Management of Mastitis Using Trans-Disciplinarily Validated Ethno-Veterinary Practices

Abstract

Mastitis is an inflammation of the mammary gland generally associated with intra-mammary infection (IMI) with certain microbes. Though common mastitis-causing bacteria are the main cause of mastitis, other organisms like viruses, fungi, yeast, certain microscopic algae (Prototheca spp.) and even physical injury might cause mastitis. The University of Transdisciplinary Health Science and Technology (TDU) conducted a participatory documentation of ethno-veterinary practice (EVP) and transdisciplinary assessment involving Siddha-Ayurveda, and Western pharmacology and established the safety and efficacy of EVP. In-vitro antimicrobial activity, clinical and molecular docking studies indicated anti-inflammatory and anti-microbial properties of EVP formulation for mastitis. The microbiome of milk from the cows with clinical mastitis indicates that after 6 days of treatment with herbal formulations, the average abundance of microbes causing mastitis was reduced substantially. Microbiome abundance in the milk of EVP-treated cows is almost similar to that of the control after 6 days. However, microbiome abundance in the milk from the antibiotic-treated cow varied substantially from the milk of the control. The clinical intervention with EVM preparations on 181,252 cows in a multicentric field study for a period of 5 years on the efficacy of EVP for subclinical, clinical and chronic mastitis with multifactorial aetiology resulted in a clinical recovery in 84.9% of cases.

1. Introduction

Mastitis is a perpetual problem of all milk-producing animals. Even though common bacteria are the main cause of mastitis, other organisms like Mycoplasma spp. [1, 2] yeast, [3] viruses and fungi might also be associated with the syndrome [4, 5]. Fungi such as Aspergillus fumigatus; A. midulus; Candida spp.; Trichosporon spp. and certain microscopic algae (Prototheca spp.) can cause mastitis. Physical or chemical injuries to the mammary region, poor hygiene and/or trauma also cause mastitis. Mycotoxins in cattle feed can suppress the immune system deepening negative energy balance and increasing the risk of metabolic disorders and infectious diseases [6, 7]. Indeed, other causes of immunosuppression also may be present.

1.1 Economic loss

The prevalence of subclinical mastitis in bovines in India ranges from 9.9 to 86.9%. The average drop in income due to mastitis is from Rs. 306–335 to 413–458 per cow per day [8]. Mastitis also causes permanent loss of productive ability of the cattle, reduces milk by 21% and butter and fat by 25%. The economic losses in mastitis are multifactorial and result a decrease in milk production, poor quality of milk, premature culling, higher veterinary service and labour cost [9, 10]. Subclinical mastitis accounts for 60–70% of the total economic losses by all mastitis types and thus causes three times more production losses than clinical mastitis [11, 12, 13]. In the last five decades, the loss due to mastitis in India has increased 115 times. The economic loss due to mastitis alone was 71,655 million Indian Rupees in the year 2012. The prevalence of the mastitis increased to more than 60% [14].

1.2 Conventional mastitis management and AMR

Non-infectious mastitis can usually be resolved without the use of antibiotics. Mastitis therapy protocols nearly always include antibiotics. The proportion of defined annual daily doses of antimicrobial per cow for mastitis alone administered on conventional dairy farms is 86.3% (Figure 1) [15]. The misuse of antimicrobials led to residue in animal products such as milk and meat, the development of the Antimicrobial Resistance (AMR) which is a threat to public health [16, 17].

Figure 1.

Daily doses of antimicrobial per cow for mastitis alone per year administered on conventional dairy farms [15].

2. Documentation and rapid assessment of EVP

The Trans-disciplinary University (TDU), more than a decade ago, developed a participatory documentation process of ethno-veterinary practice and a rapid assessment methodology to establish the safety and efficacy of EVP [18, 19, 20]. The veterinary dimension of Ayurveda or Siddha is used as a tool for studying folk/ethno-veterinary knowledge to assess the safety and efficacy of folk/EVP knowledge [21]. There is an inherent relation between the classical textual knowledge such as in Ayurveda or Siddha, which is classical Indian veterinary science and the folk knowledge which forms the empirical foundation of theory and practice in the classical texts of Ayurveda and Siddha. This relationship forms the basis of the rapid method developed by TDU. The assessment is an essentially participatory method by cross-cultural triangulation of local experience with Ayurveda, Siddha as well as Western pharmacology. It involves the community, vaidyas (healers) and medical practitioners from various systems of medicine, pharmacologists, botanists and the facilitators like non-government organisations (NGOs) and People’s Organisations [18]. Recording of the use of EVP from 24 locations in 10 states was done and rapidly assessed using Ayurveda/Siddha and established that 353 formulations out of 441 are safe and efficacious [20].

3. Standardisation of the ethno-veterinary formulation and the SOP for subclinical, clinical and chronic mastitis

3.1 EVP treatment protocol for subclinical and clinical mastitis

See (Table 1).

No.	Botanical name	Common name	Parts used	Figure	Amount
1	Aloe vera	Aloe	Leaves	2	250 g
2	Curcuma longa	Turmeric	Rhizome	3	50 g
3	—	Slaked lime	Powder	4	10 g
4	Cissus quadrangularis*	Veldt grape, winged tree bine	Stem	5	Pieces 2

Table 1.

The botanical, local names and the ratio of ingredients in the formulation.

*

In case of chronic mastitis.

See (Figures 2–5).

Figure 2.

Aloe vera.

Figure 3.

Curcuma longa–rhizome.

Figure 4.

Calcium hydroxide.

Figure 5.

Cissus quadrangularis.

3.2 Preparation and application

Method 1: A fine paste of Aloe vera leaves, turmeric and calcium hydroxide (Figure 6) is applied externally all over the udder with a circular massage after washing and removing the milk completely from the udder (Figures 7–9). Repeat the application after every 1 hour for ten times in a day for 5 days. Feed the affected animal with two lemons twice daily for 5 days besides the external application.

Figure 6.

Preparation of the herbal formulation for mastitis.

Figure 7.

Herbal formulation for mastitis.

Figure 8.

Application of the herbal formulation for mastitis on the udder.

Figure 9.

Application of the herbal formulation for mastitis on the udder.

Method 2: A combination of Aloe vera 250 gr, Curcuma longa 50 gr. and calcium hydroxide 10 gr blended to form a reddish paste. Mix 75 grams of this paste with 150 ml sesame (Gingelly) or mustard oil. Clean the mastitis-affected udder with water and remove the milk completely from the udder. Dry the udder well and apply the mixture with your hand on all quarters (affected as well as non-affected) thoroughly with a circular massage. Apply this paste five times a day for 5 days. Cut two lemons into halves and feed the affected cattle twice daily for 5 days. Both these protocols can be used during dry periods to prevent mastitis [22].

3.3 Blood in milk

A paste of curry leaves (30 g) and jaggery (100 g) is also given to the animals having clinical mastitis with blood in milk till the condition is resolved.

Method 3: Chronic mastitis: A combination of Aloe vera 250 grams, Curcuma longa 50 grams, calcium hydroxide 10–15 grams and two pieces of Cissus quadrangularis blended to form a reddish paste. Mix 75 grams of this paste with 150 ml of water to dilute it. Clean the mastitis-affected udder with water and milk the animal completely. Apply the mixture with your hand on all quarters (affected as well as non-affected) thoroughly with a circular massage. Apply this paste 8–10 times a day for 15 days or till the condition is resolved. You can also use 150 ml sesame (Gingelly) or mustard oil instead of water and apply five times a day till the condition is cured. Cut two lemons into halves and feed the affected cattle twice daily for 7 days.

4. Transdisciplinary assessment of the EVP for mastitis

4.1 Ayurveda biology conceptual framework of the pathophysiology and management of mastitis in dairy animals

“Sthanya (breast) is considered as upadhatu of Rasa dhatu (lymphatic system) and mainly consists of Twak (skin), Kandara (connective tissues), Mamsa (muscle tissue), Rasavahini (lacticiferous ducts) and Granthis (glandular lobes or acini). The disease afflicting to all these components is Sthanavidhradhi (mastitis). Sthana is also a site for Shotha (inflammation), Vrana (ulcers), Granthis (benign tumours) and Arbuda (malignant tumours).” [23] The dosha (Vata, pitha and Kapha) signifies the dushanasvabhàva (the nature of vitiation) and is the basic triggering factor in the disease causation. These, in turn, vitiate the tissue elements such as blood, muscle fat, etc., and manifest in the form of diseases. The same doŝa can produce several diseases [23].

Aloe vera L. (Kumari) has healing properties. It has the properties of Deepana (digestive), Pachana (carminative), cold in potency (pacifies pitta), Pitta and Rakthashamaka (pacifies Pitta and Raktha), Krimighna (reduce microbial load), Vranashodaka (cleanses wounds), Vranaropaka (promotes wound healing) and Shothahara (anti-inflammatory) [23].

Curcuma longa (Haridra) is Deepana (digestive), Pachana (carminative), Uttejaka (stimulant), Rakthashodaka (blood purifier), Shothahara (anti-inflammatory), Krimighna (anti-microbial), Vranashodaka cleanses wounds and Vranaropaka (promotes wound healing). Calcium hydroxide has the properties of Srotoshodaka(channel cleanser), Shothahara (anti-inflammatory), Raktashodhaka (blood purifier), Vranashodaka (Wound cleanser) and Vranaropaka (promotes wound healing) [23].

Mastitis can be compared with sthanavidhradi as described in Ayurveda which is a disease of pitta origin. The formulation consists of Aloe vera, Curcuma longa and Calcium hydroxide which are potent pitta and Raktha shamaka (Pacifies pitta humour). The formulation is Agni deepana (digestive), Amapachana (Carminative), Krimihara (reduce microbial load), Puti rodhaka (anti-infective) very good Shothahara (anti-inflammatory), Srotoshodaka (detoxifier), Vranashodaka (Wound cleanser) and Vranaropaka (promotes wound healing). Therefore, Mastitis (Sthanavidradhi) can be efficiently managed with this formulation [23].

4.2 Western science-based assessment

The in-vitro antimicrobial activity of aqueous, ethanol and ethyl acetate extracts of Aloe vera and Curcuma longa (turmeric) using agar well diffusion method exhibited antimicrobial activity against Escherichia coli, Staphylococcus aureus and Pseudomonas aurogenosa [24]. A clinical study using the mastitis combination indicated that the selected parameters like pH, Somatic Cell Count (SCC) and Electric Conductivity (EC) of milk of the mastitis-affected animals became normal within 6–7 days of treatment. The milk production returned to near normal to the pre-mastitis level [25] (Figure 10).

Figure 10.

The figure shows the reduction pH, electrical conductivity and somatic cell count of milk from mastitis-affected cows before and after treatment with an herbal formulation in comparison with values of normal cow: Pre-treatment – blue, post-treatment – red, normal cow – green bars.

Through an in-silico approach, bioactive compounds were tested for their effect against the target proteins of S. aureus using molecular docking studies [26]. Many bioactive components of Aloe vera and turmeric interact with the target protein. The pharmacodynamics study using the online server PASS reveals that the compound in the preparation possesses anti-inflammatory and anti-microbial properties. The antimicrobial activity of Aloe vera is attributed to the anthraquinones (aloin and emodin), flavonoids, tannins (active against MRSA), saponins, p-coumaric acid, ascorbic acid, pyrocatechol and cinnamic acid. Curcumin also possesses immunomodulatory and antioxidant activity [27, 28].

Alkaloids, tannins, phenolics, terpenoids, phytosterols, saponins, flavonoids, glycosides, fatty acids such as palmitoleic acid and α-turmerone in fixed oils of Curcuma longa also possess antimicrobial activity against a wide range of bacteria. Curcuminoids have antioxidant, anti-inflammatory, antiviral and antifungal properties [29, 30]. Calcium hydroxide is known to possess anti-inflammatory action and reduces oedema formation. Thus all three ingredients in the formulation act at various steps in the inflammatory pathway and synergistically produce an anti-inflammatory effect [25, 29, 30, 31, 32].

4.2.1 Changes in the microbiome of the milk from the cows with clinical mastitis before and after treatment with EVP

A preliminary study on the changes in the microbiome of milk from cows with clinical mastitis (The mastitis was confirmed with the California Mastitis Test) before treatment and after 6 days of treatment with ethno-veterinary herbal formulations. Abundance of Streptococcus, Pseudomonas, Pseudomonaceae family, Klebsiella, and Enterobacteriaceae family in the milk of mastitis-affected cows reduced to a minimum (Figure 11) [33].

Figure 11.

The figure shows abundance of Streptococcus, Staphylococcus, Enterobacteriaceae family and Pseudomonas of control, mastitis-affected and EVP-treated cows. BT-before treatment, after 3 days of treatment, after 6 days of treatment and control.

Principal coordinates analysis (PCoA) indicates that the microbiome abundance in the milk of EVP-treated cows is almost similar to the control after 6 days. However, the microbiome in the milk from the antibiotic-treated cow varied substantially from the control animal (Figure 12).

Figure 12.

(A) Principal coordinates analysis (PCoA) derived from one of the EVP-treated samples at different stages. Before EVP treatment, after 3 days, after 6 days of EVP treatment and control samples of F2 udder. Coloured dots are representative of the 4 samples. Red – F1 before EVP treatment; blue – After 3 days of EVP treatment; Orange – After 6 days of EVP treatment; green – Control. (B) Principal coordinates analysis (PCoA) derived from one of the antimicrobial-treated samples at different stages; before antimicrobial treatment, after 3 days of antimicrobial treatment, after 6 days of antimicrobial treatment and control samples. Coloured dots are representative of the 4 samples. Red – Control; blue – F1 before antimicrobial treatment; Orange – After 3 days of antimicrobial treatment; green – After 6 days of antimicrobial treatment.

5. Outcome of intervention of herbal formulation to prevent and cure mastitis as an alternative to antimicrobials

Eighty-eight per cent of subclinical mastitis cows turned CMT negative after application of the above oil-based ethno-veterinary herbal preparation (EVHP). The recovered animals produced 605 ml (7.3%) of additional milk per day. Ninety-two per cent of 3703 cattle and buffaloes treated with EVHP for clinical mastitis were clinically recovered after 5 days of treatment [34]. The application of EVHP shows a high clinical success rate of subclinical and clinical mastitis indicating the effectiveness of Aloe vera, turmeric and calcium hydroxide combination in mastitis management caused by a wide range of bacterial agents [35]. Ethno-veterinary herbal preparations comprising of Zambian grown Aloe vera and Curcuma longa, possess gram-positive antibacterial and antifungal spectrum of activity on bovine mastitis and other pathogenic microorganisms in-vitro [36].

The clinical intervention with EVM preparations on 181,252 cows in a multicentre field study for a period of 5 years from NDDB, Abbott and TDU on the efficacy of EVP in a stand-alone mode for subclinical, clinical and chronic mastitis with multi-factorial aetiology and prolonged illness from 2017 to 2018 to 2021–2022, the recovery is 84.9.00% which is a reasonably high outcome by any standard from the clinical point of view (Table 2).

S. no	Mastitis	Total treated cases	Total clinical recovery	% Clinical recovery
1	Acute Mastitis	104,475	82,878	79.3
2	Chronic mastitis	52,791	41,502	78.6
3	Sub-clinical Mastitis	23,986	19,780	82.5
4	Mastitis (Abbott)	1692	1563	92.38
5	Mastitis (TDU)	1561	1432	91.7
	Total	184, 505	147, 155	84.9

Table 2.

Feedback from various milk societies from NDDB through INAPH, Abbott and TDU on the efficacy of EVP for mastitis in cattle from 2017 to 2018 to 2021–2022.

An intervention impact study indicated an 87.9% reduction of antibiotic residue in the milk and 12.1% of samples showed residues of beta-lactams or sulphonamides [37]. These 17 farmers used antibiotics along with EVP [37]. The incidence of mastitis was reduced to 83% in a selected area from 2016 to 2019 (Table 3).

Year	2016	2018	2019
Average incidence of mastitis per union	65.63	36.5	10.6
Per cent reduction from 2016 to 2019		44.4	83.8

Table 3.

Reduction of the incidence of mastitis in cattle in the area selected for the studies when EVP was used from 2016 to 2019.

The average expenditure for the treatment of mastitis with conventional medicine was Rs. 3324. EVP treatment has reduced expenditure for the management of mastitis from Rs.3324 to 120. The farmers saved an average of Rs 3204 for each episode of mastitis. The average milk production loss during 6 days during the treatment with Western medicine was 19.92 litres (Rs.518) and EVP was 3.6 litres (Rs. 93.6) (Table 4).

S. No	Mastitis	n	Average loss of milk/day (L)	Average loss for 6 days (L)	Financial loss in Rs.
1	Allopathic treatment	76	3.32	19.92	518
2	EVP treatment	76	0.6	3.6	93.6

Table 4.

Cost impact (production loss) when Western medicine and herbal formulations are used.

Cost of Rs 26 per litre

A little reflection on the data would suggest that the herbal formulations cannot possibly be arbitrary combinations because of their high effectiveness indicates that they have been obviously designed with some pharmacological logic. The logic is based on Ayurveda pharmacology called “Dravya Guna Shastra.” Revalidating this formulation based on Western pharmacology is a cross-cultural or Trans Disciplinary exercise. It would require extensive studies on advanced combinatorial chemistry as polyherbals are involved. It would further need systemic experimental pharmacology studies with innovative bioassays because conventional bioassays are far too fractured and inadequate to detect simultaneous changes on multiple targets and sensitively designed modes of action studies.

The current approach of creating objective and verifiable standards for traditional knowledge products and concepts is one-sided and therefore, an intercultural (subjective and objective) approach involving consultation between traditional and Western health sciences is necessary to promote mutual understanding which could create relevant quality standards [38, 39, 40].

In case of mastitis in over one hundred eighty thousand cases of dairy cattle, the recovery rate is 84.9% with EVM in a stand-alone mode (Field observational study and not double-blind controlled clinical study). The above data shows a reasonably high outcome by any standard from the clinical point of view, in a multicentre field study for a period of 5 years. Of course, we need further systematic studies. The field is wide open for exploitation of traditional herbal knowledge to benefit the farming community and save our precious animal wealth and a solution for the alarming misuse of antimicrobials and the related AMR issue.

6. Conclusion

Mastitis is a perpetual problem of all milk-producing animals. Mastitis therapy protocol includes a high percentage of antibiotics. The application of EVHP showed a high clinical success rate of subclinical and clinical mastitis indicating the effectiveness of Aloe vera, Curcuma longa and calcium hydroxide combination in mastitis management caused by a wide range of microorganisms. This formulation was validated using transdisciplinary understanding. Adopting ethno-veterinary science and practices to combat mastitis has been identified and tested as a key alternative in reducing the use of antibiotic(s). It is also indicated that the EVP formulations are cost-effective and could be prepared and used by the farmers themselves whilst being extremely helpful to prevent and manage mastitis of their cattle.

Acknowledgments

We acknowledge the traditional knowledge holders, colleagues and our collaborators/partners – Tamil Nadu Veterinary and Animal Sciences University (TANUVAS), National Dairy Development Board (NDDB), Kerala State Veterinary Council (KSVC), milk unions from 14 states, several trained veterinarians, village resource persons, officers from the milk unions, GLOHMSIWA Research Labs, farmers and well-wishers who were directly or indirectly associated with developing and mainstreaming Ethno-veterinary Science and Practices. Department of Science and Technology (DST) Government of India, Eco Technology Culture (ETC) Netherlands, Natural Livestock Farming (NLF) foundation for financial support for this study.

Conflict of interest

The authors declare no conflict of interest.