Vol 55, No 2 May 2002 International Journal of Dairy Technology

ORI GI NAL
RESEARCH
*Author for
correspondence. E-mail:
david.mccleery@
dardni.gov.uk
2002 Society of
Dairy Technology
100
Blackwell Science, Ltd Oxford, UK International Journal of Dairy Technology 1364-727X Society of Dairy Technology 2002 May 2002 55 2Original research Vol 55 No 2 May 2002
Aerobic spore-forming bacteria in bulk raw milk: factors
inuencing the numbers of psychrotrophic, mesophilic and
thermophilic Bacillus spores
JAMES T M MCGUIGGAN,
2
DAVI D R MCCLEERY,
1, 2
* ALAN HANNAN
2

and ARTHUR GI LMOUR
1, 2
1
Department of Food Science (Food Microbiology), Queens University Belfast and
2
Department of Agriculture and Rural
Development for Northern Ireland, Newforge Lane, Belfast, Northern Ireland
Psychrotrophic, mesophilic and thermophilic spore concentrations of Bacillus spp. were determined on
a weekly basis in bulk raw milk samples obtained from a central processing facility, over one calendar
year. These data were correlated with concentrations of metal ions, free amino acids and somatic cell
counts obtained from the same samples, as well as local meteorological mean temperature and relative
humidity measurements relating to the same test period. A heat treatment of 80C for 20 min followed by
the addition of L-alanine to the milk at 0.1% w/v and incubation at 55C for 7 days gave optimal recovery
conditions for thermophilic spores. Free amino acids, metal ions, somatic cell counts, temperature, and
relative humidity measurements were each signicantly correlated with the recovery of spores of Bacillus
spp. from bulk raw milk, although no single factor was shown to demonstrate a consistent effect with the
psychrotrophic, mesophilic and thermophilic spore groups studied.
Keywords Bulk raw milk, Interaction with milk components, Meteorological factors, Recovery of
Bacillus spp.
*Author for correspondence. E-mail: david.mccleery@dardni.gov.uk
I NTRODUCTI ON
Aerobic spore-forming bacteria of the genus
Bacillus are commonly present in raw milk. Their
spores survive pasteurization and subsequently
germinate, outgrow, and multiply. They have been
responsible for the spoilage of pasteurized milk
and milk products
13
and UHT products.
4,5
Indeed, the presence of Bacillus spp. in foods
that contain milk or milk products is also undesir-
able because a number of species have been impli-
cated in food-borne disease.
6,7
Bacillus cereus was
incriminated in a large food poisoning outbreak
attributed to pasteurized milk
8
and WHO gures
indicate that 510% of reported food-borne dis-
ease is caused by this organism.
9
The dairy industry is interested in strategies
whereby the numbers of spore-formers in raw
milk can be controlled or reduced.
10
Germination
followed by a heat treatment to kill the resultant
vegetative cells is one method that has been postu-
lated.
1
However, there are many factors that could
possibly inuence the germination and outgrowth
of Bacillus spores in a complex medium such as
raw milk and may therefore limit the desired effect
of such a treatment. Nutrient germinants such as
amino acids
1114
and unsaturated fatty acids,
15
levels of some indigenous antibacterial factors
such as lysozyme, lactoferrin and lactoperoxi-
dase,
1517
somatic cells
18
and metal ions
19
have
been suggested as factors inuencing the ger-
mination of Bacillus species. Additionally, further
effects may result from storage temperature, heat
treatment
10,20
and even antagonism between the
sporeformers themselves.
21
Such factors can act alone or in combination and
may either stimulate or inhibit germination.
1113,22
Indeed, a compound that causes germination in one
Bacillus species may have little effect on a different
species.
23
Additional parameters inuencing uc-
tuations in the levels of Bacillus spp. may be the
nature of the milk produced as inuenced by the
stage of lactation and the dietary intake of the cow.
Although each of these factors may have an
effect on the numbers and types of Bacillus spp.
occurring in raw milk, their individual relative sig-
nicance is unclear. Moreover, spoilage problems
caused by spore-forming bacteria are more preva-
lent at certain times of the year.
1
McGuiggan et al.
24
previously examined factors
inuencing the recovery of psychrotrophic, mes-
ophilic and thermophilic spores of Bacillus spp.
from bulk raw milk and developed methods for
their optimal detection and isolation. The aim of
2002 Society of Dairy Technology 101
Vol 55, No 2 May 2002
the study reported here was to apply these devel-
oped recovery methods in an investigation of the
interactions between raw milk components, envi-
ronmental factors and the recovery of spores of
Bacillus spp. Consequently, free amino acids, metal
ions, somatic cells counts, meteorological temper-
ature and relative humidity measurements were each
compared with the recovery of psychrotrophic,
mesophilic and thermophilic spores of Bacillus
spp. from bulk raw milk, over a 1-year period.
MATERI ALS AND METHODS
Collection of raw milk
Fifty samples of raw milk (2.0-L volumes) were
collected on a weekly basis over one calendar year
JanuaryDecember (excluding weeks 6 and 51)
from a bulked source at a local milk processing
plant and were maintained at chill temperature
(< 5C) during transfer to the laboratory. The milk
was taken from a source (usually the balance tank)
as close to the pasteurizer as possible. All samples
were tested within 2 h of sampling.
Preparation of skimmed milk medium,
detection of inhibitory substances in raw milk
and preparation of L-alanine solution
Ultra high temperature (UHT) skimmed milk was
dispensed in appropriate volumes (9 or 10 mL)
and L-alanine solutions (10% w/v) were prepared
as previously described by McGuiggan et al.
24
Detection of inhibitory substances in raw and
skimmed milk was performed using the miniatur-
ized Oxoid (AIM) test.
25
Recovery of psychrotrophic, mesophilic and
thermophilic spores of Bacillus spp. from bulk
raw milk
Sample treatment
A most probable number technique (MPN) was
used to enumerate Bacillus spores in raw milk,
as previously documented.
24
Briey, raw milk
samples were thoroughly mixed before 10-mL
volumes were individually dispensed into ve sterile
test tubes, 1-mL aliquots were individually added
to ve test tubes containing 9 mL of the prepared
sterile skimmed milk medium and similarly
0.1-mL volumes were added to 10 mL of the sterile
skimmed milk medium. Where spore numbers
were expected to be high, further decimal dilutions
of the raw milk samples were examined by adding
0.1 mL of the appropriate dilution(s) (peptone
diluent) to ve test tubes containing 10 mL of ster-
ile skimmed milk. Tubes were then heat treated at
80C for a holding time of 10 min (psychrotrophic
and mesophilic spore counts only) or 20 and
30 min (thermophilic spore counts). One hundred
microlitres of L-alanine solution (10% w/v) were
added to each tube after heat treatment, to give a
nal concentration of 0.1% w/v L-alanine in the
sample. A duplicate set of MPN tubes without
L-alanine was included for the enumeration of
thermophilic spores. Test tubes were incubated
before sampling, as shown in Table 1. Psychrotrophic
spores were enumerated by incubating the MPN
tubes at 6.5C for 15 days; mesophilic spore popu-
lations were detected using an incubation temper-
ature of 30C for 15 days; and thermophilic spore
counts were performed by incubating the tubes at
55C for 7 days. After incubation, each MPN tube
was thoroughly mixed and 10 L were transferred
to a slope of milk plate count agar (mPCA; Oxoid
CM 325) and incubated under the same conditions
as those that had been used for the original MPN
tubes to conrm growth, as summarized in Table 1.
Spore conrmation
Conrmation for the presence of spores was car-
ried out by subculturing from mPCA onto sporula-
tion agar
26
and incubating at 25C overnight.
Samples that showed no signs of growth after this
time were reincubated and examined after a further
24 h. Smears were examined for the presence of
spores using phase contrast microscopy.
27
Somatic cell count
A subsample of each raw milk sample was trans-
ported (at 47C), on the same day as sampling,
to the laboratories of the United Dairy Farmers,
Antrim Road, Belfast. The number of somatic cells
per mL of milk was determined within 4 h of
Table 1 Heat treatments, incubation temperatures and times used for the recovery of psychrotrophic, mesophilic and
thermophilic Bacillus spores from bulk raw milk by an MPN method and subsequent conrmation on milk plate count
agar slopes

Holding time
(minutes at 80C)
Incubation
temperature (C) Incubation time (days)
Psychrotrophic spores 10 6.5 15
Mesophilic spores 10 30 15
Thermophilic spores 20, 30 55 7
Vol 55, No 2 May 2002
2002 Society of Dairy Technology
102
receipt using a Fossomatic instrument [Foss Elec-
tric (Ireland) Ltd, Dublin].
Metal ions concentration
Triplicate 25-mL aliquots of each raw milk sample
were combined with an equal volume of 20% (w/v)
trichloroacetic acid (TCA) before incubating at
room temperature for 30 min with mixing by inver-
sion at 5-min intervals. The samples were centri-
fuged at 2300 g for 15 min and each supernatant
was analysed in triplicate for copper, iron and
manganese using a Perkin-Elmer Model AA 5000
atomic absorption spectrophotometer (Perkin-
Elmer, England) in accordance with the manu-
facturers instructions.
Free amino acids concentrations
Duplicate milk samples were defatted by centri-
fuging at 130 g for 10 min. Defatted milk (5 mL)
was mixed with 25 mL of a 1% v/v picric acid
solution to precipitate the protein and centrifuged
for 20 min at 1000 g. The supernatant was passed
through a prepared Amberlite IRA-410 ion
exchange resin column using 0.02 N HCl and
distilled water aliquots (Pharmacia). The eluent
obtained was rotary evaporated with two water
washings to dryness and dissolved in 1.0 mL of a
lithium citrate buffer solution (pH 2.2; Pharmacia)
containing a known concentration of the internal
standard norleucine. Eighty microlitres of these
test solutions were used for cation exchange chro-
matographic separation of the constituent amino
acids on an LKB 4400 amino acid analyser
(Amersham Pharmacia Biotech, UK). Results
were calculated using a Trivector, Trilab 3 com-
puting integrator. The free amino acids detected are
listed in Table 3.
Temperature and relative humidity
measurement
Temperature (C) and relative humidity (%) data
were obtained for the corresponding 1-year period
of milk sampling from The Meteorological Ofce,
Belfast Weather Centre, 1 College Square East,
Belfast BT1 6BQ. These data comprised weekly
mean values of combined daily observations from
eight stations, within a 20-mile radius of the milk
processing centre.
Statistical analyses
Analysis of variance was carried out according to
the two methods two durations of heat factorial
design used. This tested for the main effects of the
methods used to enumerate thermophilic spores
(with and without L-alanine) and the duration of
heat treatment with associated interactions. Cor-
relation coefcients were calculated to test for
relationships between the somatic cell counts,
temperature, relative humidity, free amino acids
and metal ions concentrations with the recovery of
the three test groups of Bacillus spores isolated
from bulk raw milk.
RESULTS AND DI SCUSSI ON
Recovery method for thermophilic spores
In a previous study by McGuiggan et al.,
24
two of
the developed protocols demonstrated equivalent
optimal recoveries for thermophilic Bacillus spp.
isolated from raw milk, leading to an inability by
these researchers to recommend a single denitive
method for the recovery of such spores. Conse-
quently, in the study reported here, these recovery
protocols for thermophilic spores were further
evaluated by applying them in parallel to a larger
number of raw milk samples over a longer time
period.
In the study reported here, the effect of the dura-
tion of the heat treatment (80C) and the presence
of L-alanine in the recovery of thermophilic Bacil-
lus spp., were each found to inuence the numbers
of spores recorded from individual raw milk
samples (Table 2). The addition of L-alanine was
shown to enhance the recovery of these spores.
Indeed, the highest mean recoveries of ther-
mophilic spores, throughout the course of the
experiment, were obtained using a 20-min heat
treatment in the presence of L-alanine and conse-
quently, unless otherwise stated, the data presented
here were obtained using this method.
Psychrotrophic spores
The concentrations of psychrotrophic spores of
Bacillus spp. recovered from raw milk, over the 1-
year test period, ranged from 0.02 to 3.5 spores/mL
(Figure 1) with an overall mean level of 0.8 spores/
mL. Although variation in these recovery rates was
evident throughout the test period, with very low
levels being measured from weeks 1826 (May
June), this pattern did not appear to be related to
meteorological temperature and relative humidity
data or to somatic cells (all P > 0.05; Table 3).
These data are comparable with those reported
by McKinnon and Pettipher,
28
who recorded similar
Table 2 Effect of the duration of heat treatment and addition of L-alanine on the
recovery of thermophilic spores of Bacillus spp. from bulk raw milk

Duration of heat
treatment at 80C (min) With L-alanine Without L-alanine
20 3.98 2.02
30 2.94 2.51
Average 3.46 2.27
Figures are spores/mL, mean of 47 replications
Standard error of the mean (SEM) = 0.416
2002 Society of Dairy Technology 103
Vol 55, No 2 May 2002
spore concentrations in creamery silos, and also
to data from raw milk samples reported by Meer
et al.
2
In contrast, however, Sutherland and
Murdoch
21
detected psychrotrophic species only in
the summerautumn months and Phillips and
Grifths
29
recovered higher numbers of psychro-
trophic spores in creamery silos during the summer
autumn months than in the winter.
It is possible that the variations between the
results reported in these studies and the data pre-
sented here may be related to differences in the
methods used to recover the spore populations. It is
important to note that the method used in the study
reported here was developed to optimize the recov-
ery of psychrotrophic Bacillus spp. from milk
24
and may therefore demonstrate greater sensitivity
in comparison with other procedures. Moreover,
regional differences in husbandry practices may
have led to disparities between these studies
because Slaghuis et al.
30
suggested that pasturing
Figure 1 Recovery of spores of psychrotrophic Bacillus spp.
from bulk raw milk over one calendar year.
Table 3 Correlation between the concentration of free amino acids, metal ions, meteorological data, somatic cell counts
and the recovery of spores of Bacillus spp. isolated from bulk raw milk

Raw milk
component
Psychrotrophic
spores
Mesophilic
spores
Thermophilic
spores*
alanine 0.415
b
0.039 0.356
b
-alanine 0.359
b
0.364
b
0.048
arginine 0.008 0.537
c
0.102
aspartic acid 0.143 0.082 0.497
c
ethanolamine 0.048 0.144 0.089
glutamic acid 0.234 0.271
a
0.104
glycine 0.011 0.187 0.323
a
histidine 0.200 0.111 0.195
isoleucine 0.034 0.161 0.256
a
leucine 0.075 0.033 0.239
a
lysine 0.038 0.266
a
0.175
ornithine 0.169 0.056 0.001
phenylalanine 0.147 0.349
b
0.050
phosphoethanolamine 0.068 0.126 0.272
a
proline 0.055 0.532
c
0.238
a
serine 0.144 0.150 0.057
taurine 0.432
c
0.268
a
0.329
b
threonine 0.070 0.033 0.044
tyrosine 0.038 0.227 0.195
urea 0.038 0.430
c
0.018
valine 0.017 0.376
b
0.000
Copper 0.032 0.115 0.117
Manganese 0.198 0.120 0.017
Iron 0.267
a
0.006 0.028
Relative humidity 0.028 0.010 0.174
Temperature 0.059 0.212 0.208
Somatic cell count 0.101 0.424
b
0.268
a
Data presented are correlation coefcients relating recovery of groups of Bacillus spp. with listed milk component
concentrations, measured in the same samples
*See Table 1 for recovery methods used to enumerate different groups of Bacillus spp. from bulk raw milk (thermophilic
spores were recovered using a heat treatment of 80C for 20 min followed by the addition of 0.1% w/v L-alanine and
incubation at 55C for 7 days)
Weekly mean values of combined daily meteorological observations from eight weather stations within a 20-mile radius
of the milk processing centre
a
Statistically signicant relationship where P 0.05
b
Statistically signicant relationship where P 0.01
c
Statistically signicant relationship where P 0.001
Vol 55, No 2 May 2002
2002 Society of Dairy Technology
104
effects and effect of housing type may be more
inuential than seasonal factors.
The concentrations of individual milk compon-
ents in the bulk raw samples in comparison with
psychrotrophic spore counts (Table 3) demonstrated
a statistically signicant positive association
(P < 0.01) between recovery of this group of spores
and free amino acid concentration of alanine only.
Conversely, a signicant negative correlation was
observed with -alanine and taurine concentrations
(P < 0.01 and P < 0.001, respectively) and spore
recovery, suggesting that elevated concentrations
of these milk components may have a possible role
in the inhibition of sporulation by psychrotrophic
Bacillus spp. A similar negative correlation (P < 0.05)
was obtained with the iron levels recorded.
Mesophilic spores
Considerable variation in the recovery of mes-
ophilic spores over the 1-year test period was
observed (Figure 2), with their concentrations
ranging from 1.4 10
1
to 2.4 10
5
spores/mL and
an overall mean value of 7.6 10
3
spores/mL
being recorded. These levels are therefore similar
to those reported by Litopoulou-Tzanetaki et al.,
31
who recorded mesophilic spore concentrations of 1
to > 10
4
spores/mL in raw milk samples collected
from three factories in Greece.
Considerably lower mesophilic spore concen-
trations in raw milk samples were recorded in a
10-month study conducted by Sutherland and
Murdoch,
21
where spore populations were enumer-
ated by direct plating of the samples, following a
heat treatment (80C for 10 min). These researchers
detected mesophilic spore concentrations of
between 0.8 and 440 spores/mL in raw milk
samples obtained from ve dairy silos. Seasonal
variation recorded by Sutherland and Murdoch
21
demonstrated that the mesophilic spore counts
were highest during the winter and lowest during
the summer/autumn months, although the months
of April and May were not investigated. Thus these
data clearly contrast with those recorded in the
study reported here, not only in respect of numbers
but also in that recovery of mesophilic spores was
lowest during May and, in general, tended to be
highest in the mid to late summer.
Addition of L-alanine to media has previously
been shown to signicantly enhance (P < 0.05) the
recovery of mesophilic spores, through its pro-
posed involvement in the events triggering ger-
mination.
24
Consequently, it is plausible that
the inclusion of L-alanine in the MPN method
applied in the study reported here may have led to
enhanced recovery of mesophilic spore popula-
tions and resulted in the observed disparities
between the data of the current study and those
presented by Sutherland and Murdock.
21
In the
authors opinion it is unlikely, however, that this
factor alone could explain the marked differences
in the spore counts evident between the two stud-
ies. It is possible that other inuences may have
been involved in this effect and should be the sub-
ject of further scrutiny.
Although a statistically signicant relationship
was not observed between the meteorological data
and the concentrations of mesophilic Bacillus
spores recovered (P > 0.05; Table 3), it is interest-
ing to note that in general, higher recovery of these
spores was associated with elevated mean environ-
mental temperatures. This is suggestive of a pos-
sible trend worthy of further investigation over a
more extended study.
Enhanced recovery of mesophilic spores was
found to be positively correlated (Table 3) with the
concentrations of -alanine (P < 0.01), arginine
(P < 0.001), lysine (P < 0.05), phenylalanine (P <
0.01), proline (P < 0.001), taurine (P < 0.05),
urea (P < 0.001) and valine (P < 0.01). These data
are comparable with those reported by Shevtsov
et al.,
14
Jayachandran et al.
32
and Patel et al.
33
who
observed elevated sporulation in the presence of
arginine, lysine and valine, respectively, providing
further credence to the role of these amino acids as
germinants for Bacillus spp.
In contrast, signicantly lower concentrations of
mesophilic spores of Bacillus spp. were recovered
from milk samples with elevated levels of glutamic
acid (P < 0.05) in the study reported here. This
negative correlation between these two factors is
surprising because other studies have reported that
higher concentrations of glutamic acid are actually
associated with enhanced sporulation levels.
14,34
Thus, it is important to interpret such relationships
between potential spore levels and amino acid con-
centrations with caution until comprehensively
validated.
A highly signicant correlation was obtained
between the concentrations of mesophilic spores
recovered and the levels of somatic cells detected
(P < 0.01; Table 3). This supports the observations
of Davies,
18
who found that the level of germinant
for B. cereus was at least partly related to the
somatic cell count of milk.
A lack of signicant correlation between the
recovery of mesophilic Bacillus spores and the

Figure 2 Recovery of spores of mesophilic Bacillus spp.
from bulk raw milk over one calendar year.
2002 Society of Dairy Technology 105
Vol 55, No 2 May 2002
levels of metal ions (P > 0.05; Table 3) may not
have been unexpected because Foerster and
Foster
23
failed to demonstrate consistent effects
between 15 ionic agents and 46 Bacillus strains.
Indeed, White et al.
19
noted that ferric iron in-
hibited the germination of Bacillus licheniformis
spores at low concentrations but actually stimu-
lated germination at higher concentrations. Thus
it is feasible that in the study reported here the
species- or strain-specic nature of the interactions
between metal ion concentrations and germination
may have led to inconclusive results being
recorded when applied to the mixed populations
recovered from raw milk.
Thermophilic spores
The concentrations of thermophilic spores of
Bacillus spp. over the 1-year test period varied
between 0.08 and 54 spores/mL, with a mean
level of 3.98 spores/mL (Figure 3). This value is
therefore similar to the range of thermophilic
spore concentrations of 1100 spores/mL reported
by Litopoulou-Tzanetake et al.,
31
but consider-
ably lower than the mean value of 20 spores/mL
recorded in factory milk samples by Kamei et al.
35
It is important to note, however, that the high therm-
ophilic Bacillus spore concentrations recorded on
occasion in the study reported here may be of con-
cern in milk destined for the manufacture of UHT
milks and other long life products.
Relating these data to the meteorological meas-
urements of relative humidity and temperature
demonstrated no statistically signicant correla-
tion between these factors (Table 3). Interestingly,
however, although not optimal for the recovery of
this group of spores, the populations enumerated
using the alternative heating time of 30 min dem-
onstrated statistically signicant associations with
these meteorological data (results not shown).
Indeed, a positive effect was in evidence between
the mean relative humidity gures and the numbers
of thermophilic spores recovered (P < 0.001 and
P < 0.01; with and without the addition of L-
alanine, respectively), with an inverse relationship
being demonstrated between the mean temperature
data and the recovery of thermophilic Bacillus
spores (P < 0.05 and P < 0.001; with and without
the addition of L-alanine, respectively). These data
may therefore suggest that different populations of
thermophilic Bacillus spores were detected using
the various recovery conditions.
A signicant direct relationship (Table 3;
20 min heating with L-alanine) was observed
between the recovery of thermophilic spores and
the concentrations of a number of free amino
acids measured in the raw milk viz. aspartic acid
(P < 0.001), glycine (P < 0.05), phosphoeth-
anolamine (P < 0.05), and taurine (P < 0.01).
Aspartic acid has also been associated with ele-
vated spore levels in other investigations,
31,32
thus
demonstrating a possible role for this amino acid
as a marker of potential spore concentrations.
Conversely, a signicant inverse relationship
was demonstrated between the levels of alanine
(P < 0.01), isoleucine (P < 0.05), leucine (P < 0.01),
proline (P < 0.05) and somatic cell counts (P < 0.05)
when compared with the numbers of thermophilic
spores of Bacillus spp. recovered from bulk raw
milk. This inverse relationship between alanine con-
centration and spore recovery contrasts with that
reported by Jayachandran et al.,
32
who observed
elevated sporulation of Bacillus subtilis in the pres-
ence of this amino acid. This disparity between the
two investigations may be related to differences
in the spore populations investigated, demonstrat-
ing a lack of consistency in the relationship
between sporulation by Bacillus spp. and alanine
concentrations.
In general therefore, the correlation of the
recovery of psychrotrophic, mesophilic and
thermophilic spores of Bacillus spp. with various
parameters differed considerably. This is not un-
expected because Foerster and Foster
23
reported that
amino acids did not induce germination of all spore
strains studied, observing a marked difference
even among strains of the same species. Moreover,
Christiansson et al.
36
found that temperature was
not signicantly correlated with the B. cereus spore
content of milk and Slaghuis et al.
30
noted that
the incidence of B. cereus in raw milk was higher
when cows were outside, rather than being directly
linked with prevailing weather conditions.
The signicant interactions between the somatic
cell counts and the concentrations of mesophilic
and thermophilic Bacillus spores in raw milk were
found to be direct and inverse relationships,
respectively. Thus, the variable nature of the inter-
actions between the somatic cell counts and other
factors investigated in this study may indicate that
they are of limited value as a marker of potential
spore levels in raw milk samples. It is possible that
other uninvestigated parameters may have been
more inuential in determining Bacillus spore
concentrations, such as the effect of husbandry
practices, which would necessitate extending
the investigation to individual farms.
Figure 3 Recovery of spores of thermophilic Bacillus spp.
from bulk raw milk over one calendar year.
Vol 55, No 2 May 2002
2002 Society of Dairy Technology
106
ACKNOWLEDGEMENTS
The authors wish to thank Mr D. Reid and Dr Leslie
Dickson for excellent technical assistance; Dr
D. Kilpatrick, Biometrics Division for statistical
analyses; United Dairy Farmers for providing
somatic cell counts; and Mr P. Smyth, Belfast Weather
Centre, for the provision meteorological data.
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