Mastitis, considered one of the significant issues in the dairy production chain in Brazil and worldwide, significantly impacts the economy and milk quality due to the presence of antibiotic residues, early animal culling and the development of multi-resistant bacteria (Langoni et al., Reference Langoni, Oliveira, Junqueira, Menozzi and Joaquim2017; Caliman et al., Reference Caliman, Gasparotto and Ribeiro2023; Lima et al., Reference Lima, Gouveia, Praia, Vieira, Rego, Moura, Riff, Leite, Silva and Neves2023). The indiscriminate use of antibiotics in mastitis treatment leads to milk contamination, rendering it unsuitable for human consumption and potentially causing allergic reactions or poisoning, thereby negatively interfering with the entire production process (Lobato and De Los Santos, Reference Lobato and De Los Santos2019). Furthermore, the use of drugs generates high production costs, which brings with it the imminent need to seek other prevention and treatment methods with alternative or integrative therapies (Nóbrega et al., Reference Nóbrega, Langoni, Joaquim, da Silva, Faccioli, de Matos and Menozzi2009).
Alternative and homeopathic therapies for mastitis prevention and treatment in dairy herds are gaining traction in the agricultural sector to reduce economic losses and environmental pollution (Nóbrega et al., Reference Nóbrega, Langoni, Joaquim, da Silva, Faccioli, de Matos and Menozzi2009). In recent years, the organic products market has experienced a growth of 30% in 2020 and 12% in 2021 in Brazil. The organic market has shown great potential for development, emerging due to consumers’ interest in healthier foods and concern about reducing environmental impacts (SEBRAE, 2022).
Ozone therapy is increasingly used in veterinary medicine to integrate therapies and assist treatment processes, reducing treatment time (Ogata and Nagahata, Reference Ogata and Nagahata2000) and is already widely used as a sanitizing product in major hospitals (Garcia et al., Reference Garcia, Stanziola, IdeS, JHFdeF and Neves2008). It decomposes into O2 after coming in contact with the biological material, leaving no residue. The use of ozonated water in dairy production is an alternative to reduce antimicrobial use and milk disposal time due to allopathic therapy (Fuentes et al., Reference Fuentes, Achy, da Silva, Graboschii, JdeO, Joaquim, Fraga and Escodro2023; Oliveira et al., Reference Oliveira, MddeM, JCdosS and Brito2023).
The main preventive methodologies for mastitis occur moments before and immediately after milking, using pre- and post-dipping (Rather et al., Reference Rather, Muhee, Bhat, Haq, Parray, Taifa, Nisar, Nabi, Hussain and Beigh2020). Considering that ozone has been shown to be very effective at eliminating mastitis-causing bacteria in vitro, this study tested ozonated water in pre- and post-dipping in dairy cows, comparing it with a commercial sanitizer to evaluate its effect on mastitis prevention and somatic cell count (SCC).
Materials and methods
This research was conducted at Fazenda Lagoa da Jurema in Major Izidoro, Alagoas, Brazil and is part of a research project of the Alagoas Research Support Foundation, approved by the Ethics Committee (CEUA-UFAL) under protocol number 04/2020.
Animals and management
The study involved 58 crossbred Holstein and Gir lactating dairy cows with an average age of 4.9 ± 1.58 years, all negative for mastitis based on black-bottom cup tests and California mastitis test (CMT). The cows were randomly divided into two groups: the control group (CG, n = 23), where pre- and post-dipping were done with hydrogen peroxide at 0.5 ml/100 ml, and the ozone group (O3G, n = 35), where ozonated water at 2 µg/ml was used. This concentration corresponds to the level of ozone that the equipment allows to be maintained stably in the water during the sanitization process (Ozone&Life, São José dos Campos – SP). The average milk production was 25.1 kg/animal/day in CG and 27.6 kg/animal/day in O3G.
Cows were kept in an extensive system, fed post-milking with forage cactus (35 kg/animal/day), bagasse (7 kg/animal/day), cottonseed (2 kg/animal/day) and barley (10 kg/animal/day), divided into two meals per day, with water and mineral supplementation ad libitum. Milking was performed twice daily, with all samples collected during the first milking.
Methodological procedures and experimental design
In CG, the farm's standard sanitary management was performed using a hydrogen peroxide solution at 0.5 ml/100 ml (Prima™ DeLaval, Jaguariúna – SP) in pre-dipping and post-dipping. In O3G, pre- and post-dipping were performed with ozonated water. Ozonation was done immediately before each milking using an ozone generator model O&L JET 2 (Ozone&Life, São José dos Campos – SP) coupled to a 5 LPM oxygen generator (Longfian®, Hebei Province, China) with a capacity of 10 g/h, combined with a 20-l mineral water container via a diffuser pipe at an oxygen flow of 3 l for 5 minutes, achieving a concentration of 49 µg/ml totalling 2 µg O3/ml. The solution was immediately placed in a non-return applicator bottle for pre- and post-dipping.
At the beginning of milking, the teats were cleaned using running water and paper towels, and the pre-dipping solution was applied. This was followed by wiping before cluster attachment, and then post-dipping with the respective solutions according to the group. Daily black-bottom cup tests and clinical udder and teat examinations were conducted. CMT was performed, and the degree of gelation was assessed as negative, traces, grade 1, grade 2 or grade 3, according to the gelatinous formation presented by each sample (Rebhun, Reference Rebhun2000).
A sample of 25 mL milk was collected before the cluster placement in a sterile plastic tube with lid and analysed for the efficacy of the ozonated solution through somatic cell count (SCC), pH analysis, milk composition analysis and microbiological analysis of teats at time points M0 (pre-experiment), M1 (72 hours after experiment start), M2 (7 days after experiment start) and M3 (14 days after experiment start).
SCC was measured using the Somaticell CCS® kit (Somaticell Diagnósticos, Jundiaí – SP) as per the manufacturer's instructions, where 2 mL of collected milk was homogenized with 2 mL of the kit reagent in a graduated tube for somatic cell count per millilitre. After homogenization, the tube was rotated 180º for 20 seconds, where the mixture flowed through a hole in the tube cap. After this time, the tube returned to its original position for reading the somatic cell count based on the agglutination of somatic cells with the reagent (Avanço et al., Reference Avanço, Santos and Ponsano2011). The Somaticell CCS® kit also allows pH analysis according to a colorimetric scale after homogenization and reading, classified as normal milk (dark orange), alkaline milk (reddish) and acidic milk (yellow) (Avanço et al., Reference Avanço, Santos and Ponsano2011). For milk composition analysis, 2 ml samples were filtered through 80 g and 90 mm filter paper and analysed with an automatic milk analyser Master Mini – AKSO®. The analyser provided results for fat content (F), non-fat solids (S), density (D), protein (P), lactose (L), salts (SI), added water (Aq), freezing point (FP) and temperature (Temp). The suction nozzle of the analyser was washed twice consecutively between each sample to avoid cross-contamination (Sivestrin et al., Reference Sivestrin, LWdeB and APde2022).
For the microbiological analysis of the teat, ten animals were randomly selected from each sample group corresponding to a substantial fraction of the population studied (43.5% of the control group and 28.6% of the ozone group), and is therefore considered representative for the assessment of the microbiological variables of milk.
The number of ten animals per group was determined based on experimental feasibility, since the experiment was conducted during the farm's milking hours so as not to interfere or delay the usual milking routine, as well as on the limited resources available for laboratory analyses and the ethical management of the animals, while maintaining a sample size compatible with the total number of animals in the study. Samples were collected after pre-dipping as standard procedure from the right rear teat using swabs containing Stuart medium in circular movements around the teat sphincter. The material was stored in polystyrene foam boxes containing isothermal ice and sent for laboratory analysis, where it was seeded in brain–heart infusion (BHI) medium for 4 hours, and subsequently seeded on plates with blood agar, chocolate agar, McConkey agar and mannitol agar media, kept for 72 hours at 35ºC for subsequent identification of microorganisms, according to the technique described by Abreu et al. (Reference Abreu, Medeiros and Santos2011).
Statistical analysis
The mean and standard deviation of the profile of the selected animals in each group were calculated. The variables studied, such as pH, SCC, milk composition – fat, solids, salts, protein, lactose, freezing point, density and water, were subjected to the Shapiro–Wilk normality test. The variables that met the assumption of normality were subjected to the t-test for comparison between the groups. The Mann–Whitney test was applied to the milk composition variables and to the variables that did not present normal distribution, such as pH, SCC, microbiological growth and fat. The CMT variable was subjected to the chi-square (2 × 2) test of independence.
Results
The results of the black bottom cup test and physical examination of the udder did not show any changes in the results of the animals studied throughout the experiment, in both groups. Likewise, no animal developed clinical mastitis or needed to receive any treatment, and none needed to be excluded from the experiment.
When analysing the CMT, the comparative results between the groups and between the moments did not demonstrate a significant association, being: M1 = χ2 (1) = 1.054, P = 0.304, φ = 0.14; M2 = χ2 (1) = 0.178, P = 0.673, φ = 0.06; M3 = χ2 (1) = 0.1.353, P = 0.245, φ = − 0.15. At the end of the experiment (M3), in the CMT analysis between the groups, it was observed that 56% of the animals in the control group and 57% of the animals in the ozone group presented results within the normal range. Grades 1 and 2 gelation changes of subclinical mastitis were observed in few mammary quarters, without progression to clinical mastitis and without the need for treatment in the animals.
The SCC of both groups was within the normal range of values established by Normative Instruction 76 of the Brazilian Ministry of Agriculture, Livestock and Food Supply (IN 76) (Brazil, 2018), which recommends a maximum value of 500 × 103 cells/ml as safe values for human consumption. The average SCC between the groups, at the times studied, showed no significant difference in any of the analyses (P > 0.05, Table 1).
Concentration of somatic cells (×103 cells/ml) evaluated at moments M0 (pre), M1 (72 hours), M2 (7 days) and M3 (14 days) in the control groups and in the ozone group
Table 1 Long description
The table reports mean somatic cell concentration with standard deviation, in thousands of cells per milliliter, measured at baseline, 72 hours, 7 days, and 14 days for a control group and an ozone group. In the control group, the mean increased from 278.26 at baseline to 363.04 at 72 hours, then decreased to 250.00 at 7 days and 241.30 at 14 days. In the ozone group, means were 324.29 at baseline, 327.14 at 72 hours, 295.71 at 7 days, and 304.29 at 14 days, indicating little overall change. At 72 hours, the control group mean is marked as different from its baseline value, while no such marker appears for the ozone group. Variability is moderate in the control group and very large in the ozone group at all time points, which may limit confidence in small between-group differences.
* a is significantly different than b.
In the pH analysis, it was observed that all samples presented a value within the range stipulated by IN 76 (Brazil, 2018), which recommends a maximum pH value of 6.8. The average pH value between the groups studied did not present a significant difference in any of the analyses of the samples studied (P > 0.05) (Table 2).
pH values at moments M0 (pre), M1 (72 hours), M2 (7 days) and M3 (14 days) in the control group and in the ozone group
Table 2 Long description
The table reports pH measurements at four time points (baseline, 72 hours, 7 days, 14 days) for a control group and an ozone group, summarized by mean with standard deviation plus minimum, maximum, and median. In the control group, mean pH is 6.24 at baseline, 6.36 at 72 hours, 6.29 at 7 days, and 6.16 at 14 days, indicating a slight rise at 72 hours followed by a small decline by day 14. Control group medians are 6.4 at baseline, 72 hours, and 7 days, then 6.0 at 14 days; minima are 6.0 throughout, and maxima are 6.8 through 7 days and 7.0 at 14 days. In the ozone group, mean pH is 6.27 at baseline, 6.43 at 72 hours, 6.26 at 7 days, and 6.22 at 14 days, showing a small peak at 72 hours and then a return close to baseline. Ozone group medians are 6.4 at baseline and 72 hours, then 6.0 at 7 and 14 days; minima are 6.0 throughout, and maxima are 6.8 at baseline, 7.5 at 72 hours and 7 days, and 7.0 at 14 days. Overall, both groups remain in a similar pH range with modest time-related fluctuations, and the ozone group shows higher upper-end values at 72 hours and 7 days. No statistical testing is provided, so differences should be interpreted as descriptive only.
Milk composition variables such as fat, non-fat solids, density, protein, lactose, salts, added water, freezing point and temperature did not show any noteworthy changes in any of the groups and moments, being within the standards required by Normative Instruction No. 76 (Brazil, 2018).
Regarding the microbiological analysis of the teat samples, eight different species of microorganisms were isolated. At all times, pathogens that cause environmental mastitis and infectious mastitis were found. In M0, bacteria of the species Escherichia coli, Staphylococcus epidermidis, Staphylococcus chromogenes and Staphylococcus aureus were identified in both groups. In M1, in addition to the bacteria observed in M0, the bacterial population presented Enterobacter spp., Klebisiella sp. and Enterococcus spp. In M2, a difference was observed between the percentage of the bacterial population, with a decrease in the population of E. coli, S. aureus, S. epidermidis, S. chromogenes and an increase in Enterobacter spp., Klebisiella spp. and Enterococcus spp. In M3, only the species E. coli, Bacillus spp., Enterobacter spp., Klebisiella spp. and Enterococcus spp. were found as demonstrated in the table (Table 3).
Percentage of pathogens found by group at the specific times evaluated
Table 3 Long description
The table reports the percentage of isolates by pathogen for two groups, GC and GO3, measured at four timepoints labeled M0 through M3. Escherichia coli dominates in both groups throughout: GC is 52.94 at M0, 35 at M1, 36.84 at M2, and 41.17 at M3; GO3 is 47.36 at M0, 52.63 at M1, 30.30 at M2, and 50 at M3. Several staphylococci appear mainly at M0 and then drop out: in GC, Staphylococcus epidermidis is 23.52 at M0 and Staphylococcus aureus is 5.88 at M0, with no later values shown; in GO3, S. aureus is 5.26 at M0, rises to 10 at M1, is 3.03 at M2, and is absent at M3, while S. epidermidis declines from 10.52 at M0 to 5 at M1 and 6.06 at M2, then is absent at M3. Bacillus spp. stays low in GC but increases slightly from 5.88 at M0 to 11.76 at M3; in GO3 it is 5.26 at M0 and 5 at M1, with no values shown at M2 or M3. Enterobacter spp. and Enterococcus spp. emerge after M0 in GC, reaching 23.52 each by M3; in GO3, Enterobacter spp. is higher than GC from M1 onward, rising from 26.31 at M1 to 35 at M3, while Enterococcus spp. appears only at M2 at 12.12 and is absent at other timepoints. Klebsiella spp. appears from M1 onward in both groups, with GO3 reaching 15 at M3 while GC has no value shown at M3. Dashes indicate missing or not detected values, so comparisons at those cells should be interpreted cautiously.
Discussion
CMT is a low-cost test that is easily performed before milking, as its results are immediate for the diagnosis of subclinical mastitis (Ferreira et al., Reference Ferreira, Guirro, Blagitz and Ammpd2015). Its evaluation is performed by gelatinizing the sample of milk homogenized with the reagent and, indicatively, through viscosity, demonstrating a higher number of somatic cells (Brazil, 2012). According to Rebhun (Reference Rebhun2000), gelation changes are correlated to approximate somatic cell values, being considered negative up to 200 × 103 cells/ml of milk, traces 150–500 × 103 cells/ml or subclinical mastitis divided into grades 1, 2 or 3, according to the amount of somatic cells found in the milk, being grade 1 from 400 × 103 to 1,000 × 103 cells/ml, grade 2 from 800 × 103 to 5,000 × 103 cells/ml and grade 3 above 5,000 × 103 cells/ml of milk.There was no significant difference in the results between the groups studied at any of the sample collection times in the CMT test, showing the same behaviour between the groups.
In the animals studied in both groups, there was no significant difference in the SCC count, as these were groups of individuals of similar age and reproductive period. When the pH was analysed, the average was within normal values. Fresh milk has a slightly acidic pH, ranging from 6.6 to 6.8, with an average of 6.7 at 20°C or 6.6 at 25°C. When there is inflammation in the mammary glands, as in mastitis, the milk becomes more alkaline, with an average pH between 7.3 and 7.5 (Brazil, 2018).
The alkaline pH in cases of mastitis occurs due to the vasodilation that the inflammatory process causes in the mammary gland; the increase in blood cells also increases the pH in the milk. In addition to the inflammatory process altering the pH, bacterial proliferation in mastitis, depending on the type of bacteria, is also capable of modifying the pH of the milk (Manzi and Langoni, Reference Manzi and Langoni2017).
The results show that the ozonated solution did not interfere with the composition of the milk, there was no difference between the values found and the values established in relation to CCS, pH, and milk composition according to the current Normative Instruction IN 76, 2018 (Brazil, 2018) and considering the ozone's property of decomposing into O2 when it comes in contact with biological materials, no residues were found, corroborating Ogata and Nagahata (Reference Ogata and Nagahata2000), Arévalo et al. (Reference Arévalo, Silva, Graboschii, Brito and Escodro2021), Fuentes et al. (Reference Fuentes, DFda, Joaquim, Fraga and Escodro2022) and Fuentes et al. (Reference Fuentes, Achy, da Silva, Graboschii, JdeO, Joaquim, Fraga and Escodro2023). The main source of antibiotic residues in milk comes from erroneous handling of drugs in an attempt to control mastitis (Jones, Reference Jones2009). These drugs have a withdrawal period during which they are secreted into the milk, making it necessary to discard this production (Hillerton et al., Reference Hillerton, Halley, Neaves and Rose1999; Van Schaik et al., Reference Van Schaik, Lotem and Schukken2002)
Regarding the possible long-term effects of ozone exposure, it is important to consider both the skin of the mammary quarters and the milk components. In controlled topical use, ozone induces moderate oxidative stress, capable of activating endogenous antioxidant pathways (such as the Nrf2 pathway – a cell signalling route that regulates the body's response to oxidative and inflammatory stress) and modulating the inflammatory response, which explains its application in different skin conditions in veterinary medicine, when dose and contact time are respected (Sciorsci et al., Reference Sciorsci, Lillo, Occhiogrosso and Rizzo2020). On the other hand, excessive or prolonged exposure can theoretically lead to dryness, irritation, or damage to the teat epithelium. For this reason, in the present protocol, a low concentration (2 µg/ml) and short contact time (30–60 s) were chosen, followed by careful drying.
The most commonly described pathogens causing mastitis in the literature are S. aureus, E. coli, Klebsiella sp., and Enterobacter sp., among others (Langoni et al., Reference Langoni, Silva, Cabral and Domingues1998; Fonseca and Santos, Reference Fonseca and Santos2001; Martins et al., Reference Martins, Silva, Nakazato, Dutra and Almeida Filho2010). The same pathogens were found in this study in the groups studied.
It was observed that the pathogens of the genus Staphylococcus spp. found at moments M0, M1 and M2 were not found at moment M3 in any of the groups studied. The same result was not found by Moreira et al. (Reference Moreira, Moretti, Lima, Kozusny-Andreani and Rodriguez2014), where the experiment with intramammary ozone gas at a concentration of 10 µg/ml in a single dose did not demonstrate efficacy against the pathogen. The author raises the hypothesis that the concentration of ozone was not sufficient against the bacteria.
Considering the aetiology of the pathogens causing mastitis, Hoe and Ruegg (Reference Hoe and Ruegg2005) observed that in relation to herds where there were cases of mastitis caused by S. agalactiae and S. aureus, and it was controlled, describing the increase of isolated microorganisms causing environmental mastitis due to being opportunistic, such as Escherichia coli and Klebsiella spp. Similar to what was observed in this study, according to the continuous use of ozonated water and sanitizer in the respective groups, no bacteria of the genus Staphylococcus sp. were isolated after the treatments in both groups, but environmental bacteria such as E. coli and Klebsiella spp. remained.
A study by Tormin et al. (Reference Tormin, Navarini, Almeida, Travassos, Negri and Silva2016) demonstrated good results in relation to the infusion of ozone in a broth containing bacteria at a concentration of 10 µg/ml for 4 min. The same was not observed in the same study with different concentrations and times of exposure to ozone. In this study, the time of exposure of ozonated water on the teats may have influenced the presence of some pathogens; however, their growth remained controlled.
The results observed with the use of ozonated water can be interpreted based on the mechanism of action of ozone on microorganisms. Ozone has a high oxidative power, capable of reacting directly with membrane lipids and surface proteins, promoting lipid peroxidation, alteration of structural and transport proteins, as well as oxidative damage to nucleic acids (Thanomsub et al., Reference Thanomsub, Anupunpisit, Chanphetch, Watcharachaipong, Poonkhum and Srisukonth2002; Cenci et al., Reference Cenci, Macchia, La Sorsa, Sbarigia, Di Donna and Pietraforte2022; Travagli and Iorio, Reference Travagli and Iorio2023). Consequently, there is a loss of membrane integrity, increased permeability, leakage of intracellular contents, and ultimately, cell death. This multi-target action explains why, even at relatively low concentrations and short contact times, ozonated water exhibits a relevant antimicrobial effect on biological surfaces. In the context of the present study, the lower frequency of isolation of potentially pathogenic bacteria after the application of the ozone protocol is consistent with this mechanism of cumulative oxidative damage to the microbial cells present on the teats’ skin.
Fuentes et al. (Reference Fuentes, Achy, da Silva, Graboschii, JdeO, Joaquim, Fraga and Escodro2023) observed that the treatment of clinical and subclinical mastitis with the use of intramammary ozonated gas is effective against bacteria of the genus Staphylococcus sp. Massote et al. (Reference Massote, Zanateli, Alves, Gonçalves and Guedes2019) mention the importance of environmental factors in relation to bovine mastitis. Cleaning of milking teat cups, milking room and teats. Cleaning of teat cups was not possible on the farm of this study between milkings of each animal, which may have contributed to the persistence of some pathogens isolated in the study, but still with controlled growth, not evolving into the disease.
Several studies evaluating the direct ozonation of milk report that higher ozone concentrations and prolonged exposure times can alter fat and protein fractions, as well as some sensory attributes, due to lipid oxidation and modifications in protein structure (Varga and Szigeti, Reference Varga and Szigeti2016; Afonso et al., Reference Afonso, Moreira and Almeida2022; Vashisht et al., Reference Vashisht, Verma, Pradeep Raja Charles, Singh Saini, Sharma, Singh, Mahanta, Mahanta, Singha and Gaurav2023). It is essential to note, however, that in this study, ozone was applied externally to the teat skin at a low concentration and for a brief period, and the excess solution was removed by drying before attaching the teat cups, thereby minimizing the possibility of direct contact with the milk inside the mammary gland. Although this study did not include a detailed evaluation of the physicochemical composition of the milk, the protocol adopted and the short half-life of ozone in aqueous medium suggest that the probability of a relevant impact on milk components is low. Nevertheless, future studies are suggested that include serial evaluation of teat epithelial integrity and compositional analysis of milk after prolonged use of ozonated water.
With the increase in consumers seeking healthier and more sustainable foods, the production of milk and organic foods has been growing exponentially, but a major obstacle is still the production costs (SEBRAE, 2022). Researchers are looking for ways to more easily apply ozone, mainly via the intramammary route, such as probes, which are low-cost according to data from a prospecting study by Arévalo et al. (Reference Arévalo, Almeida and Escodro2022). Ozone therapy might be implemented as a viable and sustainable alternative in the milk production chain because it is a cheaper sanitizing option, but more studies are needed.
Conclusion
The ozonized solution did not promote physicochemical changes in the milk, nor in the somatic cell count, showing similar effectiveness to hydrogen peroxide in controlling and maintaining somatic cell count and controlling mastitis, thus maintaining the expected quality of the product. Although the study was successful regarding the effectiveness of ozonized water in pre- and post-dipping use in the prevention of mastitis in dairy cows, more research on dosage, application time and effectiveness should be carried out to consolidate the technique.
Acknowledgements
The authors thank the Federal University of Alagoas and the Alagoas Research Support Foundation for their support and funding. Special thanks to the staff at Fazenda Lagoa da Jurema for their assistance and cooperation throughout the study.
Funding statement
This study was funded by the Alagoas Research Support Foundation (FAPEAL), grant number 04/2020.
Conflicts of interest
The authors declare no conflicts of interest related to this study.
