https://doi.org/10.52973/rcfcv-e34466

Received: 05/06/2024 Accepted: 22/07/2024 Published: 24/09/2024

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Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34466

ABSTRACT

Subclinical mastitis is a signicant issue in dairy farming due to its

impact on milk production and quality, leading to economic losses

despite the absence of visible symptoms. The present study aimed to

investigate the potential use of intramammary hypochlorous acid (HOCl)

treatment as a viable substitute to antibiotics for the treatment of

subclinical mastitis (SCM) in dairy cows. A total of 232 Holstein–Friesian

dairy cows with 928 udder quarters were subjected to the California

Mastitis Test (CMT) to identify the SCM. The results indicated that 141

cows had SCM in at least one udder quarter, with a rate of 60.78% in the

herd. Among these cows, 259 udder quarters showed varying degrees

of CMT–positive. Before the treatment, somatic cell counts (SCC)

were determined, and bacterial cultures were performed on randomly

selected 74 CMT–positive udder quarters. The HOCl was administered

intramammary to these quarters for 5 days immediately after milking.

The CMT, SCC, and bacterial culture were repeated on the 3

and 5

days of the treatment. An increase in SCC was observed on the 3

and

day of the treatment compared to before treatment (P<0.001). The

bacterial growth reduced from 64.86% before treatment to 49.95% on

the 3

day and 22.97% on the 5

day of the treatment. Staphylococcus

aureus was the most prevalent bacterium before the treatment. On the

day of the treatment, bacterial growth rate, particularly in Candida

spp., decreased compared to before the treatment. However, on the 5

day of the treatment, S. aureus and the combination of S. aureus with

Candida spp. continued to show high growth rates. In conclusion, this

study underscores that HOCl is a potential alternative to antibiotics

for treating SCM in dairy cows. Further research covering both clinical

and subclinical mastitis is recommended, along with studies aiming to

prolong the presence of HOCl in the udder, determine its ideal dose,

and increase its impact on more cells.

Key words: Dairy cow; hypochlorous acid; subclinical mastitis;

treatment

RESUMEN

La mastitis subclínica es un problema signicativo en la producción

lechera debido a su impacto en la producción y calidad de la leche, lo

que lleva a pérdidas económicas a pesar de la ausencia de síntomas

visibles. El presente estudio tuvo como objetivo investigar el uso

potencial del tratamiento intramamario con ácido hipocloroso (HOCl)

como un sustituto viable a los antibióticos para el tratamiento de

la mastitis subclínica (MSC) en vacas lecheras. Un total de 232

vacas lecheras Holstein–Friesian con 928 cuartos mamarios fueron

sometidas a la prueba de mastitis de California (CMT) para identicar

la MSC. Los resultados indicaron que 141 vacas tenían MSC en al menos

un cuarto de la ubre, con una tasa del 60.78% en el rebaño. Entre

estas vacas, 259 cuartos mamarios mostraron diversos grados de

CMT positivo. Antes del tratamiento, se determinaron los conteos

de células somáticas (SCC) y se realizaron cultivos bacterianos en

74 cuartos mamarios CMT positivos seleccionados al azar. El HOCl

se administró por vía intramamaria a estos cuartos durante 5 días

inmediatamente después del ordeño. El CMT, el SCC y el cultivo

bacteriano se repitieron en el tercer y quinto día del tratamiento. Se

observó un aumento en el SCC en el tercer y quinto día del tratamiento

en comparación con antes del tratamiento (P<0.001). El crecimiento

bacteriano se redujo del 64.86% antes del tratamiento al 49,95% en el

tercer día y al 22,97% en el quinto día del tratamiento. Staphylococcus

aureus fue la bacteria más prevalente antes del tratamiento. En

el tercer día del tratamiento, la tasa de crecimiento bacteriano,

particularmente en Candida spp., disminuyó en comparación con

antes del tratamiento. Sin embargo, en el quinto día del tratamiento,

S. aureus y la combinación de S. aureus con Candida spp. continuaron

mostrando altas tasas de crecimiento. En conclusión, este estudio

subraya que el HOCl es una alternativa potencial a los antibióticos

para tratar la MSC en vacas lecheras. Se recomienda realizar más

investigaciones que abarquen tanto la mastitis clínica como la

subclínica, junto con estudios que apunten a prolongar la presencia

de HOCl en la ubre, determinar su dosis ideal y aumentar su impacto

en más células.

Palabras clave: Ácido hipocloroso; mastitis subclínica; tratamiento;

vaca lechera

Effect of Intramammary Hypochlorous Acid Administration on Subclinical

Mastitis in Dairy Cows

Efecto de la Administración Intramamaria de Ácido Hipocloroso

en la Mastitis Subclínica en Vacas Lecheras

Eyyup Hakan Ucar* , Cevdet Peker

Aydin Adnan Menderes University, Faculty of Veterinary Medicine, Department of Obstetrics and Gynecology. Aydin, Türkiye.

*Corresponding author: hakan.ucar@adu.edu.tr

TABLE I

Experimental design

Day 0 Day 1 Day 2 Day 3 Day 4 Day 5

12 h

1. milking 3. milking 5. milking 7. milking 9. milking

HOCl HOCl HOCl HOCl HOCl

2. milking 4. milking 6. milking 8. milking 10. milking

1. CMT

2. SCC

3. Sampling

4. Milking

HOCl HOCl

1. CMT

2. SCC

3. Sampling

4. Milking

5. HOCl

HOCl

1. CMT

2. SCC

3. Sampling

4. Milking

HOCl: Hypochlorous Acid, CMT: California Mastitis Test, SCC: Somatic Cell Count, Sampling: Sample

collection for bacterial culture

Subclinical mastitis in dairy cows / Ucar and Peker __________________________________________________________________________________

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INTRODUCTION

Mastitis is an inammatory condition of mammary tissue and the

surrounding connective structures. It is one of the most important

economic problems in dairy cattle farming [1]. Due to its highly complex

aetiology, it is impossible to eradicate under current conditions.

Mastitis leads to economic losses, including reduced milk yield, milk

loss, decreased milk quality, treatment costs, decreased value of the

cow, culling of the mastitic cows, and costs of protection and control

measures [2]. Clinical mastitis (CM) is typically identiable through

conspicuous symptoms like udder swelling and milk clot formation,

but subclinical mastitis (SCM) manifests without visible changes in

the udder or milk [3]. The SCM is reported to be 15–40 times more

prevalent than its clinical counterpart [4]. This subtle form, often

unnoticed by farmers, has the potential to infect healthy animals within

the herd and cause greater economic losses than the clinical form [5].

It is reported that 70–80% of the nancial losses due to mastitis are

caused by the SCM [1]. From a public health perspective, there is a

risk of pathogenic microorganisms in mastitic milk and dairy products

being transmitted to humans. Additionally, the presence of antibiotic

residues in milk due to the treatment of mastitis poses another public

health threat and diminishes the milk quality [6]. Microorganisms in

milk can lead to food toxicities in humans, while antibiotic residues can

cause allergic reactions. The use of antibiotics may also contribute to

the development of resistant bacterial strains in milk, and consuming

such milk may result in antibiotic resistance [7, 8].

Subclinical mastitis can be detected by tests such as the California

Mastitis Test (CMT) or Somatic Cell Count (SCC) measurement [3].

The SCC is a crucial parameter used in assessing milk quality and

diagnosing SCM [9, 10, 11, 12]. Somatic cells are dened as epithelial

cells that pass from the mammary tissue and inammatory cells that

pass from the systemic bloodstream into the milk. Most somatic

cells are macrophages, neutrophils, and leukocytes [13]. High SCC

in milk results, indicating intramammary infection primarily, also

leads to decreased industrial eciency and shorter shelf life of

dairy products [14, 15, 16, 17]. The CMT is a practical method used

to interpret the somatic cells in udder quarters, and an increase in

CMT score indicates high SCC in the milk [18].

It needs to devise a strategy that detects mastitis at earlier stages

and can be treated with the lowest–cost alternative medicines

regardless of severity. Such measures can increase the milking life

of dairy animals and help ght antibiotic resistance, which is an

important factor threatening human health. For this purpose, research

is being conducted on alternative mastitis treatment approaches

that do not leave residues in milk.

Hypochlorous acid (HOCl), a naturally produced compound by

neutrophils, has exhibited antimicrobial and wound–healing properties

without cytotoxicity for the last 15 years [19]. It is considered a

physiological substance that is non–ammable and non–irritating,

and compatible with the tissues and blood pH. It can be applied to

all mucous membranes and enter the systemic circulation. It has

been reported that it does not cause any toxic or adverse reactions

and provides asepsis of infected areas. It has been used in various

surgical procedures, such as perforated appendicitis, sternotomy,

mediastinitis, and peritoneal lavage [20, 21]. Hypochlorous acid,

reported to have a broad spectrum of antimicrobial effects, is effective

in as short as 12 seconds and has antibacterial, antiviral, antifungal,

antiparasitic, and anti–inammatory effects [21, 22, 23, 24]. Due to

its ability to reduce bacterial load on the periocular skin surface, it

has been used in the treatment of blepharitis [25], cleaning biolm–

contaminated implant surfaces [26], intraperitoneal treatment [27],

open wound treatment [21, 28], and as a potent antiseptic [29]. It has

also been reported to be effective against viruses [30, 31].

This study aimed to investigate the safety and ecacy of HOCl, a

promising alternative to antibiotics with no known side effects that

can be used safely in human medicine to treat SCM in dairy cows.

MATERIALS AND METHODS

Animal material

The study was carried out on 232 Holstein–Friesian dairy cows on

a dairy farm with integrated facility conditions (shared ration and

maintenance conditions).

Clinical procedures

The sequence of procedures on udders with the SCM included the

CMT test, SCC measurement, milk sampling for bacterial isolation,

milking, and HOCl administration. The experimental design is

presented in TABLE I.

California mastitis test (CMT)

To detect the SCM, the CMT was performed on all quarters after

cleaning and following standard farm procedures. The rst milk was

discarded from each quarter, and the next 2–3 mL milk sample was

placed in four sections of the CMT test cup. The test cup was tilted at

a 45° angle to ensure that an equal amount of milk (2 mL) remained in

each test area, and 2 mL of CMT solution (Bovivet CMT Liquid, Kruuse,

Denmark) was added to each area. The test cup was circularly rotated

for 10 seconds. The presence and severity of the SCM were evaluated

based on the reaction observed in the test cup’s sections, according

to the method described by Roy et al. [32]. The reactions were scored

from 0 to 3. If the mixture showed a homogeneous distribution (score

0) or slight thickening (trace), it was considered healthy. If slime or

gel formed, it was recorded as SCM positive and scored based on

severity (scores 1–3) [33].

FIGURE 1. The distribution of cows (A) and udder quarters (B) diagnosed with

subclinical mastitis (SCM) on the farm

A B

TABLE II

The distribution of aected udder quarters in

cows with subclinical mastitis (SCM)

Udder quarter Number of cows

Total quarters

(cow / %)

Blind quarters

(cow/quarter)

1 quarter 72 72 / 27.79% 6 / 7

2 quarters 36 72 / 27.79% 4 / 5

3 quarters 17 51 / 19.69% 2 / 2

All quarters 16 64 / 24.71% –

Total 141 259 / 100.00% 12 / 14

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Collection of milk samples

In the presented study, 74 of 141 cows with a positive CMT result in at

least one udder lobe at different degrees were randomly determined,

and one udder lobe of these cows (74 udder lobes) was selected

for HOCl treatment. The selected cows were aged between 3 to 6

years old, had at least one calving, did not have any local/systemic

infections, and had not received intramammary or systemic antibiotic

treatment within the last week. Milk samples were taken from all

quarters included in the study on days 0, 3, and 5 before evening

milking to determine the SCC and perform bacteriological isolation.

The teats were cleaned with 70% alcohol and were allowed to dry. The

initial milk, at risk of high SCC and bacterial count, was discarded,

and 5 mL of milk was aseptically collected in sterile tubes from each

quarter. In cases where SCM was observed in more than one–quarter

of the same cow, the rear quarters were preferred for microbiological

sampling. However, HOCl treatment was applied to all subclinical

mastitic quarters.

The milk samples designated for bacteriological isolation were

promptly refrigerated at +4°C (RT59QBPN, Samsung, South Korea)

delivered to the laboratory, and subjected to isolation procedures

without delay.

Determination of somatic cell count

Separate milk samples were also taken before the microbiological

sampling on days 0, 3, and 5 to measure the SCC. Rapid SCC

measurements were conducted using a portable automatic somatic

cell counting device [DeLaval (DCC), Tumba, Sweden].

Intramammary hypochlorous acid administration

Intramammary HOCl was administered for 5 days immediately after

the morning and evening milkings. The milk in the relevant quarter was

completely removed before infusion of the HOCl [0.02% Hypochlorous

Acid, Crystalin Animal Health, NHP, Turkey]. A total of 100 mL HOCl was

applied to each quarter using modied syringes for the teat channel.

In our previous preliminary study [34], we determined that success in

the treatment of SCM could be achieved with 3 applications of 100 ml

HOCl at 12–hour intervals. The solution was distributed to the upper

tissue parts of the quarter via careful massaging.

Bacteriological culture and isolation

For bacteriological isolation, the frozen milk samples were

brought to room temperature and gently inverted 10–12 times for

homogenization. Initially, 10 µL of each sample was streaked onto

Müller Hinton Agar. Subsequently, to identify the microorganisms

thriving under aerobic conditions both general media (sheep blood

agar) and selective media (MacConkey Agar, Mannitol Salt Agar, BBL

Bile Esculin Agar, Candida Selective Agar), along with Sabouraud

Dextrose Agar (SDA) for fungi cultivation were used. The bacterial

and yeast cultures were incubated (Nuve EN 400, Nuve, Turkey) under

aerobic conditions at 37°C for 24–48 hours for identication. After

establishing the Gram morphology of the isolated microorganisms,

cultural and biochemical properties were characterised [35]. The

cultivated SDA plates were incubated for one month to evaluate

fungal growth. Growth status and macroscopic colony morphologies

were assessed.

Statistical analyses

Statistical analysis of the study was performed using the SPSS

22.0 packet program (SPSS, IBM SPSS Statistics, Chicago, IL, USA).

We employed a Paired Samples T–Test to compare the mean SHS

values. Additionally, McNemar’s test was applied to evaluate the

isolation results. Graphs were created using GraphPad Software, Inc

8.0. Statistical signicance was indicated as follows: * for P<0.05, **

for P<0.01, and *** for P<0.001.

RESULTS AND DISCUSSION

California mastitis test

As a result of the CMT conducted on 232 Holstein–Friesian dairy cows

on the farm, 141 cows had subclinical infection in at least one–quarter,

and the SCM percentage was 60.78%. The proportion of healthy cows

was 39.22% (91 of 232) (FIG. 1A). At the quarter level, 27.91% (259 of

928) were positive for varying degrees of SCM. The proportion of

CMT–negative healthy and blind quarters was 69.94% (649 of 928)

and 2.15% (20 of 928), respectively. Six of the blind quarters were

in healthy cows, and 14 were in cows diagnosed with SCM (FIG. 1B).

The number of affected udder quarters among the 141 cows with

SCM is presented in TABLE II. The 141 cows with SCM were found to

have 259 infected udder quarters, 14 blind udder quarters, and 291

CMT–negative udder quarters.

When the CMT results were evaluated according to udder quarter

localisation, the distribution of infected quarters was 69 (26.64%),

72 (27.80%), 57 (22.01%), and 61 (23.55%) in the left frontal, right

TABLE III

The distribution of aected udder quarter localization and California Mastitis

Test (CMT) results in cows with subclinical mastitis (SCM)

Udder quarter CMT (+) CMT (++) CMT (+++) CMT (Total quarter / %) CMT (–) Blind Total

Right front 50 16 6 72 / 27.80% 157 3 232

Right rear 39 17 5 61 / 23.55% 166 5 232

Left front 39 27 3 69 / 26.64% 160 3 232

Left rear 28 21 8 57 / 22.01% 166 9 232

Total number 156 81 22 259 / 100.00% 649 20 928

Total % 60.23% 31.27% 8.49% 27.91% 69.93% 2.15% 100.00%

CMT: California Mastitis Test

FIGURE 2. The mean Somatic Cell Count (SCC) values before Hypochlorous Acid (HOCl)

administration (Day 0), on the 3

(Day 3), and 5

days (Day 5) of HOCl treatment.

Shape (***) indicates the statistical dierence between groups, with P<0.001

FIGURE 3. Distribution of bacterial isolation responses of cows included in the

study. Day 0: Before intramammary Hypochlorous Acid (HOCl) treatment, Day 3:

day of HOCl treatment, Day 5: 5

day of HOCl treatment. Statistical analyses

were conducted using SPSS followed by the McNemar test. Dierent numbers

of shapes (*) between columns indicate statistical dierences between days of

bacterial isolation, P values *; P<0.05, **; P<0.01, ***; P<0.001

Subclinical mastitis in dairy cows / Ucar and Peker __________________________________________________________________________________

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frontal, left rear and right rear quarters, respectively (TABLE III).

When categorizing front and rear quarters in total, it was observed

treatment, 64.86% (48) of the cows exhibited microbial growth, while

there was no microbial growth in 26 samples (35.14%). On the 3

day

of the HOCl treatment (before the 6

milking), the percentage reduced

to 45.95%. On the 5

day of the treatment (before the 10

milking),

microbial growth was further reduced to 22.97%. The bacterial

isolation responses of milk samples obtained from udder quarters

with SCM on days 0, 3, and 5 in the study are presented in FIG. 3.

that 54.44% (141) were in the front quarters, and 45.56% (118) were

in the rear quarters (TABLE III).

Somatic cell count

The 74 udder quarters included in the study, initially showed

varying degrees of SCM based on CMT results with an average SCC

of 629.279 ± 68.555 before HOCl administration. The average SCC on

the 3

(before 6

milking) and 5

(before 10

milking) days of treatment

were determined as 1.136,864 ± 91.009 and 1.180,000 ± 72.427,

respectively. The mean SCC of cows was signicantly higher following

the treatment when compared to before HOCl treatment (P<0.001).

The average SCC values measured in the milk samples taken on days

0, 3, and 5 in the study are shown in FIG. 2.

Bacteriological Results

In the present study, 74 random samples were taken for

microbiological culture among the 259 CMT–positive udder quarters

(representing 27.91% of the total). Before the intramammary HOCl

The percentage of isolated microorganisms was sporadic. Before

intramammary HOCl treatment, 35.14% (26 samples) of the milk

samples showed no bacterial growth. By the 3

day of treatment,

this increased to 54.05% (40 samples), and by the 5

day, 77.03%

FIGURE 4. Bacteriological isolation results before the intramammary Hypochlorous Acid (HOCl) treatment (A), 3

(B), and 5

HOCl treatment. (CNS: Coagulase–Negative, S. aureus: Staphylococcus aureus, E. faecalis: Enterococcus faecalis)

A B C

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(57 samples) of the samples showed no bacterial growth. Before the

intramammary HOCl treatment, the bacterial growth in milk samples

was as follows: Staphylococcus aureus (S. aureus) was present in 17.57%

(13 samples), Coagulase–Negative Staphylococci (CNS) in 12.16% (9

samples), Corynebacterium spp. in 9.46% (7 samples), Candida spp.

in 2.7% (2 samples), and Enterococcus spp. in 1.35% (1 sample). Mixed

cultures included S. aureus with Candida spp. in 12.16% (9 samples),

CNS with Candida spp., and CNS with S. aureus in 2.70% (2 samples

each), and CNS with Corynebacterium spp., CNS with Micrococcus

spp., Candida spp. with Corynebacterium spp., and S. aureus with

Enterococcus faecalis in 1.35% (1 sample each). The isolation results

before the HOCl treatment are presented in FIG. 4A. On the 3

day

of intramammary HOCl treatment, S. aureus was found in 9.46% (7

samples), CNS in 8.11% (6 samples), Candida spp. in 4.05% (3 samples),

and Streptococcus spp. in 1.35% (1 sample). Mixed cultures were

observed as follows: S. aureus and Candida spp. in 14.86% (11 samples),

Candida spp. and Corynebacterium spp. in 2.70% (2 samples), and CNS

and Corynebacterium spp., CNS and S. aureus, CNS and Candida spp. in

1.35% (1 sample each). The isolation results on the 3

day of the HOCl

treatment are presented in FIG. 4B. On the 5

day of intramammary

HOCl treatment, S. aureus was isolated in 9.46% (7 samples) and CNS

in 1.35% (1 sample). Mixed cultures of S. aureus and Candida spp. were

found in 12.16% (9 samples). The bacterial isolation results on the 5

day of the HOCl treatment are presented in FIG. 4C.

Subclinical mastitis in dairy cows / Ucar and Peker __________________________________________________________________________________

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In milk–producing operations, the most critical factor determining

the milk quality is the animal’s and udder’s health. Inammation of

the mammary gland is referred to as mastitis, regardless of its cause

[36]. In the subclinical form of mastitis, even though the animal’s and

udder’s health may appear normal, the decrease in milk yield and

quality is often unnoticed by the breeder, leading to underestimated

economic losses it causes or will cause [37, 38]. The SCM can also

spread to healthy cows within the herd if left untreated. The incidence

of SCM in cows has been reported to range between 19.20% and 85%

[3, 39]. This poses a signicant concern for the global dairy industry

and represents a more widespread problem than clinical mastitis

[40]. The present study found an SCM rate of 60.78% (FIG. 1A). This is

consistent with prior research, which reported a higher SCM among

dairy cows [10, 11, 12]. The results obtained in this study support the

conclusion that SCM is one of the signicant problems for dairy farms

worldwide. The high ratio of this disease in dairy cows can lead to

substantial nancial losses if it is not addressed.

The dairy industry must prioritize the early diagnosis and

treatment of SCM to mitigate its effects and reduce its occurrence.

Determination of the SCC is a critical point in evaluating milk quality,

detecting issues affecting udder health, and especially in diagnosing

the SCM. In this study, the CMT emerged as a valuable tool for

evaluating the SCC in milk, with the total incidence of mastitis at

the udder quarter level determined as 27.91% based on the positive

CMT in this study (FIG. 1B), aligning with the ndings of Sanford et al.

[18]. Mammary somatic cells consist of epithelial cells from secretory

tissue and leukocytes involved in infection response, as outlined by

Harmon [13]. Notably, an elevated CMT score correlates with increased

milk SCC levels, indicating its utility in diagnosing mastitis.

The study was extended to understand the distribution patterns

of SCM within the udder quarters. This may be important for the

development of targeted diagnostic and treatment strategies. Some

research has suggested that mastitis is more common in the rear

udder quarters [41, 42, 43], while others have reported it to be more

frequent in the front quarters [44]. In the current study, 54.44% of SCM

cases were found in the front udder lobes, while 45.56% were in the

rear udder lobes. Additionally, the right front and left front quarters

were the most affected, with positivity rates of 27.80% and 26.64%,

respectively. The results indicate that there is no signicant difference

in the localization of SCM in the udder lobes (TABLE III). In terms of the

number of infected udder lobes, the number of cows with SCM in one,

two, three, and four udder quarters was 72, 36, 17, and 16, respectively

(TABLE II). Various factors, including those related to the animal, herd

management, and the farmer, as well as the clinical type of mastitis,

the severity of SCM, and the diagnostic tool used, may have led to

different results between studies. It is recommended to take these

factors into account when evaluating the occurrence and distribution

of mastitis. Implementing distinctive interventions targeting mastitis

distribution patterns in udder quarters may be suggested to prevent

the progression of SCM to more severe clinical stages.

Subclinical mastitis can progress to CM if left untreated. If not

treated correctly, CM can lead to loss of milk production and udder

atrophy [45]. In various studies, the rate of blind udder quarters has

been reported to range from 3.83% to 4.84% [46, 47]. The presence

of blind udder quarters in this study accounts for 2.15% at the herd

level (FIG. 1B), along with the previous research reports.

In addition to SCM not being noticed by breeders, another important

problem is the widespread use of antibiotics for treatment [48, 49].

In eld conditions, antibiotics are generally used in the treatment of

mastitis with a trial–and–error approach. The overuse of antibiotics is

a signicant concern, leading to the selection of antibiotic–resistant

pathogens and posing challenges for treatment as well as potential

public health problems. The presence of antibiotic residues in milk

due to the use of antibiotics to treat mastitis can also negatively affect

milk quality and human health [48, 49, 50]. To overcome this problem,

there is a growing interest in exploring alternative approaches to

antibiotics for the control and treatment of mastitis, crucial for the

safety and quality of milk. In our preliminary study [34], which was

previously conducted on cows with SCM in two different dairy farms,

post–milking intramammary HOCl infusion was tested for 3 milkings

at 12–hour intervals. No adverse effects related to the treatment were

observed, and a reduction in bacterial growth in the udder quarters

was detected at the end of the treatment. Therefore, we suggested

that it could be an alternative to antibiotic use. In the presented

study, the effectiveness of intramammary administration of HOCl, a

naturally occurring substance produced by neutrophils [19] and safe

in human medicine, was investigated over a 5–day period with 12–hour

intervals to treat SCM with closely monitoring changes in somatic

cell count (SCC). Before the administration of HOCl, the mean SCC

measured in milk samples with positive CMT was 629.279 ± 68.555.

It was observed that the mean SCC increased on the 3

and 5

days

of the treatment and was 1.136,864 ± 91.009 and 1.180,000 ± 72.427,

respectively. Wellnitz et al. [51] mentioned an increase in milk SCC

treated with lipopolysaccharide (LPS) alone or prednisolone and LPS

combination, observed for up to 36 hours. In our previous preliminary

study [34], it was determined that HOCl application increased SCC and

decreased after 1 week of treatment. The increase in SCC following

intramammary treatment in the current study is believed to be a

result of the activation of favourable inammatory response to

microorganisms and immune cell migration to the mammary gland.

Mastitis can be caused by bacteria, fungi, viruses, chemicals, and

physical factors [52]. Some studies have shown that bacterial growth

was absent in 25% to 39.20% of CMT–positive milk samples from

cows with SCM [46, 53]. In this study, 35.14% of samples (a total of

74 quarters) tested negative for bacteria before the HOCl treatment.

This rate falls within the range reported in other studies.

The pre–treatment microbiological culture results showed that

64.86% of microbiological samples tested positive for bacteria.

Following treatment with HOCl, this percentage was reduced to

45.95% and 22.97% on day 3

and 5

of treatment, respectively.

The rise in the number of samples with no bacterial growth following

HOCl treatment is consistent with the known antimicrobial properties

of HOCl. It is effective at eliminating a wide range of pathogens,

including both bacteria and fungi [21]. The observed increase in the

proportion of samples showing no bacterial growth over time highlights

HOCl’s effectiveness in progressively lowering the microbial load.

In the study, bacterial isolation results from milk samples before

HOCl treatment revealed the presence of pathogens such as S. aureus,

Candida spp., Corynebacterium spp., Enterococcus spp., and CNS.

S.aureus emerged as the most prevalent bacterium in the investigation

and is considered a major infectious pathogen causing bovine mastitis.

These bacteria can spread from an infected cow to others through

various routes, such as contaminated milking equipment or the hands

of farmers. S. aureus infection becomes a signicant issue in the

dairy farming industry, particularly in cases of bovine SCM. These

bacteria can produce staphylococcal toxins and form biolms, which

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help protect them from the immune system and treatment methods.

CNS were previously thought to be harmless components of the

normal skin ora. However, they are now recognized as signicant

opportunistic pathogens causing mastitis. Corynebacterium spp.,

commonly found as a commensal bacterium in bovine mammary

glands, may provide protection against intramammary infections

caused by other mastitis pathogens. Initially thought to colonize only

the teat canal, Corynebacterium spp. has also been observed in the

teat cistern, gland cistern, and mammary parenchyma, emphasizing

its role as an indicator of milking hygiene practices [54].

On the 3

day of treatment, a reduction in the prevalence of S.

aureus and CNS were noted, with Candida spp. and Streptococcus

spp. also present but at lower rates. The mixed cultures showed some

persistence. By the 5

day, the prevalence of S. aureus remained at

9.46%, while CNS dropped to 1.35%. Mixed cultures of S. aureus and

Candida spp. were still present. This persistence of certain bacteria

and mixed cultures suggests that while HOCl effectively reduces

the overall microbial load, some bacteria, particularly S. aureus may

survive or adapt to the treatment, leading to a partial resurgence of

mixed cultures. This pattern of microbial reduction and stabilization

aligns with the understanding of HOCl’s antimicrobial properties.

HOCl is known to disrupt microbial communities by targeting a broad

spectrum of pathogens, which explains the initial decrease in bacterial

presence. However, the survival or adaptation of certain bacteria,

such as S. aureus highlights the challenge of completely eradicating

all strains, which may lead to a partial return of mixed cultures over

time. A study conducted on a dairy farm in the same region as the

present study identied Staphylococcus spp. as the most common

bacterial species, with an occurrence of 24.5%, and observed fungi

as one of the most common pathogens, which supports the current

study ndings [55]. Excessive and indiscriminate use of antibiotics

are the primary factors contributing to the increased frequency of

fungal infections [56]. In the present study, it was noted that Candida

spp. could be found in milk samples before HOCl treatment, either in

combination with other pathogens or as a single agent.

CONCLUSION

The study emphasized the need for a holistic approach to maintaining

milk production, milk quality, and the health of humans and dairy

animals against SCM. In conclusion, the study demonstrated that

bacterial growth was suppressed and the number of udder quarters,

in which microorganisms were not isolated, increased on the control

days after intramammary HOCl treatment. This suggests that HOCl

can be used as an alternative to antibiotics for treating SCM in cows.

Further research is recommended to develop more practical devices for

intramammary use, optimize HOCl delivery, determine the ideal doses,

and penetrate more effectively into secretory cells. The bacteriological

results, in particular, could be encouraging for targeting new research.

Ethics approval

This study was conducted with the approval and guidelines of the

Animal Research Local Ethics Committee of Aydin Adnan Menderes

University (64583101/2023/113).

Conict of interest

The authors declare that they have no conict of interest.

ACKNOWLEDGEMENT

The authors thanks to Prof. Dr. Süheyla Turkyilmaz for providing

support in microbiological analysis.

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