Evaluating a Non-antibiotic Treatment of Mastitis in Organic Dairy Cows

LSSCC (Somatic cell count)

The analysis of LSSCC from milk samples collected on day 0 (before-treatment) and day 30 (after-treatment) of treatment indicated that TRT group had significantly lower LSSCC (P < 0.001) after treatment when compared to cows in the CON group (Figure 1 A). In agreement with our result,  Walkenhorst et al. (2020) reported the supplementation of multiherbal feed additives containing 27 different plants with chicory, rosemary, stinging nettle, fennel, and fenugreek as a major herbal components decreased (P < 0.05) the SCC as compared to the control counterparts showing the beneficial effects of plant based products to control bovine mastitis. Hashemzadeh-Cigari et al. (2014) also supplemented phytobiotics-rich herbal mixture composed of cinnamon bark, turmeric roots, rosemary leaves, and clove buds in diets to cows with high initial SCC, which decreased SCC in the milk as compared to the non-supplemented control cows. Salem et al. (2019) reported that supplementation with an oil mix (black seed oil , chamomile oil, and oregano oil) reduced (P < 0.05) somatic cell counts in Friesian cows, compared to the control diet with no supplementation. Cho et al. (2015) found that intramammary treatment with 0.9 ml oregano essential oil ointment for subclinical bovine mastitis caused by Staphylococcus aureus and Escherichia coli significantly (P < 0.05) reduced somatic cell counts compared to both the saline-treated negative control and the antibiotic ointment-treated positive control. This demonstrates the efficacy of plant-based products in lowering SCC, consistent with our findings, although the product was used topically compared to our approach of dietary supplementation.

When stratifying the study population into groups, cows in the late lactation had lower LSSCC (P < 0.05) in the TRT group compared to the CON group; however, no significant difference in LSSCC was observed between the treatment groups for cows in early and mid-lactation (Figure 1 B, C, D). Based on the involvement of mastitis causing pathogen, cows infected with environmental pathogen did not differ in LSSSC between the treatment groups on 30 days after treatment (Figure 1 E); however, cows infected with contagious pathogen had lower LSSCC (P < 0.05) in the TRT group than in the CON group on 30 days after treatment (Figure 1 F). These findings suggest that the supplementation of plant-based antibiofilm bolus was effective in reducing the somatic cell count (a proxy of the inflammation of mammary gland tissues) in late lactation cows and in mastitis events due to contagious pathogens.

Bacterial growth and count

Gram-positive bacteria were identified in 27% of the culture samples, whereas 0.4% were gram-negative, and 6% of the samples had no growth of bacteria (Table 1). Staphylococcus aureus (13%), Staphylococcus chromogenes (5%), and Streptococcus dysgalacticae (3%) were the top three most isolated pathogens from the milk cultures. Overall, cows in the TRT groups tended to have lower cfu counts (3.3 ± 0.05 vs 3.4 ± 0.05, P = 0.06) than CON groups. However, both groups have an increment (P < 0.05) in the number of bacteria on day 30 compared with day 0 (Figure 2 A). Cows grouped in early, mid, and late lactation did not differ in bacterial counts between the groups after- treatment (Figure 2 B, C, D). We were not able to identify a statistically significant difference in bacterial counts on day 30 when cows were stratified into environmental and contagious pathogens (Figure 2 E, F). These results suggest that although the treatment was effective in reducing inflammation in the mammary gland, as reflected by lower SCC, it did not significantly reduce the free-living bacterial population. The plant-based bolus may modulate immune responses and alleviate udder inflammation, yet it lacks strong local bactericidal activity against the pathogens. This is consistent with the hypothesis that the main action of the boluses is to reduce the virulence of bacteria, including their biofilm forming capacity. Thus, the active ingredients facilitate ‘unsheltered’ and ‘disarmed’ bacteria,  allowing the cow’s immune system to deal with them, rather than being directly bactericidal. While this is likely to result in reduced clinical signs, it may not lead to the total removal of bacterial burdens.

Bacteriological cure at day 30 after treatment

The odds of bacteriological cure at day 30 were 44% higher in the TRT group compared with the CON group, however, this difference was not statistically significant (OR=1.44; 95% CI: 0.68-3.04) (Table 2). We observed no statistically significant effect (P > 0.05) of treatment on the bacteriological cure when cows were stratified into early, mid, and late lactation. Similarly, no effect of treatment (P > 0.05) was observed in cows infected with environmental and contagious pathogens. The treatment was not able to reduce the number of mastitis-causing bacteria, showing no differences in bacteriological cure. The current study found a bacterial cure rate of 38% using the plant-based bolus supplementation which is comparable to other studies using plant-based options to treat mastitis through intramammary administration. The intramammary treatment with PhytoMast^® (botanical preparation composed of extracts of Thymus vulgaris, Gaultheria procumbens, Glyrrhiza uralensis, Angelica sinensis, and vitamin E) resulted in a bacteriological cure 42% compared with 29% for no treatment at day 14 (Pinedo et al., 2013). In another study, the application of Cinnatube^® (herbal teat sealant composed of Calendula, Cinnamomum spp., Eucalyptus gobulus, Melaleuca alterniflora, beeswax) as teat sealant resulted bacteriological cure rate of 34%, application of PhytoMast as intramammary treatment resulted cure rate of 46%, and the use of Cinnatube and Phytomast in combination in organic dairy farms resulted the bacteriological cure rate of 41%  with no difference compared to the control (cure rate: 41%) (Mullen et al., 2014). However, studies have identified that bacteriological cure rate varies depending on the causative agent, time, and type of treatment (Pinedo et al., 2013). Previous studies evaluating the use of antibiotics reported bacterial cure rates ranging from 44% to 90% depending on the type and dosage of the antibiotics used for mastitis treatment (McDougall, 1998; Wilson et al., 1999; Owens et al., 2001; Tomazi et al., 2021; Svennesen et al., 2023). Our result was closer to the result of Wilson et al. (1999), who reported the bacteriological cure of 44% for the pirlimycin when treated intramammarily, considering the definition of the cure rate. While we expected the plant-based antibiofilm bolus to achieve a higher bacteriological cure rate compared to the control, our goal was not to compare the efficacy with antibiotics, which are the gold standard for treatment. While the active compounds present in the bolus may exhibit bactericidal activity at higher doses, at the investigated concentrations our results primarily demonstrate low efficacy in reducing bacterial growth and increasing bacteriological cure incidence.

Daily milk yield

Daily milk yield was not different between the overall treatment groups (Figure 3 A), nor when stratified into early, mid, and late lactation groups (Figure 3 B, C, D). Although milk yield in TRT cows was numerically higher than that of the CON after day 5 of treatment, we were not able to detect statistical significance in this study. The cows infected with the contagious pathogens had higher milk yield (P < 0.05) in TRT group than in the CON group (Figure 3 F), however, the mastitis events associated with environmental pathogens did not differ in milk yield between the groups (Figure 3 E). This suggests that the plant-based bolus may reduce the negative impact of contagious pathogens on milk yield, demonstrating the treatment's effectiveness against these pathogens.  Herrema et al. (2023) evaluated the effect of the AHV product used in this study on milk production over a ten-year period (2013-2023) in the Netherlands, reporting a 1.6% increase in average milk yield per lactation in cows treated with the bolus. While the treatment may cumulatively affect milk production in the long term, we were not able to identify the difference in the short term. As most of the contagious pathogens observed in the study were gram-positive, the positive effect of treatment against this group of bacteria may be due to the difference in the structure of the cell wall, as gram-negative bacteria exhibit lipopolysaccharides anchored in the outer membrane of the cell wall, which restricts the diffusion of hydrophobic compounds (Burt, 2004; Nazzaro et al., 2013) . 

Daily rumination time

A significant treatment-by-day interaction was detected for rumination time (P < 0.01; Figure 4A). In the TRT group, daily rumination time demonstrates an increase after day 6 up to day 23 of treatment whereas no such pattern was observed on the CON group. Rumination time has been used as an indicator of the mammary health status in dairy cattle (Pahl et al., 2014; Paudyal, 2021). Although the average rumination time of TRT cows appeared higher than that of CON cows after approximately 10 days of treatment, these differences were not statistically significant on any individual day. This indicates that the interaction effect reflects differences in the overall pattern of rumination over time rather than statistically significant differences on individual days, likely due in part to the adjustment for multiple comparisons using Tukey’s method. Similar to our finding, the supplementation of Scutellaria baicalensis extract on short term (5 days) or long term (60 days) did not result in a difference in rumination time as compared to the control groups (Olagaray et al., 2019). However, Wang et al. (2025) supplemented poly-herbal mixture composed of Astragalus root, Codonopsis root, Angelica sinensis, Rehmannia root, tangerine peel, oriental arborvitae leaves, Atractylodes rhizome, licorice root, sichuan lovage rhizome, ophiopogon root, white peony root, hawthorn fruit, radish seeds, rhubarb root, oriental wormwood to the dairy cows and reported increased in rumination time than non-supplemental group. The difference in proportions of bioactive compounds in the herbal mixture may be attributed to the difference in effects. The average daily rumination time observed in this study for TRT group and CON group was 477.04 ± 3.3 min/d and 475.67 ± 3.1 min/d respectively which is higher than reported by the study of Paudyal et al. (2018) for the cows with mastitis (378.18 ± 11.5 min/d) in conventional dairy farms. The difference in rumination time of cows might be due to the difference in the production system, as the cow in organic production system had higher rumination time compared to conventional production system (Pereira and Heins, 2019). Similar treatment × day effects were observed (P < 0.05) in cows in early lactation where the temporal pattern of rumination time differed between treatments, although no daily differences between treatments were detected. In contrast, no effect of treatment on rumination time was observed between the cows in mid and late lactation groups (Figure 4 B, C, D). We did not detect a statistically significant overall effect of treatment on rumination time in cows associated with environmental pathogens. However, a significant treatment by day interaction was observed in cows associated with contagious pathogens (P < 0.05), where the TRT group showed a trend toward higher rumination time after approximately day 12 of enrollment (Figure 4 E, F), although no daily differences between treatments were detected. While we identified that supplementation with the plant-based bolus would improve rumination time in some groups, the treatment did not have any negative effects on rumination in any cows, although it was lodged in the rumen locally.

Daily water intake

The analysis of the water intake data indicated no statistically significant difference in the water intake between the overall treatment groups (Figure 5 A). This indicates that the supplementation of plant-based bolus does not significantly improve or impair the water intake of cows. The result showed a numerical increase in water intake in TRT compared to the CON after day 5 of treatment, however the difference was not statistically significant. The cows in early lactation tended to have higher (P = 0.06) water intake in TRT group than in CON group (Figure 5 B) whereas no effect of treatment was observed in cows in mid and late lactation (Figure 5 C, D). The cows infected with contagious pathogen had higher (P < 0.05) water intake in TRT group than in the CON group (Figure 5 F) however, the effect was not observed in cows infected with environmental pathogen (Figure 5 E). This supports the higher milk yield in the cows in the TRT group infected with a contagious pathogen than in the control group, as increased milk yield is associated with increased water intake (Kume et al., 2010). Meyer et al. (2004) reported that the production of each liter of milk increased the demand for 1.3 liters of drinking water in a dairy cow. The average water intake for cows in TRT group (95.2 L/d) and CON group (91.4 L/d) in this study was higher as compared to the water intake of cow reported by Meyer et al. (2004) (81.5 ± 19.1 L/d) and Cardot et al. (2008) (83.6 ± 17.1 L/d), however the comparison of water intake of cows between these experiments need to be done cautiously. Factors like ambient temperature, dry matter intake, health status of the cows, and diet impact the intake of drinking water (Meyer et al., 2004; Cardot et al., 2008; Lukas et al., 2008).

Survival in the herd

No difference in the survival curves within the follow-up period of 90 days was observed for the TRT group and CON group (log-rank test = 0.33) when analyzed using Kaplan-Meier survival estimates. The overall cumulative incidence of removal from the herd (culling or death) due to mastitis was 0.9% for CON and 0% for TRT group, that is, no cows were removed from the herd due to mastitis in the TRT group.

Economic evaluation

The partial budget analysis utilizes a milk price of $28.00/cwt, reflecting a conservative yet realistic benchmark for the US organic dairy market (USDA-ERS, 2025; Zadoks & Fitzpatrick, 2009). While the high upfront cost of the plant-derived bolus kit results in a short-term net deficit of –$80.22 per cow, the long-term benefit over a 305-day lactation is significant. This benefit is driven by a sustained milk yield increase of 3.31 lbs./day (1.5 kg/day) and the elimination of discarded milk costs, as the organic bolus requires zero milk-withhold (Petrovski et al., 2006). In organic systems, the true economic value is found in herd longevity; avoiding a single culling event, often valued at over $1,000, far outweighs the initial treatment investment (Hagnestam-Nielsen, 2009; Tommasoni et al., 2023).