Antibiotic residues and mycotoxins in raw milk in Punjab (India): a rising concern for food safety

Abstract

The present study was envisaged with an aim to determine the occurrence of antibiotic residues; enrofloxacin, oxytetracycline, penicillin G, sulphamethoxazole and chloramphenicol as well as mycotoxins; aflatoxin M1 and ochratoxin A in raw milk samples collected from individual animals from dairy farms located in 9 districts of Punjab, India. A total of 168 raw milk samples were collected and analysed using commercially available competitive Enzyme linked immunosorbent assay kits. Out of these, 19 (11.3%) and 9 (5.4%) samples were found positive for antibiotic residues and mycotoxins, respectively. The milk samples were positive for enrofloxacin (4.8%), oxytetracycline (3.0%), penicillin G (3.0%) and sulphamethoxazole (0.6%) residues. The percentage of samples found above maximum residue limit established by Europen Union (EU)/Codex Alimentarius Commission (CAC) were 1.7%, 1.2% and 0.6% for enrofloxacin, oxytetracycline and penicillin G residues, respectively. Aflatoxin M1 was detected in 5.4% of monitored milk samples with 1.2% samples exceeded the established maximum levels of EU but were below the maximum levels established by CAC. The occurrence of antibiotic residues and mycotoxins in raw milk samples above maximum limits is a rising concern for food safety due to possibility of health risks to the consumers.

Introduction

Antibiotics are used in dairy industry for prophylactic and therapeutic purpose (Oliver et al. 2011). The use of antibiotics particularly as intramammary infusions and non adherence to withdrawal periods results in occurrence of their residues in milk (Sandholm et al. 2009; Gomes and Henriques 2015). Dairy animals also get exposed to antibiotics when low doses of antimicrobials are added in feed to act as animal growth promoters for improving feed conversion efficiency (Hao et al. 2014). The consumption of milk containing antibiotic residues may lead to potential health risks to consumers (Moudgil et al. 2017). They may cause allergic reactions, disruption of intestinal flora, carcinogenicity, mutagenicity, nephropathy, hepatotoxicity and bone marrow toxicity etc. (Nisha 2008; Madden et al. 2005). Antibiotic residues in milk also inhibit starter culture required for manufacturing of dairy products resulting in significant economic losses to producers and manufacturers of milk and milk products (Kebede et al. 2014; Padol et al. 2015). Besides above mentioned effects, the rising issue of prime concern of non-prudent use of antibiotics in animals is the emergence of antibiotic resistant bacteria, capable of causing life threatening infections in humans (Economou and Gousia 2015; Marshall and Levy 2011). According to international agencies like World Health Organization and European Centre for Disease Control, antimicrobial resistance is a major public health issue and a big challenge for the policy makers and health care providers to tackle the problem (Kumar et al. 2013).

Mycotoxins are toxic secondary metabolites naturally produced by moulds and are well known to cause toxicities in humans and animals (Reddy et al. 2010; Shetty and Jespersen 2009). These toxins are formed on crops during harvest, storage, processing or feeding and their occurrence in foods is not completely preventable even after following good manufacturing practices (Volkel et al. 2011). The methods used for preparation and storage of dairy animal feedstuffs such as grains and silage lead to contamination with mycotoxins and when animals ingest contaminated feed, mycotoxins are metabolized, bio-transformed and transferred to milk posing a risk to human health (Becker-Algeri et al. 2016).

Aflatoxins are a group of structurally related toxic compounds produced by mould Aspergillus flavus and A. parasiticus. About 1–3% ingested aflatoxin B1 is converted into aflatoxin M1. The presence of aflatoxin M1 in milk poses a major risk for humans, especially children, as it has immunosuppressive, mutagenic, teratogenic and carcinogenic effects (Sefidgar et al. 2011). Ochratoxins are metabolites synthesized mainly by toxigenic species of Aspergillus and Penicillium. The consumption of milk contaminated with ochratoxin A represents a potential risk for human health as it has nephrotoxic, hepatotoxic, carcinogenic and teratogenic effects (Denli and Perez 2010). The health and food safety related characteristics of antibiotics and mycotoxins targeted in the present study are listed in Table 1.

Table 1.

Health and food safety related characteristics of antibiotics and mycotoxins targeted in the present study

Compound	Group	Major adverse effects in humans	Withdrawal period (days) in cattle	Milk discard time	Maximum residue limit/maximum level (ng/mL)
Enrofloxacin	Fluoroquinolone	Neurotoxic, retinal degeneration, hypersensitivity reactions	–	–	100
Oxytetracycline	Tetracycline	Inhibition of bone growth, teeth staining, hepatotoxicity	28	–	100
Penicillin G	Beta lactam	Hypersensitivity reactions, gastrointestinal disturbances	10	3 days	4
Sulphamethoxazole	Sulphonamide	Renal toxicity, blood dyscariasis, hypersensitivity reactions	10	96 h	100
Chloramphenicol	Amphenicol	Bone marrow depression, aplastic anaemia	–	–	0.3
Aflatoxin M1	Mycotoxins	Immunosuppressive, mutagenic, teratogenic, carcinogenic	–	–	0.05
Ochratoxin A	Mycotoxins	Nephrotoxic, hepatotoxic, carcinogenic, teratogenic	–	–	2

Due to wide consumption of milk throughout the world, the presence of antibiotic residues and mycotoxins in milk pose a major threat to the health of consumers. Thus, the present study was envisaged with an aim to monitor the presence of antibiotic residues and mycotoxins in raw milk in Punjab, India to determine its safety for human consumption.

Materials and methods

Collection of milk samples

A total of 168 milk samples were collected from individual animals from dairy farms located in 9 districts (Ludhiana, Sangrur, Barnala, Moga, Bhatinda, Gurdaspur, Ferozepur, Kapurthala and Amritsar) of Punjab (India) (Fig. 1). All the samples were transported to laboratory on the same day of collection under chilled conditions and stored at − 20 °C. The samples were analyzed within 48 h of collection.

Fig. 1.

Districts (encircled) of Punjab state targeted in the present study

Analysis of antibiotic residues and mycotoxins

The milk samples were analyzed quantitatively for the presence of residues of enrofloxacin, penicillin G, oxytetracycline, sulphamethoxazole and chloramphenicol as well as mycotoxins; aflatoxin M1 and ochratoxin A. Chloramphenicol is banned in food producing animals in India and the purpose of its monitoring was to determine any usage of the drug in dairy cows.

The antibiotic residues and ochratoxin A were determined using commercially available MaxSignal^® competitive Enzyme linked immunosorbent assay (ELISA) kits procured from Bioo Scientific Corporation, Austin, USA. The aflatoxin M1 was screened using Aflatoxin M1 sensitive ELISA kits procured from Europroxima BV, Arnhem, Netherlands. The reagents and samples were prepared according to the manufacturer’s instructions given in the instructional manual.

ELISA protocol was followed according to the manufacturer’s instructions. The results were calculated by obtaining the optical density (O.D.) values and calculating the percent mean relative absorbance by using the following formula.

$$\text{Relative absorbance}\left(\%\right)=\frac{\text{Absorbance of standard or sample}}{\text{Absorbance of zero standard}}\times 100$$

Finally a standard curve was plotted between the standard concentrations and mean relative absorbance (%).

Statistical analysis

SPSS (version 22.0 Armonk, NY: IBM Corp.) was used to calculate range as well as mean ± SD of antibiotic residues and mycotoxins in milk samples using descriptive statistics.

Results and discussion

A total of 19 (11.3%) samples were found positive for antibiotic residues with mean ± SD of 58.3 ± 52.7. Out of 19 samples, 8 (4.8%), 5 (3.0%), 5 (3.0%) and 1 (0.6%) samples were positive for enrofloxacin, oxytetracycline, penicillin G and sulphamethoxazole, respectively. Three (1.7%), 2 (1.2%) and 1 (0.6%) samples for enrofloxacin, oxytetracycline and penicillin G, respectively were found above MRL established by EU/CAC. The chloramphenicol residues were not detected in any of the sample. The percentage of positive samples, determined mean ± SD, range as well as samples above MRLs are presented in Table 2.

Table 2.

Antibiotic residues detected in raw milk samples in Punjab, India

Antibiotic residues	n/N	% Positive	Range (ng/mL)	Mean ± SD (ng/mL)	MRLa (ng/mL)	Samples above MRL
Enrofloxacin	8/168	4.8	17.1–161.2	87.9 ± 44.0	100	3
Oxytetracycline	5/168	3.0	12.3–118.6	70.7 ± 45.9	100	2
Penicillin G	5/168	3.0	0.9–5.2	2.2 ± 1.5	4	1
Sulphamethoxazole	1/168	0.6	0–39.6	–	100	Nil
Chloramphenicolb	0/168	–	–	–	0.3	Nil

^aMRL established by EU/CAC

^bIn MRL column the indicated value is MRPL

For aflatoxin M1, a total of 9 (5.4%) samples were found positive with mean ± SD of 0.03 ± 0.05. Two (1.2%) samples exceeded the maximum levels (ML) established by EU (0.05 ng/mL), but were below the ML established by CAC (0.5 ng/mL). None of the sample was found positive for ochratoxin A. The percentage of positive samples, mean ± SD, range as well as samples above maximum levels are presented in Table 3.

Table 3.

Mycotoxins detected in raw milk samples in Punjab, India

Mycotoxins	n/N	% Positive	Range (ng/mL)	Mean ± SD (ng/mL)	ML (ng/mL)	Samples above ML
Aflatoxin M1	9/168	5.4	0.006–0.13	0.03 ± 0.05	0.05/0.5a	2
Ochratoxin A	0/168	–	–	–	2b	Nil

^aML (Maximum levels) established by EU-0.05 ng/mL, Maximum levels established by CAC-0.5 ng/mL

^bML established by EU

In the present study, the most commonly observed antibiotic residues in milk samples were enrofloxacin, oxytetracycline, penicillin G and sulphomethoxazole. The previous studies conducted in India as well as in various parts of world also reported the occurrence of enrofloxacin, oxytetracycline, penicillin G and sulphamethoxazole residues in milk. Sudershan and Bhat (1995) revealed the presence of oxytetracycline residues in 73% of individual animal milk samples collected from Hyderabad, India. Elizabeta et al. (2011) screened 497 raw milk samples and reported the presence of sulphonamides, tetracyclines and quinolones residues from Europe. Mokhtari et al. (2013) reported the presence of beta-lactam residues in 32.9% of milk samples in Iran, whereas Abebew et al. (2014) reported the presence of oxytetracycline and penicillin G residues in milk samples in Ethiopia. In the studies carried out by Moharana et al. (2015) in India and Ahlberg et al. (2016) in Kenya, 16.8% and 2.5% of milk samples were found positive for enrofloxacin and sulphonamide residues, respectively. Thus, all the aforementioned studies depicted the presence of one or more antibiotics in milk samples including enrofloxacin, oxytetracycline, penicillin G and sulphomethoxazole and are in accordance with the findings of the present study.

The chloramphenicol residues were not detected in any of the milk sample screened in the present study. The similar findings were observed by Elizabeta et al. (2011) where none of the analysed milk samples were found positive for chloramphenicol residues. During the past decade, the presence of chloramphenicol residues has been reported in milk and dairy products mainly from European countries; Estonia, Latvia and Lithuania (RASFF 2011). Due to suspected carcinogenicity and linkages with the development of aplastic anaemia in humans, chloramphenicol has been banned for use in food-producing animals in many countries. For chloramphenicol residues in foods of animal origin including milk, the EC established a minimum required performance level (MRPL) of 0.3 ng/mL to ensure consumers safety (EC 2002).

The various studies conducted in the past also reported the presence of aflatoxin M1 in milk samples. Boudra et al. (2007) reported the occurrence of aflatoxin M1 in 3.4% of raw milk samples in France whereas in a study conducted by Elzupir et al. (2009) in Sudan, 60% of milk samples were found to contain aflatoxin M1. Iqbal et al. (2011) and Rahimi et al. (2010) also reported the occurrence of aflatoxin M1 in milk samples in Pakistan and Iran, respectively. The mean concentration of aflatoxin M1 in both studies was found to be approximately 0.04 ng/mL, which was almost comparable to the findings of current study. Where as in another study carried out by Bilandzic et al. (2016) the mean concentration of aflatoxin M1 in milk samples were ranged from 0.0031 to 0.0041 ng/mL which was very less as compared to concentration reported in the present study. In contrary to aflatoxin M1, ochratoxin A was not detected in any of the milk sample in the present study. However, the various studies conducted in past have reported the occurrence of ochratoxin A in milk samples (Boudra et al. 2007; Younis et al. 2016).

The present study determined the occurrence of antibiotic residues and mycotoxins in raw milk in Punjab, India, where some of the analysed samples were found above maximum levels. The occurrence of antibiotic residues and mycotoxins in milk beyond a given residue levels is a rising concern for food safety as these contaminants pose hazards to the health of consumers.

The continuous efforts have been made by Government of India in the past for controlling the usage of antibiotics in food animals such as National Policy for Containment of Antimicrobial Resistance, 2011, introduction of new category of drugs H1 in the Drugs and Cosmetics Rules, 2013, Food Safety and Standards (Contaminants, Toxins and Residues) Amendment Regulations, 2017 etc. Along with these by adopting good husbandry and management practices, occurrence of antibiotic residues may be prevented in milk. These include prevention of diseases in dairy animals to minimize the use of antibiotics (by employing good managerial and biosecurity measures at the farm, early and accurate diagnosis of sick animals in the herd, timely and routine vaccination of all animals in the herd), segregation and testing of newly purchased animals before introducing them into milking herd, milking of sick and treated animals at last and their milk should not be mixed with milk of healthy animals, maintenance of good treatment records of antibiotic treated animals, timely training of persons involved in the treatment of dairy cattle and regular monitoring of antibiotic residues in milk using rapid screening tests,

The occurrence of mycotoxins in the milk can be reduced by controlling fungal contamination of cattle feed at pre-harvest stage by using fungicides for preventing fungal growth, removal of cereal grains and seeds contaminated with fungal spores, use of fungal resistant varieties of crops or at post-harvest stage by storing grains at low temperature and humidity for preventing fungal growth thus inhibiting mycotoxin production, use of approved insecticides for preventing insect infestation in stored bulk grains and detoxification of mycotoxins formed in stored feed and grains.

The occurrence of antibiotic residues and mycotoxins in milk is a matter of public health concern and causes severe economic loss to the dairy industry. There is a need to generate awareness among dairy farmers for adopting good managerial practices as well as judicious use of antibiotics in dairy cattle to prevent occurrence of antibiotic residues and mycotoxins in milk, thus, preventing unacceptable health risks to consumers. In addition to this, there is a need for framing and implementing strict regulations/legislations for controlling the occurrence of antibiotic residues in foods of animal origin.