Prevalence of antibiotic residues in commercial milk and its variation by season and thermal processing methods
Fathollah Aalipour1, Maryam Mirlohi2, Mohammd Jalali2
1 Department of Food Science and Technology, Isfahan University of Medical Sciences, Isfahan, Iran
2 Food Security Research Center, and School of Nutrition and Food Sciences, Isfahan University of Medical Sciences, Isfahan, Iran
|Date of Web Publication||29-Nov-2013|
Food Security Research Center, School of Nutrition and Food Sciences, Hezar Jerib Avenue, Isfahan University of Medical Science, Isfahan
Source of Support: Isfahan University of Medical Sciences, Conflict of Interest: None
Aims: In this study, the prevalence of antibiotic residues in pasteurized and sterilized commercial milk available in Shahre-kourd, Iran, was investigated. In addition, the influence of seasonal temperature changes on the prevalence of contamination was studied.
Materials and Methods: Commercial milk samples of 187, including 154 pasteurized and 33 sterilized, milk samples were collected from the market between early January 2012 and late July of the same year. The presence of antibiotic residues was detected using the microbiological detection test kit, Eclipse 100, as a semi-quantitative method.
Results: The results showed that 37 of the samples (19.8%) have contained antibiotic residues above the European Union Maximum Residues Limits (EU-MRLs), of which 28 samples (14.97%) were found to be contaminated but at the concentrations below the EU-MRLs. There was no significant difference between the contamination rate of pasteurized and Ultra High Temperature (UHT)-sterilized samples. Similarly, variation of weather temperature with seasons had no effect on the contamination prevalence of milk samples ( P > 0.05).
Conclusion: Based on the result of this study, antibiotics residues were present in the majority of milk samples. Neither the season nor the type of thermal processing of the commercial milks had noticeable impact on the prevalence level of the milk samples. However, an increasing trend of prevalence level for antibiotic residues was observed with increasing the temperature through the warm season.
Keywords: Antibiotic residues, milk, prevalence, season, thermal processing
|How to cite this article:|
Aalipour F, Mirlohi M, Jalali M. Prevalence of antibiotic residues in commercial milk and its variation by season and thermal processing methods. Int J Env Health Eng 2013;2:41
|How to cite this URL:|
Aalipour F, Mirlohi M, Jalali M. Prevalence of antibiotic residues in commercial milk and its variation by season and thermal processing methods. Int J Env Health Eng [serial online] 2013 [cited 2019 Jun 16];2:41. Available from: http://www.ijehe.org/text.asp?2013/2/1/41/122429
| Introduction|| |
Antibiotics are commonly used in animal husbandry. These substances are widely used at high dosage due to medicinal purposes for terms of therapy. They are also added to the dairy cattle's feed at low dosage among a long period for prophylactic and growth promotion purposes. In either cases, the antibiotic residue can be found in milk that is considered as a human health hazard in terms of allergic reactions and development of bacterial resistance, and causes failure in dairy fermentation industry. , Moreover, long-term antibiotic residue intake through food consumption may pose some specific adverse health effects such as gastrointestinal and liver implications.
Antibiotic residues in milk as well as other animal-originated foods are regulated by safety legislation and standard agencies in developed countries. Safe limits have been established by international organizations such as the Food and Agriculture Organization, World Health Organization, Codex Alimentarious and Scientific Committee of Food of European Union (EU). The antibiotics residue concentration in milk should not exceed the maximum residue levels suggested by the above-mentioned organizations. 
In Iran, despite the lack of monitoring program for detecting and determining the antibiotic residues in milk, several research studies were conducted on this issue in different regions of the country at both the farm and the market level. Based on these researches, a violation rate of 5-24% and 4.7% was reported for raw and thermal processed milk samples, respectively. ,, The appearance of antibiotics in milk has been attributed to season, type of market agent and location, in which the last one has been considered as the most influential factor. , It has been reported that weather conditions, specifically temperature, had a great impact on antibiotic residues in milk. Hence, season was considered as an independent variant when antibiotic detection in milk was of interest. ,
Besides, it is suggested that antibiotic substances tend to be destroyed in thermal processing.  Due to this, application of thermal processing and the intensity and severity of heat applied to milk during processing have been supposed to be influential parameters reducing the concentration of the antibiotic residues in milk.
In the urban society of Iran, distribution of raw milk is not legislated and processed milk in the forms of pasteurized and sterilized is quiet prevalent. The purpose of this study was to detect the prevalence of antibiotic contamination of commercially available milk and to assess the impact of season and different thermal processing on the prevalence of contaminated milk samples that carry an unacceptable level of antibiotic residues.
| Materials and Methods|| |
Collection of milk samples
Commercial milk samples (n = 187) were collected from the market during the cold season, from January to March 2012 (n = 90; 19 sterilized and 71 pasteurized) and warm season, from April to July of the same year (n = 97; 14 sterilized and 83 pasteurized). All the collected samples were produced in less than 1 week of the sampling date. The number of pasteurized products picked up in each sampling time were planned to be three to four times more than the UHT-sterilized samples. It was due to the distribution frequency and the sales ratio of 3-4 for pasteurized versus UHT-sterilized commercial products. Random sampling was conducted among the 24 various brands distributed in the Shahre-kord outlet from January to July 2012. Samples were sent to the food laboratory of the Shahre-kourd University of Medical Science, where the experiments were performed in less than 24 h from the sampling time.
Detection of antibiotic residues
The Eclipse 100-kit (Zeu-Inmunotec, Zaragoza, Spain) containing 96 wells micro- titer plate was used to detect the presence of antimicrobial residues. Detection is based on the microbial activity of Geobacillus stearothermophilus var. calidolactis, which can be inhibited in the presence of antibiotics. Sensitivity and specificity of the used detection kit for permitted level of different antibiotics in milk are shown in [Table 1]. ,,
|Table 1: Limits of detection for antibiotic residues in milk described for Eclipse test|
Click here to view
Sample preparation and test
Each well of the kits was filled with 0.10 mL of the milk sample. The wells were left at room temperature (approximately 20-24°C) for 1 h, allowing the milk to diffuse through the well. The wells were washed using 0.3 mL of distilled water for about three to four times. The wells were carefully sealed with an adhesive sheet and incubated at 65 ± 1°C for 150 min. At the end of the incubation period, the results were recorded. Based on the instruction of the kit, when the purple color of the media, which was filled in the wells completely, changed to yellow, the test was considered negative. However, if the color remained purple, it was counted as positive for antibiotic residue. However, when the color was slightly changed into yellow-purple color, the test result was recorded as partially positive, meaning that the concentration of the inhibitory substances was below the detection limit of the test.
The prevalence of antibiotic residues in milk in two different sampling times (winter and summer) and in two different groups of thermally processed commercial milk (pasteurized and UHT sterilized) was compared using the Statistical Package for Social Science software version-18. Chi-square test was conducted for comparison of the variants. All statistical tests for significance were performed two-tailed and interpreted at an alfa level of 0.05. Microsoft Office Excel version 2003 was used to provide diagrams.
| Results|| |
The prevalence of antimicrobial residues in the different target groups of milk samples is presented in [Table 2] and [Figure 1]. Sixty-four of a total of 187 examined milk samples (34.8%) were tested positive. The prevalence rate of the contaminated samples in pasteurized and UHT-sterilized milk was almost the same (33 vs. 34). It is noteworthy that there was no significant difference between the contamination rates of samples in both types of thermal processed milk when compared in terms of high or low antibiotic residue levels. Comparison of the contamination rate between the milk samples regarding the seasons, which they were produced in cold and warm season, revealed that there was no significant difference in the contamination rate. Thus, season had no impact in this regard (P > 0.05). However, distribution of the contamination rate during the total sampling time period within 7 months of the study showed that the highest contamination rate occurred in February (45%), followed by July (41%) and June (40%) [Figure 2]. Instead, a noticeable decline in contamination was observed during March to May.
|Figure 1: Antibiotic contamination rate among the thermally processed milk samples marketed in Iran in the year 2012|
Click here to view
|Figure 2: Contamination percentage of the processed milk samples with antibiotic residues during the study period (6 months‑2012)|
Click here to view
| Discussion|| |
Milk is one of the most notorious articles of our diet, which is designed to be just a food in nature. Because the most sensitive age groups of human beings, children, are always advised to have plenty of milk during a day, it is far more important to provide a safe supply of milk in the society as regards chemicals as well as microbiological aspects. Several studies conducted in different parts of the world showed that antibiotic residues could be found in cow milk, which is the first choice of milk for human use worldwide. However, the prevalence of contamination was reported to be widely different based on the location, season and milk type. The results of this study showed that 34% of the processed milk marketed in Shahre-kord was contaminated with antibiotic residues. Among them, 20% were shown to carry concentrations above the safe limit. This is the highest prevalence rate of such contamination compared with the reports of similar studies in different parts of the country.
Previously, a study conducted in Parsbad, Iran, from March to May 2009 showed that 14% of the raw milk samples was positive in antibiotic residues.  Based on the results of the present study, a contamination rate of 11.6% was observed during March-May. A higher contamination rate (24%) was stated by another study in Bostanabad, Iran, in which raw milk samples were tested for antibiotic residue from April to September 2010.  Consistently, in the present study, the prevalence of contamination during the latter period (April-June) was revealed to be 18.5%, which was more than that of the former period. It could be concluded that despite the insignificant effect of season, in the warm season of the year, along with increase in weather temperature, the prevalence of contaminated samples was raised. It is probably due to the higher incidences of diarrheal diseases in the chattels, which result in the enhancement of antibiotic administration to the cattle.
In a Kenyan study, difference between the dry and wet season had a great effect on the prevalence of antibacterial residues in milk in a way that contamination frequency during the dry season was found to be 1.6 times more than that of the wet season. The higher incidence of diarrheal diseases in the dry season was referred to as the cause of this effect.  Considering the results of two other studies carried out in African countries, a very different rate of prevalence (3.1% and 36%) was seen in this part of the world. Nevertheless, season was not considered in any of them as an influential parameter. , However, in a single Ghanaian study, it was shown that being in a dry or wet season, there was no significant difference in the prevalence of raw milk samples with antibiotic residues. In that study, during a whole study period, a prevalence rate of 35.5% was found among the tested samples. 
Some researchers have reported the rate of antibiotic prevalence in the countries nearby Iran (with almost climate condition). In Turkey, a contamination rate of 1.25-44% was found by several studies. Although seasonal difference was not focused in these pieces of research studies, they mostly presented a lower contamination rate than that reported in Iranian studies.  In Pakistan, a study showed that among 137 unprocessed market milk samples, 36.50% had ß-lactam antibiotic residues at the levels above EU maximum residues limits. 
It has been known that there is a slight decrease in the concentration of antimicrobial agents during thermal processing, especially in low-temperature-long-time treatment (conventional sterilization). ,,, In a field area of antibiotic residues in milk, some studies assessed the prevalence of contaminated samples between raw and thermal processed. ,, In Iran, raw milk has not been offered in the formal market and milk is just supplied as pasteurized or UHT sterilized. Based on the results of this study, there was no significant difference between the contamination rates of these products, which implies that antibiotic compounds used in veterinary practices in Iran resist the sever temperature of UHT processing. In line with these results, another Iranian study reported on the almost equal contamination rate of 4.7% and 5% for ultra-high temperature and raw milk samples with β-lactam antibiotics. 
The degree of contamination of milk and dairy products with antibiotics residues differs, depending on the level of legislation and effectiveness of the methods in different countries.  It is well known that milk containing antimicrobial residues poses several health hazards to consumers from which resistance to antimicrobials is regarded as a serious problem, with increasing evidences in recent studies in Iran.  Based on the results of a study in which Escherichia More Details coli and Streptococcus strains involved in mastitis infection were examined for antibiotic resistance, 52-84% of E. coli isolates and 13-20% of Streptococcus strains were found to be resistant to penicillin, oxy-tetracycline, streptomycin, erythromycin, and colistin.  Moreover, there are substantial evidences on the antibiotic resistance bacteria among human isolates in Iran. This, partly, could be due to the chronic antibiotic consumption via animal products. 
Unfortunately, in Iran, there is no national program to ensure that milk is free of antibiotic residue and monitoring of milk safety concerning chemical residues has been restricted to research studies. It seems that the contamination rate of milk by antimicrobials is being increased during the recent years relying upon the results of these studies. This results show that a control program for this issue needs to be developed.
Based on the results of this study, neither the season nor the type of thermal processing of the commercially marketed milks had a significant impact on the prevalence level of contaminated samples in the food outlet. However, a trend of increasing the prevalence level for residues can be observed with increasing the temperature through the warm seasons.
As a limitation of this study, visual justification of the test results might not provide a precise border of detection, in particular when sight color changes took place. Using the spectrophotometric devices would have a more accurate result. Furthermore, the possible interference of naturally occurring antimicrobial agents can be pointed out, which may impose falsely positive results. While the microbial inhibition method or some immunological methods are used for detection of drug, residues of such biases are inevitable. However, the advantage of such methods is that the presence of a wide spectrum of antibiotic agents in milk can be monitored.
| Acknowledgment|| |
This article is the result of an MSc thesis approved in the Isfahan University of Medical Sciences (IUMS). The authors wish to acknowledge the Vice Chancellery of Research of IUMS for the financial support, Research Project No. 390579.
| References|| |
|1.||Al-Wabel NA. The pharmacokinetics and milk residual behaviour of tylosin in lactating Najdi ewes. Iran J Vet Res Shiraz Univ 2008;9:23. |
|2.||Nisha AR. Antibiotic residues: A global health hazard. Vet World 2012;1:375-7. |
|3.||ECC. Council Regulation: No. 2377/90 of June 1990 laying down a commnity procedure for the establishment of maximum residue limits of veterinary medicine products in foodstuffs of animal prigi. Eur Comm 1990;224:1-8. |
|4.||Movassagh MH. Karami AR. Determination of antibiotic residues in bovine milk in Tabriz, Iran. Global Vet 2010;5:195-7. |
|5.||Movassagh MH. Detection of antibiotics residues in cow raw milk in Bostanabad region, Iran Res Opin Anim Vet Sci 2012;2:1-3. |
|6.||Movassagh MH. Study of antibiotics residues in cow raw milk by copan milk test in Parsabad region, Ardabil Province, Iran. Ann Biol Res 2011;2:355-9. |
|7.||Kang'ethe EK, Aboge GO, Arimi SM, Kanja LW, Omore AO, McDermott JJ. Investigation of the risk of consuming marketed milk with antimicrobial residues in Kenya. Food Control 2005;16:349-55. |
|8.||Aning KG, Donkor ES, Omore A, Nurah GK, Osafo EL, Staal S. Risk of exposure to marketed milk with antimicrobial drug residues in Ghana. Open Food Sci J 2007;1:1-5. |
|9.||Kathleen P. Martindale: The Complete Drug Reference. 32 th ed. Antibacterials. UK, London: The Pharmaceutical press; Vol 1. 1999; p. 115-32. |
|10.||Zeu-inmunotec C. Technical Report-E100. Test for detection of inhibitors in milk. Zaragoza Spain Rev 2003;1:1-38. |
|11.||Joint FAO/WHO Expert Meeting on Dietary Exposure Assessment methodologies for residues of veterinary drugs., Draft Report including Report of Stakeholder Meeting. Rome, Italy. Available from: http://www.who.int/foodsafety/chem/jecfa/FAO-WHO [Last accessed on 2011 Nov 7-11]. |
|12.||Addo KK, Mensah GI, Aning KG, Nartey N, Nipah GK, Bonsu C, et al. Microbiological quality and antibiotic residues in informally marketed raw cow milk within the coastal savannah zone of Ghana. Trop Med Int Health 2011;16:227-32. |
|13.||Brueggeman R, Druka A, Nirmala J, Cavileer T, Drader T, Rostoks N, et al. The stem rust resistance gene Rpg5 encodes a protein with nucleotide-binding-site, leucine-rich, and protein kinase domains. Proceedings of the National Academy of Sciences of the United States of America (PANS). 2008;105(39):14970-5. |
|14.||Kaya SE, Filazi, A. Determination of Antibiotic Residues in Milk Samples. Kafkas Univ Vet Fak Derg 2010;16(suppl-A)(S):31-5. |
|15.||Khaskheli M, Malik RS, Arain MA, Soomro AH, Arain HH. Detection of ß-Lactam antibiotic residues in market milk. Pak J Nutr 2008;7:682-5. |
|16.||Hassani M, Lázaro R, Pérez C, Condón S, Pagán R. Thermostability of oxytetracycline, tetracycline, and doxycycline at ultrahigh temperatures. J Agric Food Chem 2008; 56:2676-80. |
|17.||Tarbin JA, Bygrave J, Bigwood T, Hardy D, Rose M, Sharman M. The effect of cooking on veterinary drug residues in food: Nicarbazin (dinitrocarbanilide component). Food Addit Contam 2005;22:1126-31. |
|18.||Xuan R, Arisi L, Wang Q, Yates SR, Biswas KC. Hydrolysis and photolysis of oxytetracycline in aqueous solution. J Envir Sci Health B 2010;45:73-81. |
|19.||Rose MD, Farrington WH, Shearer G. The effect of cooking on veterinary drug residues in food. Food Addit Contam 1998;15:157-61. |
|20.||Adesiyun AA, Stoute S, David B. Pre-processed bovine milk quality in Trinidad: Prevalence and characteristics of bacterial pathogens and occurrence of antimicrobial residues in milk from collection centres. Food Control 2007;18:312-20. |
|21.||Bando E, Oliveira RC, Ferreira GM, Machinski M Jr. Occurrence of antimicrobial residues in pasteurized milk commercialized in the state of Paraná, Brazil. J Food Prot 2009;72:911-4. |
|22.||Al-Mazeedi H, Abbas A, Alomirah H, Al-Jouhar W, Al-Mufty S, Ezzelregal M, et al. Screening for tetracycline residues in food products of animal origin in the State of Kuwait using Charm II radio-immunoassay and LC/MS/MS methods. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 2010;27:291-301. |
|23.||Ebrahimi A, Kheirabadi KH, Nikookhah F. Antimicrobial susceptibility of environmental bovine mastitis pathogens in west central Iran. Pak J Biol Sci 2007;10:3014-6. |
|24.||Firoozeh F, Shahcheraghi F, Zahraei Salehi T, Karimi V, Aslani MM. Antimicrobial resistance profile and presence of class I integrongs among Salmonella enterica serovars isolated from human clinical specimens in Tehran, Iran. Iran J Microbiol 2011;3:112-7. |
|25.||Ghorashi Z, Ghorashi S, Soltani-Ahari H, Nezami N. Demographic features and antibiotic resistance among children hospitalized for urinary tract infection in northwest Iran. Infect Drug Resist 2011;4:171-6. |
[Figure 1], [Figure 2]
[Table 1], [Table 2]
|This article has been cited by|
||Identification and dietary exposure assessment of tetracycline and penicillin residues in fluid milk, yogurt, and labneh: A cross-sectional study in Lebanon
| ||Suzanne Kabrite,Christelle Bou-Mitri,Jessy El Hayek Fares,Hussein F. Hassan,Jocelyne Matar Boumosleh |
| ||Veterinary World. 2019; 12(4): 527 |
|[Pubmed] | [DOI]|
||Human health risk assessment of antibiotic resistance associated with antibiotic residues in the environment: A review
| ||Yujie Ben,Caixia Fu,Min Hu,Lei Liu,Ming Hung Wong,Chunmiao Zheng |
| ||Environmental Research. 2019; 169: 483 |
|[Pubmed] | [DOI]|
||Assessment of Raw Cow Milk Quality in Smallholder Dairy Farms in Pemba Island Zanzibar, Tanzania
| ||S. H. Gwandu,H. E. Nonga,R. H. Mdegela,A. S. Katakweba,T. S. Suleiman,R. Ryoba |
| ||Veterinary Medicine International. 2018; 2018: 1 |
|[Pubmed] | [DOI]|
||Antibiotic residues in meat, milk and aquatic products in Shanghai and human exposure assessment
| ||Hexing Wang,Lingshuang Ren,Xin Yu,Jing Hu,Yue Chen,Gengsheng He,Qingwu Jiang |
| ||Food Control. 2017; |
|[Pubmed] | [DOI]|
||Dietary exposure to tetracycline residues through milk consumption in Iran
| ||Fathollah Aalipour,Maryam Mirlohi,Mohammad Jalali,Leila Azadbakht |
| ||Journal of Environmental Health Science and Engineering. 2015; 13(1) |
|[Pubmed] | [DOI]|
||Determination of antibiotic consumption index for animal originated foods produced in animal husbandry in Iran, 2010
| ||Fathollah Aalipour,Maryam Mirlohi,Mohammd Jalali |
| ||Journal of Environmental Health Science and Engineering. 2014; 12(1): 42 |
|[Pubmed] | [DOI]|