Goat milk is primarily destined for the production of fermented products, in particular
cheese. Therefore, the control of antibiotic residues in milk is of great importance, since
these could have negative repercussions on technological properties of the milk as well
as on the health of consumers.
In milk quality control programs, microbial inhibitor tests are widely applied to detect
antibiotics during the screening stage. However, tests are non-specific and may be
affected by substances other than antimicrobials which could inhibit the growth of the
test micro-organism, causing false positive results.
The aim of this thesis was to evaluate the interference, related to the presence of
different contaminants in goat milk, on the response of microbial inhibitor tests
commonly used in Spain to detect antibiotics (BRT MRL, Delvotest SP-NT MCS and
Eclipse 100 tests). The influence of the physicochemical characteristics of goat milk on
the false positive outcomes in microbial screening tests was also investigated.
The suitability of microbial inhibitor tests for screening antibiotics in colostrum
secretions was studied by analysing antibiotic-free colostrum and milk samples from
forty-three Murciano-Granadina goats, collected every 12 hours during the first week
post-partum. Microbial inhibitor tests were not suitable for the analysis of goat
colostrum because they presented a high percentage of doubtful and positive results
(up 37.2% in the 36 hours after partum).
To evaluate the effect of caprine colostrum on the microbial test response,
antimicrobial-free goat milk spiked with different concentrations of colostrum was
analysed to calculate the inhibitory concentrations producing 5% of positive results.
The highest interferences were obtained for the addition of colostrum from 12 to 24
hours post-partum and the colostrum concentrations producing 5% positive results
were between 5.1 and 34.6%. The BRT MRL was the test the most affected.
In another study, the interference of detergents and disinfectants used for the cleaning
of milking equipment and milk storage tanks of dairy farms was investigated.
Antimicrobial-free goat milk was spiked with eight concentrations of different cleaning
products (5 acid, 5 alkaline, 5 domestic washing-up liquids, and 1 disinfectant) and
analysed using microbial screening tests. The presence of acid detergent and
disinfectant based on sodium hypochlorite in goat milk did not affect the microbial test
response. However, alkaline detergents at concentrations ≥ 1 ml/l could lead to false
positive results in microbial inhibitor tests (up to 16.7%) and from 4 ml/l on 100%
positive results were obtained. Regarding the products used for home use, and those
used on farms and small size dairies, washing-up liquid containing sodium laureth
sulphate and ethanol had the greatest effects on microbial inhibitor tests, even starting
from a relatively low concentration (1 ml/l). On the other hand, the presence of a
relatively low concentration of detergents in goat milk (0.5 ml/l) slightly modified the
detection capability of the microbial inhibitor tests for amoxicillin, ampicillin,
benzylpenicillin, and cloxacillin, although the detection of these drugs at MRL (safe
level) was not compromised.
Antiparasitic agent residues in goat milk could be another possible cause of false
positive results in microbial screening tests. An in vitro study to evaluate the effect of
seven parasiticides commonly used in dairy goats was carried out. Further two studies,
where albendazole and ivermectin were applied to two groups of dairy goats in
lactation were performed. It should be noted that the parasiticide ivermectin is banned
for the treatment of animals producing milk for human consumption, although its
inclusion in this study was considered interesting to understand the potential effect of
their residues in milk, in the event the practice was performed illegally.
In the in vitro study, raw antibiotic-free milk from goats was spiked individually with eight
different concentrations of albendazole, closantel, diclazuril, febendazole, levamisole,
diazinon, and ivermectin. The microbial inhibitor test results showed a great variability
according to the test and the drug under study. Of the tests considered, the BRT MRL
test was the most sensitive to antiparasitic agents, with the lowest concentrations of
antiparasitic agent causing 5, 10, and 50% of positive results. Generally, closantel and
diazinon were the antiparasitic agents that produced higher interferences in all tests,
since low concentrations already resulted in positive results, while only higher
concentrations of diclazuril and ivermectin showed an inhibitory effect.
To evaluate the effect of albendazole residues on the microbial inhibitor test response,
eighteen healthy Murciano-Granadina goats in mid-lactation were treated with a single
oral administration of the commercially available albendazole registered for dairy sheep
(7.5 mg/kg b.w. of active compound) with a withdrawal period of 4 days for milk
production in ovine. Albendazole and its metabolite residues in goat milk after under
cascade treatment were not detected above MRL from the third day post-administration.
However, a high occurrence of non-compliant results was obtained for the BRT MRL test
during the first six days after treatment, suggesting that factors related to the
albendazole application other than the drug concentration are able to affect the microbial
inhibitor test response in some cases.
Regarding the ivermectin study, twenty-eight Murciano-Granadina goats infested with
Sarcoptes scabiei var. caprae were treated with a subcutaneous injection of ivermectin
(200 μg/kg b.w.), with a second dose applied seven days after the first treatment. Drug
residues in goat milk were recorded during the first fifteen days of the experiment with
concentrations ranging from 8.13 to 24.25 ng/ml. In addition, all the microbial screening
tests seem to be affected by the ivermectin treatment, with BRT MRL the most affected
(20%) compared with Delvotest SP-NT MCS and Eclipse 100 (6.6 and 5.7%,
respectively). These positive results cannot be associated with the ivermectin
concentration in goat milk, as the concentrations measured were lower than the
inhibitory concentrations as reported in a previous in vitro study for these microbial
tests. Thus, as suggested by some authors, interferences could be related to changes
or alterations caused by the application of the parasiticide agent or by the parasitic
disease itself, which could affect the immune response of the animals favouring the
presence of inhibitory substances in milk.
The study of the effect of the goat milk composition on the specificity (rate of false
positive results) of microbial inhibitor tests for screening antibiotics was also
considered. Thus, individual goat milk samples (n=200) were analysed by microbial
inhibitor tests using both visual and instrumental classification of the test results. The
highest specificity values were obtained for the instrumental interpretation of the test
results (94-99% vs 90-96%) due to the occurrence of samples with intermediate
colorations (green-yellow, yellow-blue) making the visual classification more difficult
and subjective. A relation was found between positive results in BRT MRL and Eclipse
100 tests and an elevated fat content in the goat milk. Positive outcomes in Eclipse 100
were associated with the butyric acid concentration in the milk. Further, the Delvotest
SP-NT MCS test response was affected by elevated pH values, high lactoferrrin and
myristoleic acid concentrations in the goat milk. This percentage of positive results
could be minimized by a pre-treatment prior to microbial inhibitor test analysis, such as
fat removal by centrifugation (3,100 g for 10 min at 4 ºC) and/or heating (80 ºC for 10
min).
Undoubtedly, improvements on the specificity of the microbial inhibitor tests for
screening antibiotics in goat milk are desirable to avoid the destruction of milk
compliant for human due to the occurrence of false positive results. The related
financial losses affect farmers and dairies. However, it should be noted that the
presence of contaminants in goat milk could be avoided by applying good farming
practices designed to ensure that milk is obtained from healthy animals under proper
hygienic conditions so ensuring the food safety of goat milk and related dairy products. / Romero Rueda, T. (2015). Evaluation of false positive results in microbial inhibitor tests for screening antibiotics in goat milk [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48552 / TESIS
| Identifer | oai:union.ndltd.org:upv.es/oai:riunet.upv.es:10251/48552 |
| Date | 31 March 2015 |
| Creators | Romero Rueda, Tamara |
| Contributors | Beltrán Martínez, Mª Carmen, Molina Pons, Mª Pilar, Reybroeck, Wim, Universitat Politècnica de València. Departamento de Ciencia Animal - Departament de Ciència Animal |
| Publisher | Universitat Politècnica de València |
| Source Sets | Universitat Politècnica de València |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/doctoralThesis, info:eu-repo/semantics/acceptedVersion |
| Rights | http://rightsstatements.org/vocab/InC/1.0/, info:eu-repo/semantics/openAccess |
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