Oprean L. et. al./Scientific Papers: Animal Science and Biotechnologies, 2011, 44 (1)
Pathogenic Microorganisms from Raw Milk of Different
Animals
Letiţia Oprean, Ramona Iancu, Eniko Gaşpar, Ecaterina Lengyel
„Lucian Blaga” University of Sibiu, Faculty of Agricultural Sciences, Food Industry and Environmental Protection
Abstract:
Milk is an ideal environment for microbial growth and for this reason the separation of some pathogens is very
important. The analysis of milk regarding pathogenic microorganisms is a clear indicator of hygienic quality and this
influences the dairy production. Samples of raw milk from cow, goat and sheep were analyzed for pathogens like
Staphylococcus aureus and Escherichia coli. The microorganisms found in milk directly affect the human health and
can cause a public illness if the unpasteurized milk is used in the food industry.
Keywords: cow, goat, hygienic quality, pathogenic microorganisms, raw milk, sheep
The records of the year 2009 shows that, in
Romania, 4.7 billion liters of milk are produced
annually, of which only 20% reach the
processing industry, as in only this amount of
milk is being processed under hygienic
conditions [7].
This study was realized in order to isolate S.
aureus and E. coli in raw milk of cows, sheep
and goats.
1. Introduction
The frequency of outbreaks of illness is
attributed to milk infected with pathogens that
underscores the importance of milk and milk
products as vehicles of human infection.
For these reasons, the presence of the
microbiological pathogens in unpasteurized milk
remains a public health threat [1].
It is known that a high percentage of clinically
normal animals on dairy farms can be infected
with pathogens [2, 3]. The access of these agents
in milk can be limited by considering the farm
factors regarding general hygiene [4], as the
degree of cleanliness of the facilities and animal
cleanliness of the milking equipment.
Moreover, the hygienic quality of milk depends
on the health of dairy herd [5].1Other sources of
contamination with microorganisms are by the
unclean teats and transport equipment that
contribute to the poor quality of milk [6].
2. Materials and methods
The selected farms, from where the samples for
the microbiological analysis were taken, are: the
first was a goat farm from Vâlcea with
Carpatina breed; the second was a sheep farm
from Răşinari with Ţurcana breed and the third
was a cow farm from Cristian with Holstein
breed.
From each farm there were selected 10 animals
and the study was conducted on a total number
of 30 animals.
The samples of raw milk from cows, sheep and
goat in the selected farms were examined in
term of S. aureus and E. coli.
* Corresponding author: Iancu Ramona, Tel: 0040745961898, e-mail: [email protected]
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The milk samples were collected aseptically,
from individual animals from the mentioned
species in sterile screw capped flasks and were
directly transported to the laboratory within the
Center of Research in Biotechnologies and
Microbiology of ULBS.
There were taken up to 10 samples from each
type of animal.
S. aureus was detected with the Petrifilm
method and was distinguished from other
staphylococcus with the Phosphatase test.
The number of coliforms and E. coli were
carried out through the Petrifilm method.
Isolation of S. aureus by Petrifilm method
Labeled films were used for the test.
One ml of milk homogenized sample was
transferred to the basic core of the film.
Peak film was then rolled slowly over the
sample to prevent catching the bubbles in the
film. Using a spatula, the pressure was applied
over the film to stretch the sample evenly.
For a minute films have been stretched to
solidify. Stacked, the films were incubated at
37°C for 24 hours.
Violet-red colonies indicated the presence of S.
aureus strains colonies. [5]
The confirmation of the presence of S. aureus
bacteria could be achieved using a disk which
contains blue O-toluidine in order to facilitate
the development of a pink areas around the
specific colonies. [8]
Distinguishing the S.aureus using the
Staphylase test
This test distinguishes S. aureus from other
staphyloccus that do not secrete the coagulase
enzyme, the test for the presence or absence of
the coagulation enzyme. Coagulase presence is
viewed by the fibrogen’s grouping [1]. A drop
of sample was spread on a thin film to cover the
circle on the response card.
At the tested colonies it was aimed to observe
the agglutination reactions. All positive
reactions were confirmed by repeating the same
procedure [9].
Isolation of E. coli by Petrifilm method
Labeled films were used for the test. One ml of
homogenized milk sample was transferred to the
basic center of the film. The film peak was then
rolled slowly over the sample to prevent
catching the bubbles in the film. Using a spatula,
the pressure was applied over the film to evenly
stretch the sample.
For a minute, films haven’t been stretched in
order to solidify.
Stacked, the films were incubated at 32°C for 24
hours [2]. Red colonies associated with gas,
showed coliform bacteria colonies (Figure 1).
But colonies identified by their blue color and
associated with gas are considered positive for
E. coli colonies. Colonies that were not
associated with gas were not counted [8].
Figure 1. Coliform colonies 3. Results and discussion
The results of the present study are summarized
in Table 1. According to these results the highest
E. coli contamination was recorded from the
samples of raw milk obtained from sheep and
goat, out of 10 samples 5 were contaminated
(50%), followed by cows which had the lowest
contamination, out of 10 samples only 1 resulted
positive (10%). Regarding S. aureus the highest
contamination was recorded in the cow`s milk,
out of 10 samples 7 were positive (70%),
followed by sheep`s and goat`s milk from which
only 2 samples resulted contaminated (20%).
Table 1: Raw milk analysis from different animals
concerning the pathogenic contamination
Cow’s
Sheep’s
Goat’s
milk
milk
milk
No. of
10
10
10
samples
S. aureus
E. coli
7 (70 %)
2 (20 %)
2 (20 %)
1 (10 %)
5 (50 %)
5 (50 %)
E. coli is commonly known as a frequent
occurring organism in milk [10].
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Oprean L. et. al./Scientific Papers: Animal Science and Biotechnologies, 2011, 44 (1)
pasteurization/boiling of milk is required before
collection and distribution for consumption.
The problem of bacterial contamination requires
more elaborative studies from the point of view
of milk production and consumption.
Acknowledgements
This work would not have been possible without our
advisors and we thank them for the support,
encouragement and the confidence given. We also
thanks to the ealuation committee for the relevant
recommendations.
References
Figure 2. S.aureus detected in the milk samples of
the three types of animals
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Figure 3. E. coli detected in the milk samples of the
three types of animals
Also, it may be used as an index of recent fecal
contamination [11] and suggests that other
microorganisms of fecal origin, including
pathogens, might be present such as S. aureus
[3, 12] which was found in all three types of
analyzed milk (Figure 2).
The milk sold in raw form represents a great
problem concerning the public health without
adopting hygienic measures because of
possibilities of contamination with E. coli
(Figure 3) or S. aureus [13, 14].
4. Conclusions
The results obtained in this study concluded that
the raw milk available to the consumer has a
medium pathogenic contamination.
Therefore, more strict preventive measures are
needed such as regular sterilization of dairy
equipment, washing of utensils, of milker’s
hands, udders, eradication of diseased animals,
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