Original Article

Contribution to the Identification of Antibiotics

Residues in Raw Bovine Milk in Algeria
Lyes Ouabdesselam 1,2*, Zoubir Benmaamar 3, Ali Berbar 2

1Food Science Division, Center for ABSTRACT

Research and Physical-Chemical
Analysis (CRAPC), Bou-Ismail, Tipaza, The presence of residues of veterinary drugs such as antibiotics used for the
ALGERIA treatment of animals can be dangerous for the consumer. Indeed, residues of
these drugs can end up in the form of undesirable traces in animal productions,
2National Institute of Veterinary in particular milk when there is an inappropriate use such as failure to comply
Sciences, SAAD DAHLEB University of with the waiting times between the administration of antibiotics to the animal
Blida, Route de Soumâa, BP 270, Blida and meat collection. This study is part of a global project carried out with the
RP 09000, ALGERIA aim of evaluating the presence of antibiotic residues in the raw milk of cows
3
reared in cattle farms in Blida. This study involved 200 milk samples were
Faculty of Chemistry, SAAD DAHLEB
collected in the Wilaya of Blida. A screening of the samples was carried out by
University of Blida, Route de Soumâa,
the acidification test followed by a confirmation agar diffusion test. The results
BP 270, Blida RP 09000, ALGERIA
showed residues in 20.51 % of the milk tested. The study revealed the presence
* Corresponding author: of antibiotic residues in raw cow’s milk from farms in varying proportions.
[email protected] Penicillin residues and / or tetracyclines were originally of the contamination of
93.62 % milk samples positive while residues macrolide and / or aminoglycoside
were only detected in 6.38 % of samples tested positive. The use of antibiotics
should be done by veterinarians or under their responsibility by animal health
professionals.

Keywords: raw milk, bovine, contamination, antibiotic residues, Algeria, Blida

EUROPEAN JOURNAL OF
BASIC MEDICAL SCIENCE
Eur J Basic Med Sci 2020;10(1):43-46

Received: 10 Aug 2020

Accepted: 5 Oct 2020

Copyright © 2020 by Author/s and Licensed by Veritas Publications. This is an open access article distributed under the Creative Commons Attribution 4.0
International (CC BY 4.0) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Contribution to the Identification of Antibiotics Residues in Raw Bovine Milk in Algeria

INTRODUCTION Two techniques were successively implemented: An

acidification test which is based on the demonstration
Algeria is the leading dairy consumer in the Maghreb of a possible inhibition of Bacillus stearothermophilus
with 115 liters / inhabitant / year in 2016 and a milk var. calidolactis C953 (strain C953, CIP 5281) indicated
market estimated at 5 billion liters per year [1]. This by the turn of colored indicator antibiotic residues
growing demand is a corollary to the development of which would be present in the sample. Follow-up by a
mass distribution and local processing industries. This confirmatory test, corresponding to the performance of
rapid development in demand leads to changes in two agar diffusion tests, one with Bacillus subtilis
behavior with fraudulent sanitary practices, due to a (ATCC 6633, spore suspension) at 30°C and the other
proliferation of breeding aids that are difficult to with Bacillus stearothermophilus variety calidolactis at
control, for a rapid gain. The presence of antibiotic 55 °C [4,5].
residues in milk constitutes a danger for the consumer
by provoking the development of resistance to Acidification test
antibiotics by promoting the emergence of a multi-
resistant microflora. It may December lencher toxic The milk sample is seeded with B. spores.
accidents or even rarely allergic disorders [2]. The stearothermophilus in a nutrient peptone agar
problem of residues can also be the cause of the total medium, comprising bromocresol purple and
or partial inhibition of fermentation phenomena of trimethoprim, for 2 h 30 min at 64°C. A negative control
bacterial origin in dairy products such as yoghurts and (reconstituted milk powder certified free from
cheeses, which represents an economic loss for the antibiotic residues) and a positive control (milk
dairy industry. The purpose of this study is to identify containing 0.01 IU/ml of control penicillin) are
antibiotic residues in raw milk produced in the Wilaya incubated in the presence of 0.1 ml of the test culture.
of Blida (Algeria). under the same conditions and at the same time as the
samples to be tested [5].
MATERIAL AND METHODS
Agar diffusion method
Material
The Muller Hinton agar medium, previously melted at
In this study, we realized 200 samples so divided 100°C and cooled to 55°C, is poured into Petri dishes.
homogeneous four breeding s sedentary previously The medium was then inoculated with B. subtilis and B.
identified in the region of Blida (Algeria). Two stearothermophilus spores at 103 and 105 spores / ml.
collections of 500 ml of raw milk were carried out at an The sterile filter paper discs, impregnated with the milk
interval of 15 days in each of the breeding farms. The to be tested by capillarity were then placed on the
first collection was made at least one month after the surface of the agar and the Petri dishes were incubated
administration of an antibiotic treatment ti the animals at 55°C (B. stearothermophilus) and 30°C (B.
on the basis of the declarations made by the breeders. subtilis).). After 24h of incubation, the diameters of
Other collection s are interveneds 15 days after the first the zones of inhibition were measured using a caliper
collection. The raw milk samples collected from each [5].
breeding farm were taken from the bucket or plastic
bowl in which the milk is collected. The milk was RESULTS
packaged in sealed plastic bottles, labeled and
Acidification Test Results
identified according to the farm number. In total, 200
raw milk samples were collected by area in the course The purple color of the persistent blue bromocrésol was
of the period of collection and transported under cooler recorded for 41 samples of the 200 tested (20.51%),
Laboratory. Then the milk samples were placed in the reflecting the presence of antibiotic residues. On the
freezer at –20°C for storage [3]. other hand, a color change was observed for 153
samples (76.49%) which were declared negative. It was
Methods impossible to etablisch a conclusion for 06 samples (3%)
Milk samples collected from the three wilayas will be considered as impaired (Table 1).
subjected to an acidification test to carry out an initial
screening followed by diffusion in agar [4].

44 Eur J Basic Med Sci 2020;10(1):43-46

 Dhotre et al.

Table 1. Demonstration of antibiotic residues in cow’s milk by the acidification test / Region of Blida (Algeria)
Samples Number Negatives (%) Suspect (%) Positives (%)
Total (%) 2 00 76.49 % 03% 20.51 %

Table 2. Demonstration of antibiotic residues in cow’s milk by the Agar Diffusion Confirmation test / Region of
Blida (Algeria)
Wilaya Number of samples positive For B. stearothermophilus % For B. subtilis %
Blida 47 44 93.62 3 6.38

Agar Diffusion Confirmation Test Streptococcus thermophilus as a test microorganism

which does not offer two previously cited antibiotics
See Table 2. that have respective sensitivity of 3 to 4 times the MRL
and 2-4 times the MRL. The risk of false negative results
INTERPRETATIONS AND DISCUSSION with milk with a very low concentration of antibiotic
residues close to the MRLs is thus limited. The
This study you confirmed that the contamination rate
confirmation test shows great specificity [8].
was on the Qurante Seven (47) positive samples with
antibiotic residues, a percentage of 93.62% of the CONCLUSION
samples were positive for penicillin and / or
tetracyclines. A percentage of 6.38% were positive for The growing and sometimes irrational use [9,10] of
macrolides and / or aminoglycosides (Table 2). Forty- antibiotics produced by the non-respect of time s
four (44) of the 47 tested exhibited an annular zone of waiting results in the presence of residues in milk
inhibition of B. stearothermophilus with a diameter produced by cows treated and is a real public health
greater than 12.67 mm ± 0.72 mm. These samples were problem in Algeria [9] given the danger of resistance to
considered positive for penicillin and / or tetracycline antibiotics incurred by the end consumer. This risk is
residues. Three (3) samples out of the 47 tested were low in the formal controlled circuit [11]. The dairy
found to be positive for macrolide and / or industry in Algeria remained for many years the
aminoglycoside residues by inducing a circular property of the State. The establishment of private
inhibition of 11.18 mm ± 1.09 mm of the colonies of B. collectors encouraged by the public authorities, and
subtilis. These results demonstrate the wide use of this since 2000, has not kept all its promises; since the
penicillins and tetracyclines in the treatment of quantities collected have always remained very low.
infections in dairy farming in Algeria. Particularly for The commitment of dairies to farmers is almost
mastitis by injected ions intramammary. The insignificant [12], above all. The specialization of these
microbiological analysis method implemented using farms is imprecise; informal collection is therefore very
B.stearothermophilus as a test microorganism is present and is required despite all the efforts made by
characterized by an antibiotic detection threshold as the public authorities to formalize this entire segment.
close as possible to the maximum residue limits (MRLs) It is evident that in view of the large quantities of milk
of the antibiotics most frequently used in the treatment production (over 96%) which escape formal circuits
of cattle. dairy while maintaining a broad spectrum of [13]. The art public policies in this area are ineffective.
antibiotics. In fact, the sensitivity of the new While competing with each other, the two types of
acidification technique is much better for the two sectors are highly interdependent. They meet food
families of antibiotics most widely found in the intra- needs not covered by legal businesses and largely
mammary specialties used in Algeria (beta- lactams and contribute to supplying urban populations. The control
tetracyclines). In fact, Bacillus stearothermophilus is of the sanitary quality of milk must be further
characterized by a remarkable sensitivity to beta strengthened by the State.
lactams, its growth is inhibited by a concentration of 5
ppb of ampicillin [6]. The sensitivity of this technique ACKNOWLEDGEMENTS
is particularly high for cloxacillin and relatively Our sincere thanks go to the Personnel of National
acceptable for tetracycline [7]. The acidification Institute of Veterinary Science (INSV) from SAAD
technique is thus characterized by a sensitivity close to DAHLEB University (Blida) and the Center for Research
the MRL for cloxacillin and 50-100% of the MRL for in Physico-Chemical analysis (CRAPC) sis Tipaza.
tetracycline compared to the old method using

Eur J Basic Med Sci 2020;10(1):43-46 45

Contribution to the Identification of Antibiotics Residues in Raw Bovine Milk in Algeria

Conflict of Interest: None to Declare 7. Basic Tests for Pharmaceutical dosage forms,
World Health Organization; Detection of
Source of funding: No funding Oxytetracycline (OTC) in milk supernatant, 1986; 1
& 3: 122.
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