Reduction of Antibiotic residue in milk through the use of cost effective Ethno-Veterinary Practices (EVP) for cattle health management

This study demonstrated that antibiotic residue in milk can be reduced when dairy farmers use Ethno-veterinary Practices (EVP) based on herbal alternatives to prevent and cure common clinical conditions in cattle instead of antibiotics. Of the 220 farmers selected for the study, 140 were trained and motivated to use validated herbal formulations, 80 were kept as control. Milk samples from the selected farmers (except Thirukanurpatti milk society) tested positive for antibiotic residue in the baseline survey. One year after interventions, the milk from 123 (87.86%) farmers out of 140, were without any detectable antibiotic residue, while samples from 11 farmers (7.85%) were low positive for either Beta-lactams or sulphonamides and 6 (4.29%) were positive for Beta lactams and/or sulphonamides. These 17 (11 + 6) farmers had used antibiotics along with herbal formulations. The milk samples from the control groups were positive for beta lactam and sulphonamide. There was suggestive significance of change in knowledge, attitude and practice of EVP among the farmers from Kerala and Tamil Nadu. A progressive reduction in the incidence of mastitis, enteritis, repeat breeding and cowpox were observed from 2016 to 2019 among the cows treated with EVP. Use of herbal alternative also resulted in a significant reduction in health care expenditure of cattle.


Introduction
There are many reports of antibiotic residues in milk [1][2][3][4][5][6][7][8][9][10][11][12]. It also causes side effects and allergic reactions on people who consume animal products such as milk and meat [13][14][15][16][17][18]. In many countries AMR is a threat to public health and the world is looking for safer alternatives through herbs. It is also predicted that the global consumption of antimicrobials will go up to 67 % from 2010 to 2030 [16, 18, and 20]. Tackling a Crisis for the Health and Wealth of Nations was published on the request of the UK Prime Minister to address the growing global problem of It is predicted that drug-resistant infections will kill an extra 10 million people a year worldwide by 2050 and the total cost of antimicrobial resistance on world Gross Domestic Product (GDP) will be $ 100 trillion [17,21]. One of the immediate challenges to reduce AMR is to limit the use of antibiotics in human and animal health management. As the antibiotic residues finding their way into the food chain, there is an urgent need to focus on reducing the use of antibiotics in veterinary practice. With limited or no strategic implementation of regulatory policy on controlling the misuse of antibiotics in dairy sector in India, the focus needs to be shifted to work with farmers, animal keepers and veterinarians in identifying approaches and options to use ethno-veterinary practices based on natural plant products to control microbial diseases. There are reports on the documentation of ethno-veterinary practices from several parts of India . Ethno-veterinary medicine has also been suggested as alternative to antibiotics [14, 15, and 20] The University of Trans-disciplinary Health Sciences and Technology (TDU) along with Tamil Nadu Veterinary and Animal Sciences University (TANUVAS) have documented Ethno-veterinary practices from 24 locations in 10 states of India and rapidly assessed them using Ayurveda and established that 353 formulations out of 441 are safe and efficacious [39] There are reports of validation of herbal formulations for Mastitis [40][41][42][43] and reduction of retention of placenta (ROP) [44]. This study reports change in knowledge, attitude and practice of the ethno-veterinary practices (EVP) among farmers for selected animal health conditions and significant reduction of the antibiotic residue(s) in the milk after using cost effective ethno-veterinary medicine for prevention and cure of mastitis, foot and mouth disease (FMD), diarrhoea, udder pox, repeat breeding, bloat, indigestion and maggot wounds in cattle.

Antibiotic residue analysis of the Market samples
Thirty five brands of milk sold in the Market were tested for presence of antibiotic residue. Twenty two had Beta lactams and Sulphonamides, 10 had only Sulphonamide. One brand had 4 antibiotics (Beta lactams, Sulphonamides, Gentamicin and Tetracycline) residues and 2 did not have any antibiotic residue ( Table 4).

Baseline
The milk sample from 11 milk Union's collection centres had Quinolones, Beta lactams, Tetracycline and Sulphonamides as residues ( Table 5). Gentamicin, Streptomycin, Neomycin and Chloramphenicol were absent. Quinolones, Beta lactams, Gentamicin, Sulphonamides were present as residues in the milk samples from farmers selected for the study except the milk from Thirukanurpatti, ( Table 6). Tetracycline, Streptomycin, Neomycin, Chloramphenicol were absent.

End line survey
The end line survey indicated that the milk samples from 123 farmers out of 140 (87.86%) were without any detectable antibiotic residue(s), 11 (7.85%) samples showed low positive of Beta lactams or sulphonamides and 6 (4.29%) showed positive to antibiotic residue(s) of Beta lactams and or sulphonamides (Fig 1, table  7).

Reduction of incidence of disease conditions in cattle in the area selected for the studies when EVP is used
The incidence of mastitis was 66 in 2016, reduced to 37 in 2018 and to 11 in 2019 among the cows of farmers selected for the study. The overall reduction of mastitis from 2016 to 2019 is 83.3%. There is also reduction in the incidence of enteritis (63.6%), repeat breeding (96%) and cowpox (100 %) from 2016 to 2019 ( Table 8)

The control group
The milk samples from control group had Beta lactams and/or sulphonamide (Table 9). Tetracycline, Streptomycin, Neomycin, Chloramphenicol Gentamicin were absent

Production loss
The average milk production loss was 15 to 22.2 litres for 6 days when treated with western medicines. The details are given in the table 11. However, the loss is only 0.2 to 0.5 litres when treated with EVP (Table 12). In the case of FMD there is no loss but sometimes there is an increase of production by 0.7 litres

Discussion
This study shows that veterinarians, farmers and para-vets are using antibiotics and there is antibiotic residue in the milk [1][2][3][4][5][6][7][8][9][10][11][12]. This indicates that farmers/veterinarians do not practice health ministry's withdrawal time [2, 11, 13, 14, 15 and 20]. The rule states that the antibiotics used for therapeutic purpose in animals should be labeled with the withdrawal periods i.e. "milk and eggs should be kept out of human food minimum for one week, poultry and meat products 28 days, fish and other marine products 500 degree days" (Health ministry: withdrawal

The intervention
The herbal formulations used for Mastitis, Foot and mouth disease (FMD), Diarrhoea, Udder Pox, Repeat Breeding, Bloat, Indigestion and Maggot wound, not only cure these clinical conditions but also prevent these diseases. This is evident from the substantial reduction of incidence of mastitis (83.8%), enteritis (63.6%) repeat breeding (95.8%) and udder pox (100%). The reduction of antibiotic residue is significant (87.86%). There is limited number of such studies done on the role of EVP (herbal formulation) in animal health conditions. [14,15,[39][40][41][42][43][44]. The residue in the milk from the control group after one year is largely Beta lactams and sometimes Sulphonamides indicating the widespread use of these groups of antibiotics. This intervention also has increased the knowledge, aptitude and practice among the farmers and awareness about the antimicrobial residue in the animal products and associated antimicrobial resistance.

Economic benefit
Misuse of the drug, non-adherence to withdrawal period, economic reasons, ignorance, lack of medication records are the major reasons of appearance of veterinary drug residues in the animal products [45][46][47]. The combination of improved management practices with the use of herbal formulation in animal health care evidently is cost effective. It also minimised the production loss and improved quality of dairy milk. This work also indicates the reduction of use of antimicrobials for management of cattle health and their residue in the milk. This is a step towards renewed interest in ethno-veterinary practices (herbal alternatives) among the national dairy development of India initiatives and enterprises, which face the problems related to antimicrobial residue in the dairy milk due to misuse of antibiotics.

Material and Methods
Two hundred twenty farmers were selected from eight milk societies from Kerala state, two societies in Karnataka and one in Tamil Nadu. The names of the selected milk societies and number of farmers selected for the study are given in table 13. One forty farmers were included in the intervention study group and 80 in the control group.

Table 13 Show the name of the milk society, number of farmers selected as interventions and controls group
Knowledge, attitude, and practice (KAP) survey of Ethno-veterinary practices (EVP) among the selected farmers, were undertaken. The baseline and end line surveys were conducted using a format with 1 to 10 scales and personal interview.
Thirty five market samples in triplicate were collected and tested for the presence of antibiotic residue(s). The pooled milk samples from 11 milk union collection centres and 220 farmers before and after intervention were tested for the presence of antibiotic residue(s) using Unisensor (Belgium). farmers were monitored randomly. The field support was given whenever it was necessary. Hand books were printed in local languages (Kannada, Malayalam and Tamil) and distributed to the selected farmers for reference.

Conclusion
The use of herbal preparation as alternatives to antimicrobials shows significant reduction in the antibiotic residues in the milk from cattle and increased financial saving for the farmers. Adopting the Ethno-veterinary Science and Practices to combat infectious diseases in livestock has been identified and tested as a key game changer in rationalising the use of antibiotics in veterinary health care and reducing antibiotic residue(s) in milk.

Acknowledgement
We acknowledge Department of Science and Technology Government of India (DST GOI SEEDS project) for financial support for this work. We also thank MILMA, BAMUL and AAVIN and all employs of the Union collection centres and farmers for their cooperation to conduct this study. We express our sincere appreciation to the Mr. Narayana Kaimal of KCT, Chakkampuzha, Mr Abhilash Raju, Mrs Anitha Nagaraj and Aditye Nair for the help during the field study. Our thanks to Dr K P Suresh from NIVEDI for the statistical analysis

Author Contributions
Field study at Kerala conducted by M N B Nair, Tamil Nadu by N Punniamurthy and Karnataka by Kumar SK and all authors approved the final version of the manuscript.

9.Conflicts of Interest
The authors declare that they have no Conflicts of Interest.