Environmental mastitis, a subclinical form of mastitis, is frequently caused by Escherichia coli (E. coli) in dairy cattle. Subclinical mastitis, also referred to as SCM is a serious issue that dairy animals face globally. Breeders suffer huge losses as a result, which has an impact on the nation’s GDP (Ramachandraiah et al., 1990). SCM has been described as the most difficult issue in dairy production, causing significant financial harm to the dairy sectors in developed as well as developing nations (Kovacevic et al., 2021). Escherichia coli O157:H7, which produces verocytotoxins, has emerged as a significant food borne pathogen and is currently regarded as a serious public health concern as a result of these outbreaks (McKee et al., 2003; Bettelheim, 2000) and responsible for producing major public health concern i.e. heamorrhagic colitis and haemolytic uraemic syndrome. Shiga toxins (stx) are secreted by O157:H7, which causes virulence by preventing the host cells fropm synthesising proteins and ultimately resulting in cell death. It is evidently implied that poor productivity and health issues in dairy animals are more common in economically disadvantaged and developing countries (Antanaitis et al., 2021; Bhakat et al., 2020; Dabele et al., 2021). This might be caused by, among other things, inadequate dairy infrastructure, bad husbandry techniques, inadequate nutrition and ignorance (Singh et al., 2021; Bhakat et al., 2020; Getaneh et al., 2017). Mastitis can be defined as inflammation of the mammary glands that affects the physical, chemical and bacterial characteristics of the milk and mammary gland tissues of the afflicted animal (Bhakat et al., 2020; Kumari et al., 2019; Saravanan et al., 2015; Radostits et al., 2000). SCM has a negative impact on the health and milk production of dairy animals without visual signs in contrast to clinical mastitis cases where signs and symptoms are more severe and readily apparent to the unaided eye. In SCM cases, there is a decrease in milk yield and quality (Khan et al., 2022). Additionally, health problems in animals are reflected in the multifaceted subpar performance of dairy animals. Some of the most common pathogenic microorganisms are Escherichia coli, Staphylococcus aureus, Streptococcus uberis and Streptococcus dysgalactiae (Ibrahim, 2017). Certain types of yeast may occasionally also result in mastitis. Still, the main culprits behind mastitis in dairy cows continue to be bacteria (Ezzat et al., 2014). Animals with SCM infection could end up infecting other animals in the same herd. Somatic cell counts (SCCs) in milk are generally regarded as healthy and normal when they are under 1,000,000. When SCCs exceed 2,000 000, the milk is deemed to be in an SCM case (Hasan et al., 2018). Antimicrobial resistance (AMR) is a major worry when it comes to mastitis since it causes dairy farms to utilize antibiotics and improper use of antibiotics can result in AMR (Oliveira and Ruegg, 2014).

A total number of 250 milk samples from 125 dairy cattle were collected during the period from February 2024 to November 2024 in Mathura district. Milk samples were collected in autoclaved and UV sterilized McCartney bottles directly from cattle teats then taken to the laboratory ice-cooled and processed within 12 hours of collection. Confirmation of milk samples for SCM was done by CMT and SCC methods. CMT was done by CMT Reagent Kit. Somatic cell count of milk samples were evaluated in LACTOSCAN COMBO‘s SCC based on fluorescent microscope technique of counting cells. The analysis of the milk is precise, reliable and fast. In order the somatic cell to be counted by with SCC, the sample was mixed with SOFIA GREEN dye. Now 8 µL dye was pipetted onto the single LACTOCHIP. After that, the CHIP was loaded in the device. The analysis was being conducted during a period between 10 seconds and 2 minutes and the duration was dependent on the number of filmed fields.
       
Milk samples were processed for isolation of E. coli as per OIE reference laboratory for Escherichia coli,  protocol for primary isolation of E. coli 1 ml milk sample and swab sticks of all types were directly enriched in 9 ml Soyabean Casein Digest Medium (tryptone soya broth-TSB). The samples were incubated at 37°C for 16-24hrs and for the differential plating the loopful culture growth from TSB was streaked on MacConkey Lactose Agar (MLA) and incubated at 37°C for 24 hrs, the inoculum from MLA plates was selectively streaked on Eoisin Methylene Blue (EMB) Agar and incubated at 37°C for 24 hours for selective plating. Colonies showing characteristic metallic sheen on EMB agar. The purified cultures of E. coli were stored in Nutrient Agar slant for further identification by biochemical tests and other studies. After isolation Gram’s staining was done, on Gram’s staining E. coli appeared as Gram-negative cocci with small, pink rod like structure.
       
Further the biochemical tests were carried out as per procedure described by Barrow and Feltham (1993). A single colony from each isolate was picked up from Nutrient agar and inoculated in 5 mL Brain heart infusion broth (BHI) and incubated at 37°C for 4-6 hrs until inoculum turbidity is 0.5 McFarland. The tests were performed for Indole, Methyl red, Voges-proskauer and Citrate utilization test was also performed along with IMViC.
       
The purified cultures of E. coli were streaked on HiCrome™ EC O157:H7 Agar for primary detection of O157:H7 serotype. After streaking, the plates were incubated at 37°C for 24 hours. The chromogenic substrate is specifically and selectively cleaved by a dark purple to magenta coloured moiety. E. coli forms Escherichia coli O157:H7 (VTEC) resulting in a dark purple to magenta coloured moiety. E. coli other then O157:H7 forms bluish green coloured colonies.
       
PCR analysis for the detection of virulence gene Stx1, Stx2, hlyA, eaeA was carried out as per the method described by Paton and Paton (1998) (Table 1). PCR was carried out in a final reaction volume of 25 µl containing 12.5 µl of Master mix, 3 µl of DNA template, 1 µl of each of the primers (forward and reverse) and rest DNAse free water. The PCR tubes with all the components were transferred to the thermal cycler. For gene amplification, the initial denaturation step was carried out at 95°C for 5 min followed by denaturation at 94°C for 1 min., annealing at 59°C for 1 min., extension 72°C for 1 min. and a final extension step at 72°C for 6 min. For each gene 30 amplification cycles were performed. After the amplification, amplicons were separated in 1.5% gel in tris-acetate EDTA (TAE) buffer at 80 volt for 40 min, stained with 0.5% ethidium bromide solution and visualized under ultraviolet light.


       
The bacterial isolates were subjected to in vitro antibiotic sensitivity test as per the method Bauer et al. (1966) on Mueller Hinton agar with a opacity of the broth tube was matched with that of standard 0.5 Mc Farlands tube no 0.5 (1.5×10⁶ organisms/ml). A sterile cotton swab was dipped into the broth culture and streaked on entire agar surface. Antibiotics discs were placed on inoculated agar surface at about two cm apart. The plates were incubated at 37°C over- night and diameter of the zones of inhibition was measured. The measurements were compared with zone size interpretative chart furnished by the manufacturer and the zones were graded as sensitive and resistant. The 14 antibiotics used were Amikacin (10 µg),  Cefoperazone/Tazobactum (75/10 µg), Chloramphenicol  (50 µg), Enrofloxacin (15 µg), Gentamicin (30 µg), Ofloxacin (2 µg), Oxytetracyclin (30µg), Cefotaxime/Clavulanic acid (30/10 µg), Amoxycillin / Sulbactum (30/15 µg), Levofloxacin (5 µg), Ceftriaxone (10 µg), Sulfasomidine (300 µg), Ceftriaxone/Tazobactum (30/10 µg), Meropenam (10 µg), Ertapenam (10 µg).

In the present study, a total number of 125 dairy milch cattle were screened for subclinical mastitis, 63 animals found positive for SCM. The overall prevalence of subclinical mastitis was 50.40% (63/125) (Table 2). Abed et al., (2021) found prevalence of subclinical mastitis 44.8% from SCC which is nearly similar to the findings of this study. Jena et al., (2015) found 74.55% of prevalence of SCM from SCC which is higher to the finding of this study. 30 E. coli isolates were found positive, out of 125 milk samples. The overall percent prevalence of E. coli from milk of dairy cattle was found to be 24.00% (30/125), which is nearby similar to the findings of Rajpoot (2013) i.e. 28.75% and Kumar et al. (2021) reported 36.74% which is lower to the findings of Behera (2016) i.e. 51.66%. The overall percent of E. coli from SCM positive cattle milk was found to be 47.61 % (30 out of 63), which is nearby similar to the findings of Ahmadi et al., (2020) i.e. 43.25%, higher to the findings of Sheet et al., (2023) i.e. 36.3% and lower to the findings of Chowdhury et al., (2024) i.e. 50.54%.


       
All 30 E. coli isolates were processed for O157:H7 (Fig 1). Overall percent positivity of O157:H7 from milk was 4.00% (5 out of 125), which is nearby similar the findings of Das et al., (2008) i.e. 4-5%, higher to the findings of Rajpoot (2013) i.e. 2.5% and lower to the findings of Behera (2016) i.e. 13.33% and the overall per cent of O157:H7 from SCM positive cattle milk was 7.93% (5 out of 63), which is lower to the findings of Ahmadi et al., (2020) i.e. 22.54% and Chowdhury et al., (2024) i.e. 17.20%. Further all these 5 O157:H7 isolates of E. coli were screened by PCR to detect the presence of VTEC virulent genes: stx1, stx2, eaeA and hlyA (Fig 2). Out of which, 4 samples were found positive only for stx2 gene 80.00% (4/5), no isolates were found positive for stx1, eaeA and hlyA. Ahmadi et al. (2020) found highest percent positivity of stx2 gene i.e. 64.10% and Chowdhury et al. (2024) found highest percent positivity of stx2 gene i.e. 56.25%, which is quite similar pattern to our findings. The stx1/stx2 were detected in 2.47% samples by Nalband et al. (2020). In present study percent (%) positivity of O157:H7 was found 16.66% (5/30), which is higher than Allam et al., (2018) and Ahmadi et al., (2020) i.e. 8.8% and 7.96%, respectively.




       
On antibiogram assay (Fig 3) Chloramphenicol, Sulfasomidine, Meropenum, Cefotaxime /Clavulanic acid showed highest sensitivity (96.80%) followed by Ofloxacin (90.32%), Levofloxacin (83.87%), Amoxicillin/Sulbactum (77.41%), Ertapenam and Ceftriaxone (70.96%), Oxytetracyclin (66.12%), Ceftriaxone/Tazobactum (54.83%) rest of the antibiotics were having sensitivity below 50%. Antibiotics like Amikacin (72.58%) showed highest resistance. Gentamicin (50.00%), Ceftriaxone/Tazobactum and Ceftriaxone (19.35%), Ofloxacin and Ertapenam (9.68%), Oxytetracyclin and Levofloxacin (6.45%), Cefoperazone/Tazobactum (3.20%) followed by Sulfasomidine and Cefoperazone/Tazobactum (3.20%) showed lowest resistance. Rests of the antibiotics were having resistance among above drugs. Abed et al., (2021) found Doxycilin (90%), Amoxicillin/Clavulanic acid (88%), Gentamicin (72%) were resistance for E.coli, which is in consistent with our result. Chowdhury et al., (2024) found Amoxicillin (100%), Ampicillin (87.50%), Gentamicin (75%), Tetracyclin (68.75%) were resistance for E.coli, which is in similar to our result. In present study 23.33% (07/30) isolates was found for multi drug resisitance (MDR) pattern in milk samples which were affected from SCM. Hinthong et al. (2017) found 84.61% (22/26), MDR in milk samples which were affected with SCM, which is higher to our findings.

Subclinical mastitis in dairy cattle remains a significant concern for both animal health and milk production quality. The identification and management of subclinical mastitis, through regular monitoring and diagnostic tools such as somatic cell count (SCC) and microbiological analysis, are essential in mitigating its effects. Early molecular detection and antibiotic stewardship are key to reducing the incidence of subclinical mastitis. Studies have detected VTEC in milk samples from dairy cattle with subclinical mastitis, highlighting the need for proper hygiene practices, pasteurization and milk processing techniques to mitigate this risk.

The authors are highly thankful to Dean, College of Veterinary Science and Animal Husbandry, Uttar Pradesh Pandit Deen Dayal Upadhayay Pashu Chikitsa Vigyan Vishvidhyalaya Ewam Go-Anusandhan Sansthan (DUVASU), Mathura, U.P., India, for providing necessary funds and facilities to carry out the investigations.

The authors declare that there is no conflict of interest.