methods

2.1. Sampling

Rawmilk samples were collected each month from farm vats in fiveof themainmilk collection regions of NewZealand (Northland,Waikato,Taranaki/Manawatu, Canterbury and Southland). The aim was to test300 samples overall and approximately equal numbers from each re-gion. Sampling began in April 2007 (endof the 2006/07 dairying season)and ceased in May 2008 (end of the 2007/08 dairying season). Eachmonth, individual samples (250–400 ml) were collected aseptically

306 B. Hill et al. / International Journal of Food Microbiology 157 (2012) 305–308

from five randomly selected farm vats within each region. An effort wasmade to avoid testing any supply more than once during the study. Theoutcomewas that 297 sampleswere actually collected during the study:Northland— 54 samples from 53 suppliers;Waikato— 54 samples from48 suppliers; Taranaki/Manawatu — 60 samples from 60 suppliers;Canterbury — 64 samples from 64 suppliers; Southland — 65 samplesfrom 65 suppliers. Only seven farm vats were sampled more than onceduring the entire study.

2.2. Microbiological analyses

The samples were analysed for Staphylococcus aureus andEscherichia coli using agar enumerationmethods, and for the presenceof E. coli O157:H7, Listeria, Campylobacter and Salmonella according tostandard 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 samplesenriched using standard procedures. In this approach, 25, 10 and 1 mlportions of each milk sample were enriched using the appropriateenrichment methods (Table 1). The application of the modifiedMPN approach allowed a large number of samples to be screened todetect low numbers of pathogens. To minimise cost, all three subsam-ples (25, 10, 1 ml) were enriched; however, initially, only the 25 mlsample was screened with the appropriate rapid method (Table 1).If the 25 ml sample returned a positive result, the 10 and 1 mlenriched samples were also screened immediately. However, if the25 ml sample returned a negative result, the 10 and 1 ml enrichmentsamples were discarded. For the pathogens enumerated using thismodified MPN technique, a MPN result could be calculated from the25, 10 and 1 ml results using Table 2.

3. Results and discussion

Fig. 1 presents the enumeration results for S. aureus (n=293) andE. coli (n=297) as percentages of samples that contained colony con-centrations ranging from b1 to >103 cfu/ml in 50 cfu/ml increments.

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

Bacteria Reference method Type of assay Pr

S. aureus count ISO 6888-1 (1999) Plate count identification BaPr

E. coli count ISO 16649-2 (2001) Plate count ß-at

E. coli O157 detection AOAC 996.09 (1999)MIRINZ (2000c)

Detection and MPNestimate

TE

Identification IsoInv(CSoiro

Paton and Paton (1998) Fotox

Fields et al. (1997)Wang et al. (2002)

PC

Speirs et al. (1977)Konowalchuk et al. (1977)Karmali (1989)

To

L. monocytogenes detection AOAC 2002.09 (2003) Detection and MPNestimate

TE

FDA (2000) Identification Pras

Salmonella spp. detection AOAC 998.09 (2001) Detection and MPNestimate

TE

Identification Prpo

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

Detection and MPNestimate

BaCa

In the case of S. aureus, the results show that 60% of the raw milksamples contained b102 cfu/ml and 30% contained between 102 and103 cfu/ml. On only one occasion did a sample result exceed104 cfu/ml. It is difficult to compare the results from this study withthose conducted in other countries, because most studies expressthe results qualitatively, e.g. the prevalence of S. aureus in raw milkfrom studies conducted in other countries has ranged widely from13 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 themammary glands and a resultant sporadic shedding of S. aureuscells into the raw milk (Barkema et al., 2006). Therefore, the presenceof large concentrations of S. aureus is indicative of mastitis in a dairyherd. From a food safety perspective, it is recognised that S. aureusis an enterotoxin-producing pathogen but that the concentrationneeds to exceed 105 cfu/ml for sufficient toxin to be produced tocause human illness (Hill, 1981; Jay, 2000). None of the raw milksamples in this study contained numbers of S. aureus that wereclose to this.

In the case of E. coli, 99% of samples tested had counts b102 cfu/mland only 0.7% were >103 cfu/ml (Fig. 1). Pathogenic E. coli O157:H7was not detected in any samples tested in this study (n=296). Inother studies published internationally, this pathogen has also beenfound to be at low prevalences in raw milk (0–0.2% of samples inmost trials) with the exception being an American study (Jayarao etal., 2006) in which 2.4% of samples were found to contain E. coliO157:H7. Crump et al. (2001) have credited New Zealand's systemof pastoral agriculture and grass-fed herds for the delay of the appear-ance of this pathogen in local herds. Previous research has shown thathealthy cattle are known reservoirs of such E. coli strains in NewZealand (Cookson et al., 2006). Non-pathogenic E. coli O157 (i.e.non-H7, lacking Shiga toxins 1 and 2, eae and Hyl A genes) wasdetected in 1% of samples, but did not exceed 1 cfu/4 ml (Table 3).

Salmonella (n=294) was not detected in any sample tested dur-ing this study, and Campylobacter (n=296) was detected in only asingle sample (0.34%). This isolate was not identified further. The

ocedure

ird–Parker agar plus egg yolk tellurite (0.5% w/v) or rabbit plasma fibrinogen agar.esumptive colonies confirmed using a coagulase test.Glucuronidase-positive E. coli colonies counted on tryptone bile X-glucuronide agar37 and 44 °C.CRA O157 visual immunoassay (VIA™).

lation using immuno magnetic separation as per manufacturer's instructions (Dynabeads,itrogen USA) and plating on to MacConkey agar with sorbitol, cefixime and telluriteT-SMAC).rbitol-negative colonies with typical reactions on triple sugar iron agar and lysinen agar were screened using an E. coli O157 latex test kit (Oxoid, UK).r latex-positive colonies, multiplex PCR was carried out to confirm the presence of thein gene(s) stx1 and/or stx2, and eae and Hly A genes.R was carried out to test for the presence of H7 as described previously.

xin production was confirmed using a Vero Cell Assay as previously described.

CRA Listeria Visual Immunoassay (VIA™).

esumptive isolates were identified using phenotypical and biochemical characteristicsdescribed in the FDA bacteriological analytical manual, chapter 10.CRA Salmonella Visual Immunoassay (VIA™).

esumptive isolates were identified using slide agglutination with Salmonellalyvalent O (A-S) and polyvalent H (phases 1 and 2) antisera (Remel Europe Limited).sed on primary enrichment in Bolton broth followed by isolation on modifiedmpylobacter blood-free selective agar.

Table 2Conversion of possible MPN results into MPN/ml or equivalent 1 cfu/x ml.

Possible MPN results MPN/ml or equivalent1 cfu/x ml

Confidence intervals

1×25 ml 1×10 ml 1×1 ml Lower 95% Upper 95%

− − − b0.028(≡b1 cfu/35.7 ml)

– –

− − + 0.028a

(≡1 cfu/35.7 ml)0.003 0.27

− + − 0.033a

(≡1 cfu/30.3 ml)0.0038 0.28

+ − − 0.047(≡1 cfu/21.3 ml)

0.0069 0.33

− + + 0.067a

(≡1 cfu/14.9 ml)0.011 0.41

+ − + 0.11(≡1 cfu/9.1 ml)

0.19 0.66

+ + − 0.24(≡1 cfu/4.2 ml)

0.03 2.00

+ + + >0.24(≡>1 cfu/4.2 ml)

– –

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

Table 3Estimates of numbers of bacterial pathogens contained within positive samples accord-ing to a modified MPN method and Table 2.

Bacteria Number of positive(%) samples

Volume (ml) in which1 cfu was contained(according to MPN and Table 2)

Campylobacter 1 (0.34%) 21Non-pathogenicE. coli O157:H7

1 30a

1 (1.01%) 211 4

Listeria spp.b 2 (0.68%) 21L. innocua 8 (4.07%) 21

4 4L. monocytogenes 2 (0.68%) 4

a In relation to Table 2, this is an unusual MPN outcome. It is attributable to an initialpositive rapid screening result (performed on a 25 ml enrichment) subsequently beingconfirmed as a negative result, while the 10 ml sample was tested and resulted in apositive.

b Not identified to the species level.

307B. Hill et al. / International Journal of Food Microbiology 157 (2012) 305–308

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 etal., 2002; Rohrbach et al., 1992; Steele et al., 1997).

It is reasonable to suppose that the presence and the concentra-tion of Salmonella in bulk tank milk on farms are dependent on vari-ous factors including geographical region, herd size and subclinicalshedding, farm management practices and its presence in the envi-ronment (Ruzante et al., 2010). For example, subclinical shedding ofSalmonella is reportedly common in Ohio, USA, where the prevalenceof infected herds ranged from 1 to 97% (Huston et al., 2002). In thecase of New Zealand, outbreaks of Salmonella infections within dairyherds seem to occur sporadically, and the organism is often notdetected in herd animals or their faeces (Grinberg et al., 2005;Moriarty et al., 2008; Vermunt and Parkinson, 2000). In addition, ep-idemiological evidence has linked raw milk with very few outbreaksof nontyphoid salmonellosis in New Zealand (King et al., 2011).

When Campylobacter is detected in raw milk, it is thought tooriginate from various environmental sources including indoor cowhousing (Ellis-Iversen et al., 2009). In New Zealand, dairy cattle arepredominantly grass fed in open fields year round. Therefore, thelow prevalence of Campylobacter in raw milk in this study is not

0

10

20

30

40

50

60

70

80

<11 -

49

50 -9

9

100 -

149

150-

199

200 -

249

250 -

299

300 -

349

350 -

399

400 -

449

450-

499

500 -

549

550 -

599

600 -

649

650 -

699

700 -

749

750-

799

800 -

849

850 -

899

900 -

949

950 -

1000

>1000

cfu/ml

% o

f sa

mpl

es

Fig. 1. Counts of S. aureus (n=293; black bars) and E. coli (n=297; white bars) in NewZealand raw milk, as percentages of samples that contained colony numbers rangingfrom b1 to >1000 cfu/ml, in 50 cfu/ml increments.

unexpected and is in agreement with another publication whichindicated that Campylobacter is usually detected in only a smallpercentage of rawmilk 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 in0.68% of samples (n=295). In the case of L. monocytogenes, thequantification achieved using the MPN technique indicated that theconcentration in the two positive samples was 1 cfu/4 ml (Table 3).Various published studies indicate that some of the contributors toListeria contamination of milk include the housing of cattle indoorsall well as poorly made silage and poor on-farm hygiene (Husu etal., 1990; Sanaa et al., 1993). The previously mentioned absence ofthe practice of indoor housing of dairy cattle in New Zealand mayalso explain in part the lower rates of detection of L. monocytogenesin this study compared with those found in many of the studiesperformed 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 indi-cate that raw milk sampled from farm vats in New Zealand containedrecognised pathogens. Even so, the prevalence and concentration ofthe pathogens included in the study were relatively low. Detectionrates for Salmonella, E. coli O157:H7, Campylobacter and Listeriawere generally lower than those found in many of the studies fromother countries. This finding is perhaps not surprising, as the presenceof these pathogens in raw milk is believed to be influenced by envi-ronmental factors, such as indoor housing of cattle and poor qualityfeed, such as silage, which are less likely to occur on New Zealanddairy farms where the animals are predominantly pasture fed.Notwithstanding, the inescapable presence of such pathogens inraw milk, albeit at low levels, highlights the continued need for cor-rect pasteurisation or other equivalent destructive technique to bepractised routinely to ensure the production of safe dairy productsfor consumption. Without the maintenance of pasteurisation orother effective controls, these pathogens have the potential to causeillnesses in consumers of raw milk or products made from raw milk.

Acknowledgements

Wewould like to thank Dr. Andrew Hudson (Christchurch ScienceCentre) for his significant contributions to the planning of this studyand for his editorial input, Dr. Rob Crawford (Fonterra) for the devel-opment of the modified MPN method used in this study and also thecontributions made to the design and execution of the study byDr. Steve Hathaway, Dr. Roger Cook and Dr. Lisa Oakley (NZFSA),

308 B. Hill et al. / International Journal of Food Microbiology 157 (2012) 305–308

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 theimportant contribution of the Fonterra milk collection staff who en-sured that the required samples were collected, chilled and courieredto the analytical laboratory throughout the study.

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