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

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International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Short communication

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 i n f o a b s t r a c t

Article history: The results of this study demonstrate the occurrence of the non-spore-forming pathogens, Staphylococcus aureus,
Received 4 December 2011 Escherichia coli (total count and O157:H7), Listeria, Campylobacter and Salmonella, in New Zealand's raw milk
Received in revised form 26 March 2012 supply. Samples of raw milk were collected monthly within five major dairying regions over one year. Each
Accepted 27 March 2012
month, samples from five randomly selected farm vats in each region were collected for analysis (297 samples
Available online 9 April 2012
in total). Methods based on plate count techniques were used to enumerate S. aureus and E. coli. Enrichment
Keywords:
methods in combination with a modified most probable number detection method were used to monitor sam-
New Zealand ples for the presence of E. coli O157:H7, Listeria, Campylobacter and Salmonella. Salmonella was not detected in
Raw milk this study, and Campylobacter was isolated once (0.34%). E. coli was present at b 100 cfu/ml in 99% of samples
Microbiology 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 sizes range from approximately 40 to 100 cows), and larger volumes
of milk (total of 13 billion litres in 2010) are processed. In addition,
Bovine milk is highly nutritious — it contains lipids, proteins New Zealand dairy herds are generally not housed and are predomi-
(casein, whey), carbohydrates (lactose), amino acids, vitamins and nantly grass fed for a large part of the year. All these factors have been
minerals (calcium), essential for the nutritional requirements of the suggested to affect the prevalence of foodborne pathogens, in particular
growing calf (Haug et al., 2007). However, because of its nutritional Listeria monocytogenes, in raw milk (Husu et al., 1990; O'Donnell, 1995;
properties, milk is also a good growth matrix for a variety of spoilage Rea et al., 1992; Sanaa et al., 1993; Waak et al., 2002). As a result, previ-
and potentially pathogenic microorganisms. ously published global studies may not provide a reliable basis for pre-
A considerable amount of information about pathogens in raw milk dicting the pathogen status of raw milk within New Zealand. Thus, the
has already been published internationally (Coia et al., 2001; aim of this study was to produce a snapshot of the occurrence of select-
Desmasures et al., 1997; Heuvelink et al., 1998; Jayarao et al., 2006; ed pathogens in the New Zealand raw milk supply. The information will
Normanno et al., 2005; Rea et al., 1992; Steele et al., 1997; Waak et al., be used by the New Zealand Food Safety Authority in the development
2002). Unfortunately, none of the more comprehensive publications of dairy risk assessments relating to the risk posed by the consumption
has addressed the occurrence of food borne pathogens in New Zealand's of raw milk products.
raw milk supplies. A preliminary study by Stone (1987) investigated the
prevalences of Campylobacter jejuni, Listeria and Yersinia enterocolitica 2. Materials and methods
in a small number of New Zealand milk samples. Although studies
performed in other countries might be relevant to New Zealand, in real- 2.1. Sampling
ity these studies have generally been performed in countries in which
the milking practices are very different from those in New Zealand. Raw milk samples were collected each month from farm vats in five
For example, dairy herds in New Zealand are much larger (average of the main milk collection regions of New Zealand (Northland, Waikato,
herd size — 376 in 2009/10) than those generally seen in the EU (herd Taranaki/Manawatu, Canterbury and Southland). The aim was to test
300 samples overall and approximately equal numbers from each re-
gion. Sampling began in April 2007 (end of the 2006/07 dairying season)
⁎ Corresponding author. Tel.: + 64 6 350 4649; fax: + 64 6 356 1476. and ceased in May 2008 (end of the 2007/08 dairying season). Each
E-mail address: bruce.hill@fonterra.com (B. Hill). month, individual samples (250–400 ml) were collected aseptically

0168-1605/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2012.03.031
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 was In the case of S. aureus, the results show that 60% of the raw milk
made to avoid testing any supply more than once during the study. The samples contained b10 2 cfu/ml and 30% contained between 10 2 and
outcome was that 297 samples were actually collected during the study: 10 3 cfu/ml. On only one occasion did a sample result exceed
Northland — 54 samples from 53 suppliers; Waikato — 54 samples from 10 4 cfu/ml. It is difficult to compare the results from this study with
48 suppliers; Taranaki/Manawatu — 60 samples from 60 suppliers; those conducted in other countries, because most studies express
Canterbury — 64 samples from 64 suppliers; Southland — 65 samples the results qualitatively, e.g. the prevalence of S. aureus in raw milk
from 65 suppliers. Only seven farm vats were sampled more than once from studies conducted in other countries has ranged widely from
during the entire study. 13 to 100% (Adesiyun et al., 1998; Chye et al., 2004; Jørgensen et al.,
2005).
2.2. Microbiological analyses S. aureus is one of the causative agents of mastitis in dairy herds
(Barkema et al., 2006). This disease involves inflammation of the
The samples were analysed for Staphylococcus aureus and mammary glands and a resultant sporadic shedding of S. aureus
Escherichia coli using agar enumeration methods, and for the presence cells into the raw milk (Barkema et al., 2006). Therefore, the presence
of E. coli O157:H7, Listeria, Campylobacter and Salmonella according to of large concentrations of S. aureus is indicative of mastitis in a dairy
standard enrichment methods described in Table 1. herd. From a food safety perspective, it is recognised that S. aureus
In order to estimate concentrations of E. coli O157:H7, Listeria, is an enterotoxin-producing pathogen but that the concentration
Campylobacter and Salmonella, a modified most probable number needs to exceed 10 5 cfu/ml for sufficient toxin to be produced to
(MPN) method was developed (Table 2) and used to analyse samples cause human illness (Hill, 1981; Jay, 2000). None of the raw milk
enriched using standard procedures. In this approach, 25, 10 and 1 ml samples in this study contained numbers of S. aureus that were
portions of each milk sample were enriched using the appropriate close to this.
enrichment methods (Table 1). The application of the modified In the case of E. coli, 99% of samples tested had counts b10 2 cfu/ml
MPN approach allowed a large number of samples to be screened to and only 0.7% were >10 3 cfu/ml (Fig. 1). Pathogenic E. coli O157:H7
detect low numbers of pathogens. To minimise cost, all three subsam- was not detected in any samples tested in this study (n = 296). In
ples (25, 10, 1 ml) were enriched; however, initially, only the 25 ml other studies published internationally, this pathogen has also been
sample was screened with the appropriate rapid method (Table 1). found to be at low prevalences in raw milk (0–0.2% of samples in
If the 25 ml sample returned a positive result, the 10 and 1 ml most trials) with the exception being an American study (Jayarao et
enriched samples were also screened immediately. However, if the al., 2006) in which 2.4% of samples were found to contain E. coli
25 ml sample returned a negative result, the 10 and 1 ml enrichment O157:H7. Crump et al. (2001) have credited New Zealand's system
samples were discarded. For the pathogens enumerated using this of pastoral agriculture and grass-fed herds for the delay of the appear-
modified MPN technique, a MPN result could be calculated from the ance of this pathogen in local herds. Previous research has shown that
25, 10 and 1 ml results using Table 2. healthy cattle are known reservoirs of such E. coli strains in New
Zealand (Cookson et al., 2006). Non-pathogenic E. coli O157 (i.e.
3. Results and discussion 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).
Fig. 1 presents the enumeration results for S. aureus (n = 293) and Salmonella (n = 294) was not detected in any sample tested dur-
E. coli (n = 297) as percentages of samples that contained colony con- ing this study, and Campylobacter (n = 296) was detected in only a
centrations ranging from b1 to >10 3 cfu/ml in 50 cfu/ml increments. 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 Baird–Parker agar plus egg yolk tellurite (0.5% w/v) or rabbit plasma fibrinogen agar.
Presumptive colonies confirmed using a coagulase test.
E. coli count ISO 16649-2 (2001) Plate count ß-Glucuronidase-positive E. coli colonies counted on tryptone bile X-glucuronide agar
at 37 and 44 °C.
E. coli O157 detection AOAC 996.09 (1999) Detection and MPN TECRA O157 visual immunoassay (VIA™).
MIRINZ (2000c) estimate
Identification 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).
Paton and Paton (1998) 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.
Fields et al. (1997) PCR was carried out to test for the presence of H7 as described previously.
Wang et al. (2002)
Speirs et al. (1977) Toxin production was confirmed using a Vero Cell Assay as previously described.
Konowalchuk et al. (1977)
Karmali (1989)
L. monocytogenes detection AOAC 2002.09 (2003) Detection and MPN TECRA Listeria Visual Immunoassay (VIA™).
estimate
FDA (2000) Identification Presumptive isolates were identified using phenotypical and biochemical characteristics
as described in the FDA bacteriological analytical manual, chapter 10.
Salmonella spp. detection AOAC 998.09 (2001) Detection and MPN TECRA Salmonella Visual Immunoassay (VIA™).
estimate
Identification Presumptive isolates were identified using slide agglutination with Salmonella
polyvalent O (A-S) and polyvalent H (phases 1 and 2) antisera (Remel Europe Limited).
Campylobacter spp. detection MIRINZ (2000a) Detection and MPN Based on primary enrichment in Bolton broth followed by isolation on modified
MIRINZ (2000b) estimate Campylobacter blood-free selective agar.
Baylis et al. (2000)
 B. Hill et al. / International Journal of Food Microbiology 157 (2012) 305–308 307

Table 2 Table 3
Conversion of possible MPN results into MPN/ml or equivalent 1 cfu/x ml. Estimates of numbers of bacterial pathogens contained within positive samples accord-
ing to a modified MPN method and Table 2.
Possible MPN results MPN/ml or equivalent Confidence intervals
1 cfu/x ml Bacteria Number of positive Volume (ml) in which
1 × 25 ml 1 × 10 ml 1 × 1 ml Lower 95% Upper 95%
(%) samples 1 cfu was contained
− − − b0.028 – – (according to MPN and Table 2)
(≡b 1 cfu/35.7 ml)
Campylobacter 1 (0.34%) 21
− − + 0.028a 0.003 0.27
Non-pathogenic 1 30a
(≡1 cfu/35.7 ml)
E. coli O157:H7 1 (1.01%) 21
− + − 0.033a 0.0038 0.28
1 4
(≡1 cfu/30.3 ml)
Listeria spp.b 2 (0.68%) 21
+ − − 0.047 0.0069 0.33
L. innocua 8 (4.07%) 21
(≡1 cfu/21.3 ml)
4 4
− + + 0.067a 0.011 0.41
L. monocytogenes 2 (0.68%) 4
(≡1 cfu/14.9 ml)
a
+ − + 0.11 0.19 0.66 In relation to Table 2, this is an unusual MPN outcome. It is attributable to an initial
(≡1 cfu/9.1 ml) positive rapid screening result (performed on a 25 ml enrichment) subsequently being
+ + − 0.24 0.03 2.00 confirmed as a negative result, while the 10 ml sample was tested and resulted in a
(≡1 cfu/4.2 ml) positive.
b
+ + + >0.24 – – Not identified to the species level.
(≡>1 cfu/4.2 ml)
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. 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).
prevalence of Salmonella in raw milk has ranged from ‘not detected’ Of the 16 raw milk samples found to contain Listeria in this study,
to 8.9% in several studies published internationally (D'Amico et al., L. innocua was detected in 4% and L. monocytogenes was detected in
2008; Jayarao and Henning, 2001; Jayarao et al., 2006; Murinda et 0.68% of samples (n = 295). In the case of L. monocytogenes, the
al., 2002; Rohrbach et al., 1992; Steele et al., 1997). quantification achieved using the MPN technique indicated that the
It is reasonable to suppose that the presence and the concentra- concentration in the two positive samples was 1 cfu/4 ml (Table 3).
tion of Salmonella in bulk tank milk on farms are dependent on vari- Various published studies indicate that some of the contributors to
ous factors including geographical region, herd size and subclinical Listeria contamination of milk include the housing of cattle indoors
shedding, farm management practices and its presence in the envi- all well as poorly made silage and poor on-farm hygiene (Husu et
ronment (Ruzante et al., 2010). For example, subclinical shedding of al., 1990; Sanaa et al., 1993). The previously mentioned absence of
Salmonella is reportedly common in Ohio, USA, where the prevalence the practice of indoor housing of dairy cattle in New Zealand may
of infected herds ranged from 1 to 97% (Huston et al., 2002). In the also explain in part the lower rates of detection of L. monocytogenes
case of New Zealand, outbreaks of Salmonella infections within dairy in this study compared with those found in many of the studies
herds seem to occur sporadically, and the organism is often not performed in other countries (Fox et al., 2011; Hayes et al., 1986;
detected in herd animals or their faeces (Grinberg et al., 2005; Jayarao et al., 2006; Rohrbach et al., 1992; van Kessel et al., 2004).
Moriarty et al., 2008; Vermunt and Parkinson, 2000). In addition, ep-
idemiological evidence has linked raw milk with very few outbreaks 4. Conclusions
of nontyphoid salmonellosis in New Zealand (King et al., 2011).
When Campylobacter is detected in raw milk, it is thought to As with other studies conducted internationally, our results indi-
originate from various environmental sources including indoor cow cate that raw milk sampled from farm vats in New Zealand contained
housing (Ellis-Iversen et al., 2009). In New Zealand, dairy cattle are recognised pathogens. Even so, the prevalence and concentration of
predominantly grass fed in open fields year round. Therefore, the the pathogens included in the study were relatively low. Detection
low prevalence of Campylobacter in raw milk in this study is not rates for Salmonella, E. coli O157:H7, Campylobacter and Listeria
were generally lower than those found in many of the studies from
80 other countries. This finding is perhaps not surprising, as the presence
of these pathogens in raw milk is believed to be influenced by envi-
70 ronmental factors, such as indoor housing of cattle and poor quality
60
feed, such as silage, which are less likely to occur on New Zealand
dairy farms where the animals are predominantly pasture fed.
% of samples

50 Notwithstanding, the inescapable presence of such pathogens in

raw milk, albeit at low levels, highlights the continued need for cor-
40 rect pasteurisation or other equivalent destructive technique to be
practised routinely to ensure the production of safe dairy products
30
for consumption. Without the maintenance of pasteurisation or
20 other effective controls, these pathogens have the potential to cause
illnesses in consumers of raw milk or products made from raw milk.
10
Acknowledgements
0
>1 00
5 9
0 9
<1

0 49
0 9
0 49

35 - 3 9
40 - 3 9
0 99

50 - 4 9
0 99

60 -5 9
0 99

70 - 6 9
0 99
0 49
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0 9
95 - 9 9
0 49

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20 - 19

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85 -84

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00

We would like to thank Dr. Andrew Hudson (Christchurch Science

0
15 - 1

25 -2
30 - 2

45 - 4

55 - 5

65 - 6

75 - 7

90 -8

-1
1

Centre) for his significant contributions to the planning of this study

cfu/ml and for his editorial input, Dr. Rob Crawford (Fonterra) for the devel-
opment of the modified MPN method used in this study and also the
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 contributions made to the design and execution of the study by
from b 1 to >1000 cfu/ml, in 50 cfu/ml increments. Dr. 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), ISO, 2001. Standard 16649–2: Microbiology of Food and Animal Feeding Stuffs −
Horizontal Method for the Enumeration of Beta-Glucuronidase-Positive Escherichia
Dr. Nigel French (Massey University), Warwick Aspin (AsureQuality) coli − Part 2: Colony-Count Technique at 44 °C Using 5-Bromo-4-Chloro-3-Indolyl
and Sally Miller (Fonterra Research Centre). We also recognise the Beta-D-Glucuronide. International Organization for Standardization, Geneva.
important contribution of the Fonterra milk collection staff who en- Jay, J.M., 2000. Modern Food Microbiology, sixth ed. Aspen Publishers, Inc., Gaithersburg,
Maryland, pp. 441–459.
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