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Bovine Subclinical Mastitis: Risk Factors, Molecular Diagnosis and Therapeutic Evaluation of Cefalonium and Polyherbal Dry Cow Therapy

R
Rakesh Dangi1,*
N
Nidhi S. Choudhary1
R
Rakhi Gangil2
A
Arpita Shrivastav3
P
Pawan Maheshwari1
S
Shweta Rajoriya4
S
Supnesh Jain1
1Department of Veterinary Medicine, College of Veterinary Science and Animal Husbandry Mhow, Nanaji Deshmukh Veterinary Science University, Jabalpur-453 446, Madhya Pradesh, India.
2Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry Mhow, Nanaji Deshmukh Veterinary Science University, Jabalpur-453 446, Madhya Pradesh, India.
3Department of Pharmacology and Toxicology, College of Veterinary Science and Animal Husbandry Mhow, Nanaji Deshmukh Veterinary Science University, Jabalpur-453 446, Madhya Pradesh, India.
4Department of Veterinary Physiology and Biochemistry, College of Veterinary Science and Animal Husbandry Mhow, Nanaji Deshmukh Veterinary Science University, Jabalpur-453 446, Madhya Pradesh, India.

ABSTRACT

Background: Subclinical mastitis (SCM) is a major constraint to dairy productivity, affecting reproductive performance of the animal, particularly during the dry period when susceptibility to new intramammary infections is high. Combination of herbal products and antibiotic as a therapeutic approach along with analysis of the risk factors associated with SCM, can lessen antimicrobial resistance and help control SCM. The present study provides a methodical field valuation of antibiotic-herbal dry period approach and assesses how various risk factors affect its efficacy.

Methods: Pregnant cows (n=500) from both organized and unorganized dairy farms were screened for SCM. Staphylococcus aureus being the most common pathogen involved in mastitis were isolated and confirmed through molecular detection of nuc gene. Later SCM positive animals were subjected to treatments at drying off period using Cefalonium alone and in combination with a topical poly-herbal formulation (turmeric, aloe vera, limestone) and oral lemon supplementation.

Result: All treatment groups showed a significant reduction in SCC and normalization of milk pH compared to pre-treatment values (p<0.05). The highest cure rate was observed in combination of Cefalonium and polyherbal formulation (93.33%), followed by Cefalonium alone (73.33%) on day 45 post-calving. The present investigation also revealed that high parities and poor udder hygiene acted as common risk factors for the condition of sub clinical mastitis.

INTRODUCTION

Bovine mastitis (BM) represents a perilous infectious challenge for the worldwide dairy industry, triggering significant economic shortfalls and compromising animal welfare (Weber et al., 2021). The prevalence of subclinical mastitis is generally higher than clinical mastitis and poses a continuous challenge to dairy farmers, not only under Indian field conditions but worldwide. Subclinical mastitis in cows’ costs decrease reproductive performance, lower milk yield and quality. It also affects animal health, wellbeing, longevity and performance, leading to reduced productivity and profit to farmers (Pakrashi et al., 2023). Many studies have found that various risk factors, including age, parity, stage of lactation, milk supply, breed, previous history of mastitis, flooring type and teat disinfection, also affect the incidence of subclinical mastitis (Jadhav et al., 2018).
       
National Mastitis Council recommended five points to control mastitis including post-milking teat disinfection, comprehensive dry cow therapy, therapy of clinical cases during lactation, proper milking machine maintenance and culling problems cows. During dry period, the mammary gland of the animal undergoes a series of changes that influence the cow’s resistance to bacterial infection, thus the chances of acquiring new intramammary infections are high during this time (Jyothi et al., 2022). Thus, being the most crucial phase of the dairy cow’s lactation cycle the initiation of therapy for the control of mastitis is especially beneficial (Mourya et al., 2020).
       
Cefalonium dihydrate, a first-generation cephalosporin, is commonly used for dry cow therapy due to its prolonged activity and efficacy against major mastitis-causing pathogens. However, increasing concerns regarding antimicrobial resistance and prudent use of antibiotics have encouraged exploration of complementary and alternative approaches that can enhance therapeutic outcomes (Ramesh et al., 2021). Herbal formulations containing bioactive compounds such as turmeric (Curcuma longa) and aloe vera possess well-documented antimicrobial, anti-inflammatory and immunomodulatory properties and have shown potential benefits in mastitis management (Thangadurai et al., 2017). The present study was therefore undertaken to see efficacy of intramammary application of a standardized polyherbal formulation during the dry period along with conventional antibiotic dry cow therapy against subclinical mastitis infections and thus reducing the incidence of new post-calving intramammary infections along with the various risk factors associated during the treatment period.

MATERIALS AND METHODS

Selection of animals
 
A total of 500 pregnant cattle, including non-descript, cross-bred and exotic breeds from organized and unorganized dairy farms, were screened for SCM using the California Mastitis Test (CMT), Somatic Cell Count (SCC) and milk pH analysis. The study was conducted at the Department of Veterinary Medicine, College of Veterinary Science and Animal Husbandry, Mhow, Madhya Pradesh. The research work was carried out from October, 2023 - September, 2025. Udder and teat examinations were performed to identify abnormalities such as inflammatory swelling, fibrosis, wounds, scars, teat orifice patency and ease of milking. Milk samples from CMT-positive cows were aseptically collected at drying off into pre-labelled sterile tubes containing details of collection date, farm identification, animal number and udder quarter, following the guidelines of the National Mastitis Council (NMC) and Adkins et al., (2017).
 
California mastitis test
 
The CMT was employed as an on-site diagnostic tool for screening lactating dairy cows to SCM as per the method described by Constable et al., (2017) and Kumar et al., (2021). The test score was observed and CMT results were classified as either negative or positive, with a cow considered CMT-positive if at least one quarter exhibited a positive reaction. The scoring system was 0 (negative) and 1 to 4 (graded positive). Cows with a positive CMT score in the absence of visible abnormalities in milk or udder were classified as positive for subclinical mastitis (SCM).
 
Milk pH and somatic cell count (SCC)
 
Milk pH was estimated by briefly immersing the tip of pH indicator paper into the milk sample and the corresponding pH value was recorded. SCC was conducted on milk samples from subclinical mastitis (SCM)-positive quarters to evaluate the severity of intramammary infection. The SCC was performed using the Modified Newman’s staining technique, as described by Harmon (2001).
 
Isolation and identification of Staphylococcus
 
Milk samples found positive for SCM were processed for the isolation and identification of species as described by Barrow and Feltham (1993). Presumptive Staphylococcus colonies were identified based on morphological characteristics, Gram staining and standard biochemical tests including catalase, oxidase and coagulase tests. (Collee et al., 1996).
 
Molecular identification
 
 DNA was extracted from the isolates and the nuc gene, specific to Staphylococcus aureus, was amplified through polymerase chain reaction (PCR). PCR amplification was carried out under standardized thermal cycling conditions and the products were analysed by electrophoresis on a 1.5% agarose gel. The DNA bands were visualized under a gel documentation system and the product size was determined using a 100 bp DNA ladder (Brakstad et al., 1992) (Table 1).

Table 1: Details of primers for amplification of nuc gene.


 
Risk factor analysis
 
Parity, stage of lactation and udder hygiene were the major key factors found to play important role in SCM. Parity affecting SCM was categorized into three distinct groups, cows with 1-3 calvings were assigned a score of 1, those with 4-6 calvings received a score of 2 and cows with seven or more calvings were given a score of 3. Similarly, the stage of lactation was classified based on the number of months post-calving: Early lactation (1-3 months) was scored as 1, Mid-lactation (3-6 months) as 2 and Late lactation (³7 months) as 3. A standardized four-point scoring system by (Schreiner and Ruegg, 2003) was used to assess visible contamination on the rear udder and hind limbs, ranging from score 1 indicated a clean udder or leg, 2 denoted slight contamination (2-10% of the area covered with dirt), 3 indicated moderate contamination (10-30%) and 4 reflected severe contamination with caked-on dirt covering over 30% of the area.
 
Therapeutic efficacy of drugs
 
On the day of drying off cows screened positive by CMT, SCC and pH for subclinical mastitis were selected for the therapeutic evaluation. The infected cows were randomly divided into two groups, each comprising 15 cows along with healthy control group of 15 cows. Group T2 cows were treated with intramammary administration of Cefalonium dihydrate (Cepravin Dry Cow - MSD Animal health) @ 250 mg per teat infused in all teats for drying off. Group T3 cows received the same treatment of intramammary Cefalonium dihydrate @ 250 mg per teat along with a combination of topical herbal paste containing 50 g Turmeric powder (curcumin), 250 g Aloe vera (aloin, emodin, acemannan and flavonoids), 20-25 g Limestone (calcium hydroxide) and two fresh lemons orally thrice a day for 3 days, the herbal paste was applied topically five times a day for 5 days. The herbal paste was prepared in the laboratory under aseptic conditions as per the method described by Vijay et al., (2024) with slight modifications. Briefly fresh whole leaves of Aloe vera were collected, washed thoroughly and the thorns were removed. The leaves were then cut into small pieces and blended with turmeric powder and lime to obtain a homogeneous reddish-coloured paste. Before application, the udder and teats were washed, cleaned and all quarters, including unaffected quarters, were completely milked out. A handful of the prepared herbal paste was mixed with approximately 200 mL of clean water to obtain a thin consistency suitable for topical application. The diluted paste was gently applied over the entire udder surface.
       
The results of the curative therapy were judged by retesting the milk samples after the completion of the therapy, in the CMT, SCC and pH on the 7th, 14th and 45th days post-calving.
 
Statistical analysis
 
Changes across different treatment groups at various intervals were analysed using two-way ANOVA and the means were compared using Post Hoc, Duncan’s Multiple Range Test (Snedecor and Cochran, 1994) Data were analyzed using IBM SPSS Statistics Version 30.0 (IBM Corp., Armonk, NY, USA). Statistical significance was considered at p<0.05.

RESULTS AND DISCUSSION

The therapeutic efficacy of topical poly-herbal paste and intramammary Cefalonium during the dry period was evaluated using CMT, SCC, milk pH and bacterial culture on days 7, 14 and 45 post-calving. Milk pH at dry off (day 0) was significantly higher in SCM affected cows (T2 and T3; 7.0±0.00) compared to healthy controls (T1; 6.5±0.00). Following treatment, milk pH gradually declined toward the normal physiological range. Group T3 showed greater normalization than T2 and maintained lower pH values up to day 45 post-calving. Similar observations were reported by Zeleke et al., (2024) and Amran et al., (2025). Increased milk pH in SCM may be due to inflammation-induced permeability changes in mammary tissues, causing increased leakage of alkaline blood constituents and reduced lactose content (Table 2).

Table 2: Mean milk pH in different treatment groups at different intervals.


       
Pre-treatment SCC values were significantly higher in SCM affected groups than healthy controls. At day 0, SCC values were highest in T2 and T3, whereas T1 maintained normal levels. Post-treatment, SCC progressively declined in both treatment groups, with T3 showing a marked reduction and approaching normal values by day 45 post-calving, indicating better therapeutic response. The elevated SCC observed in this study as also reported by Alkhouly et al., (2023) may be attributed to variations in farm management practices and the occurrence of intramammary infections, these differences reflect the multifactorial nature of mastitis. An increase in SCC also indicates an inflammatory response in the mammary gland, typically initiated by bacterial invasion and serves as part of the udder’s natural defence mechanism. The extent of SCC elevation generally corresponds to the severity of the infection, thereby making it a reliable indicator for the detection of subclinical mastitis. The diagnostic significance of SCC in identifying subclinical cases has also been confirmed by Karabasanavar et al., (2019) and Jesse et al., (2023) (Table 3).

Table 3: Mean somatic cell count (105 cells/ml) in different treatment groups at different intervals.


 
Molecular detection by amplification of nuc gene using PCR
 
Bacteriological examination of CMT-positive milk samples revealed creamish-yellow colonies suggestive of Staphylococcus spp. (Fig 1). Further yellow colonies indicated mannitol fermentation on Mannitol Salt Agar (MSA), gram’s staining showed Gram-positive cocci arranged in grape-like clusters, characteristic of Staphylococcus spp. The amplified PCR products showed a 279 bp band on agarose gel electrophoresis, confirming the presence of S. aureus (Fig 2). Findings confirmed that PCR targeting the nuc gene is a reliable method for identification of Staphylococcus aureus from subclinical mastitis milk samples similar to those reported by Saeed et al., (2022) and Hoque et al., (2023). S. aureus is particularly adroit at invading mammary tissue and establishing deep-seated infections, making it a major pathogen in the development of SCM.

Fig 1: Yellow coloured colonies of Staphylococcus spp. on mannitol salt agar (MSA).



Fig 2: PCR amplification of nuc gene of Staphylococcus spp. showing 279 bp of PCR product.


 
Risk factors analysis
 
A significant association (P<0.05) was observed between SCM prevalence and parity, lactation stage and udder-leg hygiene score. Higher prevalence was recorded in cows with advanced parity (4-6 and ≥7) compared to cows with lower parity (1-3), indicating increased susceptibility with age and repeated lactations. Similar findings were reported by Kumar et al., (2021) and Sharma et al., (2023). Increased milk production, age-related decline in immunity and structural changes in the udder may contribute to the higher occurrence of SCM in multiparous animals.
       
SCM prevalence was highest during early lactation, followed by mid and late lactation stages. Early lactation is considered a critical period due to immunosuppression, oxidative stress and changes in milk composition, favouring bacterial growth. Comparable observations were reported by Patel et al., (2023) and Salamanca-Carreno et al. (2024). However, Khasanah et al., (2021) and Maalik et al., (2019) reported greater prevalence during mid-lactation.
       
Udder and leg hygiene significantly influenced SCM occurrence, with prevalence increasing from slightly dirty to very dirty animals. Poor hygiene promotes environmental contamination and teat damage, facilitating pathogen entry and increasing intramammary infections. Similar observations were reported by Ndahetuye et al., (2020) and Kilyenyi et al., (2021) (Table 4).

Table 4: Chi-squared analysis of risk factors for SCM in cow.


 
Therapeutic efficacy of drugs
 
In this study the comparative efficacy of a topical poly-herbal paste and a standard intramammary antibiotic preparation (Cefalonium), as dry cow therapy for the treatment of SCM in cattle was observed. Analysis of data revealed that the T3 treatment group (herbal paste and oral lemon therapy alongside Cefalonium), demonstrated the highest cure rate (93.33%) compared to the T2 where antibiotic alone was given (73.33%). These findings were also reported by Freu et al., (2020) and Tiwari et al., (2020), who demonstrated that Cefalonium-based dry cow therapy effectively eliminates existing intramammary infections and reduces the incidence of new infections during the dry period. Cefalonium with an initial high concentration being effective in treating existing infections, including gram positive and gram-negative bacteria (Table 5).

Table 5: Therapeutic efficacy of dry cow therapy and herbal paste in post calving cows.


       
The efficacy of herbal treatment was similar to Thangadurai et al., (2017) and Forno-Bell et al. (2021), as these authors reported that herbal formulations containing Aloe vera, turmeric and lime were effective in the management of mastitis without producing adverse effects. Aloe vera gel extract has been shown to exert antimicrobial activity by disrupting the bacterial cell membrane, resulting in lysis of up to 75% of Staphylococcus aureus isolates. Whole leaf of Aloe vera contain anthraquinones like aloein and aloe-emodin which possess antibacterial action by interfering with bacterial protein synthesis. and Acemenan present in the gel act as the macrophage activator which make the survival of Staphylococcus more difficult (Nair et al., 2017). Aloin, show its anti-inflammatory effects by inhibiting the JAK1/STAT1/3 signalling pathways (Vijay et al., 2024).
       
Turmeric contains curcumin which causes membrane damage and oxidative stress to the bacteria. Curcumin, the active ingredient in turmeric powder was also shown to decrease synthesis of IL-6 and TNF-α in S. aureus induced mastitis (Xu et al., 2020). According Panigrahi et al., (2022), turmeric powder was reported to be particularly effective in the treatment of subclinical mastitis. formulations containing turmeric were found to inhibit the growth of major mastitis-causing pathogens, including Staphylococcus aureus, Streptococcus agalactiae and Escherichia coli. Citric acid and limonene present in the lemon causes pH reduction and lipid membrane dissolution (Rath et al., 2020). Calcium Hydroxide [Ca(OH)2] was reported to have anti-microbial effect through release of hydroxyl ions in aqueous conditions. Also, it inactivates endotoxins and play immunomodulatory role hrough decreasing TNF-α production in LPS stimulated macrophages (Vijay et al., 2024). Citrate plays a crucial role in lactogenesis and the maintenance of udder health by facilitating ionic equilibrium (Hyvonen et al., 2010). The findings of the present study suggest that supplementation with lemon fruits may provide beneficial effects similar to citrate administration and could contribute to the management of mastitis.

CONCLUSION

Dry cow therapy with Cefalonium dihydrate was found to be effective in reducing subclinical mastitis (SCM) in dairy cows; however, its efficacy was significantly enhanced when combined with supportive herbal therapies. Among the different treatment regimens evaluated, the combination of Cefalonium with a poly-herbal formulation showed the highest therapeutic success, with a cure rate of 93.33% by day 45 post-calving. This group also exhibited the most pronounced reduction in SCC and normalization of milk pH, indicating improved udder health and resolution of inflammation.
       
Furthermore, the study highlights that parity, stage of lactation and udder hygiene are significant risk factors influencing the the occurrence of S. aureus infections all of which can modulate the susceptibility of cows to infection. The integration of herbal formulations with conventional antibiotic dry cow therapy could be a promising, cost-effective and sustainable approach for the management of subclinical mastitis and may also contribute in reducing antibiotic usage and mitigating antimicrobial resistance.

ACKNOWLEDGEMENT

The authors express their sincere gratitude to the Dean, College of Veterinary Science and Animal Husbandry, Mhow, NDVSU, Jabalpur, for providing the necessary facilities and support to conduct this study.

CONFLICT OF INTEREST

The authors declare that is there is no conflict of interest.

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    Full Research Article

    Bovine Subclinical Mastitis: Risk Factors, Molecular Diagnosis and Therapeutic Evaluation of Cefalonium and Polyherbal Dry Cow Therapy

    R
    Rakesh Dangi1,*
    N
    Nidhi S. Choudhary1
    R
    Rakhi Gangil2
    A
    Arpita Shrivastav3
    P
    Pawan Maheshwari1
    S
    Shweta Rajoriya4
    S
    Supnesh Jain1
    1Department of Veterinary Medicine, College of Veterinary Science and Animal Husbandry Mhow, Nanaji Deshmukh Veterinary Science University, Jabalpur-453 446, Madhya Pradesh, India.
    2Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry Mhow, Nanaji Deshmukh Veterinary Science University, Jabalpur-453 446, Madhya Pradesh, India.
    3Department of Pharmacology and Toxicology, College of Veterinary Science and Animal Husbandry Mhow, Nanaji Deshmukh Veterinary Science University, Jabalpur-453 446, Madhya Pradesh, India.
    4Department of Veterinary Physiology and Biochemistry, College of Veterinary Science and Animal Husbandry Mhow, Nanaji Deshmukh Veterinary Science University, Jabalpur-453 446, Madhya Pradesh, India.

    ABSTRACT

    Background: Subclinical mastitis (SCM) is a major constraint to dairy productivity, affecting reproductive performance of the animal, particularly during the dry period when susceptibility to new intramammary infections is high. Combination of herbal products and antibiotic as a therapeutic approach along with analysis of the risk factors associated with SCM, can lessen antimicrobial resistance and help control SCM. The present study provides a methodical field valuation of antibiotic-herbal dry period approach and assesses how various risk factors affect its efficacy.

    Methods: Pregnant cows (n=500) from both organized and unorganized dairy farms were screened for SCM. Staphylococcus aureus being the most common pathogen involved in mastitis were isolated and confirmed through molecular detection of nuc gene. Later SCM positive animals were subjected to treatments at drying off period using Cefalonium alone and in combination with a topical poly-herbal formulation (turmeric, aloe vera, limestone) and oral lemon supplementation.

    Result: All treatment groups showed a significant reduction in SCC and normalization of milk pH compared to pre-treatment values (p<0.05). The highest cure rate was observed in combination of Cefalonium and polyherbal formulation (93.33%), followed by Cefalonium alone (73.33%) on day 45 post-calving. The present investigation also revealed that high parities and poor udder hygiene acted as common risk factors for the condition of sub clinical mastitis.

    INTRODUCTION

    Bovine mastitis (BM) represents a perilous infectious challenge for the worldwide dairy industry, triggering significant economic shortfalls and compromising animal welfare (Weber et al., 2021). The prevalence of subclinical mastitis is generally higher than clinical mastitis and poses a continuous challenge to dairy farmers, not only under Indian field conditions but worldwide. Subclinical mastitis in cows’ costs decrease reproductive performance, lower milk yield and quality. It also affects animal health, wellbeing, longevity and performance, leading to reduced productivity and profit to farmers (Pakrashi et al., 2023). Many studies have found that various risk factors, including age, parity, stage of lactation, milk supply, breed, previous history of mastitis, flooring type and teat disinfection, also affect the incidence of subclinical mastitis (Jadhav et al., 2018).
           
    National Mastitis Council recommended five points to control mastitis including post-milking teat disinfection, comprehensive dry cow therapy, therapy of clinical cases during lactation, proper milking machine maintenance and culling problems cows. During dry period, the mammary gland of the animal undergoes a series of changes that influence the cow’s resistance to bacterial infection, thus the chances of acquiring new intramammary infections are high during this time (Jyothi et al., 2022). Thus, being the most crucial phase of the dairy cow’s lactation cycle the initiation of therapy for the control of mastitis is especially beneficial (Mourya et al., 2020).
           
    Cefalonium dihydrate, a first-generation cephalosporin, is commonly used for dry cow therapy due to its prolonged activity and efficacy against major mastitis-causing pathogens. However, increasing concerns regarding antimicrobial resistance and prudent use of antibiotics have encouraged exploration of complementary and alternative approaches that can enhance therapeutic outcomes (Ramesh et al., 2021). Herbal formulations containing bioactive compounds such as turmeric (Curcuma longa) and aloe vera possess well-documented antimicrobial, anti-inflammatory and immunomodulatory properties and have shown potential benefits in mastitis management (Thangadurai et al., 2017). The present study was therefore undertaken to see efficacy of intramammary application of a standardized polyherbal formulation during the dry period along with conventional antibiotic dry cow therapy against subclinical mastitis infections and thus reducing the incidence of new post-calving intramammary infections along with the various risk factors associated during the treatment period.

    MATERIALS AND METHODS

    Selection of animals
     
    A total of 500 pregnant cattle, including non-descript, cross-bred and exotic breeds from organized and unorganized dairy farms, were screened for SCM using the California Mastitis Test (CMT), Somatic Cell Count (SCC) and milk pH analysis. The study was conducted at the Department of Veterinary Medicine, College of Veterinary Science and Animal Husbandry, Mhow, Madhya Pradesh. The research work was carried out from October, 2023 - September, 2025. Udder and teat examinations were performed to identify abnormalities such as inflammatory swelling, fibrosis, wounds, scars, teat orifice patency and ease of milking. Milk samples from CMT-positive cows were aseptically collected at drying off into pre-labelled sterile tubes containing details of collection date, farm identification, animal number and udder quarter, following the guidelines of the National Mastitis Council (NMC) and Adkins et al., (2017).
     
    California mastitis test
     
    The CMT was employed as an on-site diagnostic tool for screening lactating dairy cows to SCM as per the method described by Constable et al., (2017) and Kumar et al., (2021). The test score was observed and CMT results were classified as either negative or positive, with a cow considered CMT-positive if at least one quarter exhibited a positive reaction. The scoring system was 0 (negative) and 1 to 4 (graded positive). Cows with a positive CMT score in the absence of visible abnormalities in milk or udder were classified as positive for subclinical mastitis (SCM).
     
    Milk pH and somatic cell count (SCC)
     
    Milk pH was estimated by briefly immersing the tip of pH indicator paper into the milk sample and the corresponding pH value was recorded. SCC was conducted on milk samples from subclinical mastitis (SCM)-positive quarters to evaluate the severity of intramammary infection. The SCC was performed using the Modified Newman’s staining technique, as described by Harmon (2001).
     
    Isolation and identification of Staphylococcus
     
    Milk samples found positive for SCM were processed for the isolation and identification of species as described by Barrow and Feltham (1993). Presumptive Staphylococcus colonies were identified based on morphological characteristics, Gram staining and standard biochemical tests including catalase, oxidase and coagulase tests. (Collee et al., 1996).
     
    Molecular identification
     
     DNA was extracted from the isolates and the nuc gene, specific to Staphylococcus aureus, was amplified through polymerase chain reaction (PCR). PCR amplification was carried out under standardized thermal cycling conditions and the products were analysed by electrophoresis on a 1.5% agarose gel. The DNA bands were visualized under a gel documentation system and the product size was determined using a 100 bp DNA ladder (Brakstad et al., 1992) (Table 1).

    Table 1: Details of primers for amplification of nuc gene.


     
    Risk factor analysis
     
    Parity, stage of lactation and udder hygiene were the major key factors found to play important role in SCM. Parity affecting SCM was categorized into three distinct groups, cows with 1-3 calvings were assigned a score of 1, those with 4-6 calvings received a score of 2 and cows with seven or more calvings were given a score of 3. Similarly, the stage of lactation was classified based on the number of months post-calving: Early lactation (1-3 months) was scored as 1, Mid-lactation (3-6 months) as 2 and Late lactation (³7 months) as 3. A standardized four-point scoring system by (Schreiner and Ruegg, 2003) was used to assess visible contamination on the rear udder and hind limbs, ranging from score 1 indicated a clean udder or leg, 2 denoted slight contamination (2-10% of the area covered with dirt), 3 indicated moderate contamination (10-30%) and 4 reflected severe contamination with caked-on dirt covering over 30% of the area.
     
    Therapeutic efficacy of drugs
     
    On the day of drying off cows screened positive by CMT, SCC and pH for subclinical mastitis were selected for the therapeutic evaluation. The infected cows were randomly divided into two groups, each comprising 15 cows along with healthy control group of 15 cows. Group T2 cows were treated with intramammary administration of Cefalonium dihydrate (Cepravin Dry Cow - MSD Animal health) @ 250 mg per teat infused in all teats for drying off. Group T3 cows received the same treatment of intramammary Cefalonium dihydrate @ 250 mg per teat along with a combination of topical herbal paste containing 50 g Turmeric powder (curcumin), 250 g Aloe vera (aloin, emodin, acemannan and flavonoids), 20-25 g Limestone (calcium hydroxide) and two fresh lemons orally thrice a day for 3 days, the herbal paste was applied topically five times a day for 5 days. The herbal paste was prepared in the laboratory under aseptic conditions as per the method described by Vijay et al., (2024) with slight modifications. Briefly fresh whole leaves of Aloe vera were collected, washed thoroughly and the thorns were removed. The leaves were then cut into small pieces and blended with turmeric powder and lime to obtain a homogeneous reddish-coloured paste. Before application, the udder and teats were washed, cleaned and all quarters, including unaffected quarters, were completely milked out. A handful of the prepared herbal paste was mixed with approximately 200 mL of clean water to obtain a thin consistency suitable for topical application. The diluted paste was gently applied over the entire udder surface.
           
    The results of the curative therapy were judged by retesting the milk samples after the completion of the therapy, in the CMT, SCC and pH on the 7th, 14th and 45th days post-calving.
     
    Statistical analysis
     
    Changes across different treatment groups at various intervals were analysed using two-way ANOVA and the means were compared using Post Hoc, Duncan’s Multiple Range Test (Snedecor and Cochran, 1994) Data were analyzed using IBM SPSS Statistics Version 30.0 (IBM Corp., Armonk, NY, USA). Statistical significance was considered at p<0.05.

    RESULTS AND DISCUSSION

    The therapeutic efficacy of topical poly-herbal paste and intramammary Cefalonium during the dry period was evaluated using CMT, SCC, milk pH and bacterial culture on days 7, 14 and 45 post-calving. Milk pH at dry off (day 0) was significantly higher in SCM affected cows (T2 and T3; 7.0±0.00) compared to healthy controls (T1; 6.5±0.00). Following treatment, milk pH gradually declined toward the normal physiological range. Group T3 showed greater normalization than T2 and maintained lower pH values up to day 45 post-calving. Similar observations were reported by Zeleke et al., (2024) and Amran et al., (2025). Increased milk pH in SCM may be due to inflammation-induced permeability changes in mammary tissues, causing increased leakage of alkaline blood constituents and reduced lactose content (Table 2).

    Table 2: Mean milk pH in different treatment groups at different intervals.


           
    Pre-treatment SCC values were significantly higher in SCM affected groups than healthy controls. At day 0, SCC values were highest in T2 and T3, whereas T1 maintained normal levels. Post-treatment, SCC progressively declined in both treatment groups, with T3 showing a marked reduction and approaching normal values by day 45 post-calving, indicating better therapeutic response. The elevated SCC observed in this study as also reported by Alkhouly et al., (2023) may be attributed to variations in farm management practices and the occurrence of intramammary infections, these differences reflect the multifactorial nature of mastitis. An increase in SCC also indicates an inflammatory response in the mammary gland, typically initiated by bacterial invasion and serves as part of the udder’s natural defence mechanism. The extent of SCC elevation generally corresponds to the severity of the infection, thereby making it a reliable indicator for the detection of subclinical mastitis. The diagnostic significance of SCC in identifying subclinical cases has also been confirmed by Karabasanavar et al., (2019) and Jesse et al., (2023) (Table 3).

    Table 3: Mean somatic cell count (105 cells/ml) in different treatment groups at different intervals.


     
    Molecular detection by amplification of nuc gene using PCR
     
    Bacteriological examination of CMT-positive milk samples revealed creamish-yellow colonies suggestive of Staphylococcus spp. (Fig 1). Further yellow colonies indicated mannitol fermentation on Mannitol Salt Agar (MSA), gram’s staining showed Gram-positive cocci arranged in grape-like clusters, characteristic of Staphylococcus spp. The amplified PCR products showed a 279 bp band on agarose gel electrophoresis, confirming the presence of S. aureus (Fig 2). Findings confirmed that PCR targeting the nuc gene is a reliable method for identification of Staphylococcus aureus from subclinical mastitis milk samples similar to those reported by Saeed et al., (2022) and Hoque et al., (2023). S. aureus is particularly adroit at invading mammary tissue and establishing deep-seated infections, making it a major pathogen in the development of SCM.

    Fig 1: Yellow coloured colonies of Staphylococcus spp. on mannitol salt agar (MSA).



    Fig 2: PCR amplification of nuc gene of Staphylococcus spp. showing 279 bp of PCR product.


     
    Risk factors analysis
     
    A significant association (P<0.05) was observed between SCM prevalence and parity, lactation stage and udder-leg hygiene score. Higher prevalence was recorded in cows with advanced parity (4-6 and ≥7) compared to cows with lower parity (1-3), indicating increased susceptibility with age and repeated lactations. Similar findings were reported by Kumar et al., (2021) and Sharma et al., (2023). Increased milk production, age-related decline in immunity and structural changes in the udder may contribute to the higher occurrence of SCM in multiparous animals.
           
    SCM prevalence was highest during early lactation, followed by mid and late lactation stages. Early lactation is considered a critical period due to immunosuppression, oxidative stress and changes in milk composition, favouring bacterial growth. Comparable observations were reported by Patel et al., (2023) and Salamanca-Carreno et al. (2024). However, Khasanah et al., (2021) and Maalik et al., (2019) reported greater prevalence during mid-lactation.
           
    Udder and leg hygiene significantly influenced SCM occurrence, with prevalence increasing from slightly dirty to very dirty animals. Poor hygiene promotes environmental contamination and teat damage, facilitating pathogen entry and increasing intramammary infections. Similar observations were reported by Ndahetuye et al., (2020) and Kilyenyi et al., (2021) (Table 4).

    Table 4: Chi-squared analysis of risk factors for SCM in cow.


     
    Therapeutic efficacy of drugs
     
    In this study the comparative efficacy of a topical poly-herbal paste and a standard intramammary antibiotic preparation (Cefalonium), as dry cow therapy for the treatment of SCM in cattle was observed. Analysis of data revealed that the T3 treatment group (herbal paste and oral lemon therapy alongside Cefalonium), demonstrated the highest cure rate (93.33%) compared to the T2 where antibiotic alone was given (73.33%). These findings were also reported by Freu et al., (2020) and Tiwari et al., (2020), who demonstrated that Cefalonium-based dry cow therapy effectively eliminates existing intramammary infections and reduces the incidence of new infections during the dry period. Cefalonium with an initial high concentration being effective in treating existing infections, including gram positive and gram-negative bacteria (Table 5).

    Table 5: Therapeutic efficacy of dry cow therapy and herbal paste in post calving cows.


           
    The efficacy of herbal treatment was similar to Thangadurai et al., (2017) and Forno-Bell et al. (2021), as these authors reported that herbal formulations containing Aloe vera, turmeric and lime were effective in the management of mastitis without producing adverse effects. Aloe vera gel extract has been shown to exert antimicrobial activity by disrupting the bacterial cell membrane, resulting in lysis of up to 75% of Staphylococcus aureus isolates. Whole leaf of Aloe vera contain anthraquinones like aloein and aloe-emodin which possess antibacterial action by interfering with bacterial protein synthesis. and Acemenan present in the gel act as the macrophage activator which make the survival of Staphylococcus more difficult (Nair et al., 2017). Aloin, show its anti-inflammatory effects by inhibiting the JAK1/STAT1/3 signalling pathways (Vijay et al., 2024).
           
    Turmeric contains curcumin which causes membrane damage and oxidative stress to the bacteria. Curcumin, the active ingredient in turmeric powder was also shown to decrease synthesis of IL-6 and TNF-α in S. aureus induced mastitis (Xu et al., 2020). According Panigrahi et al., (2022), turmeric powder was reported to be particularly effective in the treatment of subclinical mastitis. formulations containing turmeric were found to inhibit the growth of major mastitis-causing pathogens, including Staphylococcus aureus, Streptococcus agalactiae and Escherichia coli. Citric acid and limonene present in the lemon causes pH reduction and lipid membrane dissolution (Rath et al., 2020). Calcium Hydroxide [Ca(OH)2] was reported to have anti-microbial effect through release of hydroxyl ions in aqueous conditions. Also, it inactivates endotoxins and play immunomodulatory role hrough decreasing TNF-α production in LPS stimulated macrophages (Vijay et al., 2024). Citrate plays a crucial role in lactogenesis and the maintenance of udder health by facilitating ionic equilibrium (Hyvonen et al., 2010). The findings of the present study suggest that supplementation with lemon fruits may provide beneficial effects similar to citrate administration and could contribute to the management of mastitis.

    CONCLUSION

    Dry cow therapy with Cefalonium dihydrate was found to be effective in reducing subclinical mastitis (SCM) in dairy cows; however, its efficacy was significantly enhanced when combined with supportive herbal therapies. Among the different treatment regimens evaluated, the combination of Cefalonium with a poly-herbal formulation showed the highest therapeutic success, with a cure rate of 93.33% by day 45 post-calving. This group also exhibited the most pronounced reduction in SCC and normalization of milk pH, indicating improved udder health and resolution of inflammation.
           
    Furthermore, the study highlights that parity, stage of lactation and udder hygiene are significant risk factors influencing the the occurrence of S. aureus infections all of which can modulate the susceptibility of cows to infection. The integration of herbal formulations with conventional antibiotic dry cow therapy could be a promising, cost-effective and sustainable approach for the management of subclinical mastitis and may also contribute in reducing antibiotic usage and mitigating antimicrobial resistance.

    ACKNOWLEDGEMENT

    The authors express their sincere gratitude to the Dean, College of Veterinary Science and Animal Husbandry, Mhow, NDVSU, Jabalpur, for providing the necessary facilities and support to conduct this study.

    CONFLICT OF INTEREST

    The authors declare that is there is no conflict of interest.

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