Total viable count and ABST of milk in cows with subclinical mastitis

Introduction

Subclinical mastitis is an important disease affecting dairy animals and is associated with reduced milk quality and economic losses. Unlike clinical mastitis, the disease may occur without visible abnormalities in milk or the udder, making early detection and preventive management particularly important. Post-milking teat disinfection is an important component of mastitis prevention. Conventional chemical disinfectants such as iodine are commonly used to reduce microbial contamination and minimise the risk of new intramammary infections. However, increasing interest in natural products has encouraged the investigation of plant-derived compounds as alternative antimicrobial agents. Essential oils contain biologically active compounds that may exhibit antimicrobial activity against a variety of microorganisms. Eucalyptus, lavender, peppermint and tea tree oils have individually been investigated for their antimicrobial properties. Their incorporation into a post-milking teat dip may therefore provide a plant-based approach for reducing bacterial contamination associated with the teat surface and milk. Total viable count is commonly used as an indicator of the viable bacterial population present in milk. Therefore, the present study was undertaken to evaluate the effect of an essential oil-based post-milking teat dip on the total viable count of milk from crossbred cows affected with subclinical mastitis. The antibacterial activity of the essential oil blend against Streptococcus spp. and Escherichia coli was also evaluated.

Materials and Methods

Experimental animals and study period

The experiment was conducted at Shri Gaurakshan Sanstha, Akola, Maharashtra, for 90 days from 13 November 2024 to 17 February 2025. Thirty-six lactating crossbred cows affected with subclinical mastitis were selected for the experiment. The selected cows were randomly divided into three groups containing 12 animals each.

Experimental treatments

The treatment groups were as follows T₀: Distilled water post-milking teat dip T₁: 1% iodine post-milking teat dip T₂: Essential oil-based post-milking teat dip The essential oil-based teat dip was prepared using 0.5 ml each of eucalyptus, lavender, peppermint and tea tree oils. The oils were mixed with 2 ml liquid soap and two drops of glycerine, and the volume was made up to 100 ml with distilled water.

Collection of milk samples

Milk samples were collected aseptically from the experimental animals. The initial streams of milk were discarded, and approximately 50 ml of milk was collected in sterile containers. Samples were labelled and transported under suitable conditions for microbiological examination.

Estimation of total viable count

Total viable count was estimated by serial ten-fold dilution and pour plate technique. Milk samples were serially diluted, and aliquots from the selected dilutions were inoculated into sterile Petri plates. Molten agar was added to the plates, mixed thoroughly and allowed to solidify. The plates were incubated at 35-37 °C for 24 h. Following incubation, visible colonies were counted. Total viable count observations were recorded at 10⁻³, 10⁻⁴ and 10⁻⁵ dilutions for pre-treatment and post-treatment milk samples.

Antibacterial activity of the essential oil blend

The antibacterial activity of the essential oil blend was evaluated against Streptococcus spp. and Escherichia coli. The blend was tested in undiluted form and at 1:2, 1:4, 1:8, 1:16 and 1:32 dilutions. Antibacterial activity was assessed by measuring the zone of inhibition.

Statistical analysis

The experimental data were analysed using IBM SPSS Statistics version 22. The data were subjected to appropriate statistical analysis, and mean values with their standard errors were calculated. Analysis of variance and Duncan's multiple range test were used for comparison among treatment means. Pre-treatment and post-treatment observations were also compared using the t-test.

Results

Total viable count

The total viable counts observed before and after treatment at different dilutions are presented in Table 1. At the 10⁻³ dilution, the pre-treatment total viable counts were 28.91 ± 0.25, 29.66 ± 0.28 and 29.33 ± 0.35 in T₀, T₁ and T₂, respectively. The post-treatment counts were 36.00 ± 0.32, 25.83 ± 0.20 and 24.08 ± 0.22, respectively. The lowest post-treatment count was recorded in T₂. At the 10⁻⁴ dilution, the pre-treatment total viable counts were 27.33 ± 0.25, 27.25 ± 0.17 and 27.83 ± 0.38 in T₀, T₁ and T₂, respectively. The corresponding post-treatment counts were 34.16 ± 0.27, 24.16 ± 0.20 and 22.16 ± 0.20. The lowest post-treatment count was again recorded in T₂. At the 10⁻⁵ dilution, the pre-treatment counts were 25.91 ± 0.25, 25.83 ± 0.20 and 25.16 ± 0.24 in T₀, T₁ and T₂, respectively. The post-treatment counts were 32.33 ± 0.25, 21.91 ± 0.31 and 20.75 ± 0.25, respectively. The essential oil-treated group recorded the lowest post-treatment count. The post-treatment values differed significantly among the treatment groups at all three dilutions. However, the paired comparison between pre-treatment and post-treatment observations within the individual groups was reported as non-significant.

Antibacterial activity of the essential oil blend

The antibacterial activity of the essential oil blend against Streptococcus spp. and Escherichia coli is presented in Table 2. The undiluted essential oil blend produced the maximum zone of inhibition against Streptococcus spp. and Escherichia coli, measuring 15.50 ± 0.42 mm and 14.50 ± 0.42 mm, respectively. The inhibition zones decreased progressively with increasing dilution of the essential oil blend. Against Streptococcus spp., inhibition zones of 12.50 ± 0.42, 10.50 ± 0.42 and 8.83 ± 0.30 mm were observed at 1:2, 1:4 and 1:8 dilutions, respectively. No zone of inhibition was observed at 1:16 and 1:32 dilutions. Against Escherichia coli, inhibition zones of 10.50 ± 0.42, 8.50 ± 0.42 and 7.00 ± 0.25 mm were observed at 1:2, 1:4 and 1:8 dilutions, respectively. No zone of inhibition was observed at 1:16 and 1:32 dilutions.

Discussion

The present study demonstrated lower post-treatment total viable counts in the iodine-treated and essential oil-treated groups compared with the distilled water control. Among the treatment groups, the essential oil-based teat dip consistently recorded the lowest post-treatment count at 10⁻³, 10⁻⁴ and 10⁻⁵ dilutions. The increase in total viable count in the distilled water control group indicated that distilled water alone did not provide an antimicrobial effect. In contrast, the lower posttreatment counts in the iodine and essential oil groups suggested an association between post-milking antimicrobial teat treatment and reduced viable bacterial counts. The lowest counts recorded in the essential oil-treated group may be associated with the antimicrobial properties of the constituent essential oils. The formulation contained eucalyptus, lavender, peppermint and tea tree oils, which together demonstrated measurable antibacterial activity against both Streptococcus spp. and Escherichia coli in the present investigation. The in vitro findings supported the microbiological observations. The undiluted essential oil blend produced the largest zones of inhibition against both tested bacterial organisms. The progressive reduction in the zone of inhibition with increasing dilution demonstrated a concentration-dependent antibacterial response. Previous studies cited in the thesis have also evaluated plant-derived and essential oil-based teat preparations for their potential role in reducing microbial contamination and improving udder health. The present results were consistent with the concept that plant-derived antimicrobial preparations may have potential application as post-milking teat disinfectants. An important consideration in interpreting the present findings is the statistical outcome of the pre-treatment and post-treatment comparison. Although lower numerical values were consistently recorded in the essential oil-treated group after treatment, the paired t-test comparison within the groups was reported as non-significant. Therefore, the results indicate a favourable treatment-associated pattern rather than establishing a statistically significant pretreatment to post-treatment reduction within the essential oil group.

Conclusion

The essential oil-based teat dip containing eucalyptus, lavender, peppermint and tea tree oils recorded the lowest post-treatment total viable counts among the experimental groups at 10⁻³, 10⁻⁴ and 10⁻⁵ dilutions. The essential oil blend also demonstrated concentration-dependent antibacterial activity against Streptococcus spp. and Escherichia coli. The findings indicated the potential of the tested essential oil formulation as a natural post-milking teat disinfectant. However, because the paired pre-treatment and posttreatment comparisons within the groups were reported as non-significant, further studies involving larger sample sizes and repeated observations are required to confirm its microbiological efficacy.

Acknowledgement

AcknowledgmentsThe authors are thankful to the authorities of the Post Graduate Institute of Veterinary and Animal Sciences, Akola, Maharashtra, and Shri Gaurakshan Sanstha, Akola, for providing the facilities and support required for conducting the research work.

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