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 signicant 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
rd
and 5
th
days of the treatment. An increase in SCC was observed on the 3
rd
and
5
th
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
rd
day and 22.97% on the 5
th
day of the treatment. Staphylococcus
aureus was the most prevalent bacterium before the treatment. On the
3
rd
day of the treatment, bacterial growth rate, particularly in Candida
spp., decreased compared to before the treatment. However, on the 5
th
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 signicativo 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 identicar
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 inammatory 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 identiable 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 dened as epithelial
cells that pass from the mammary tissue and inammatory 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 eciency 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–inammatory 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 biolm–
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 ecacy 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 aected 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 modied 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 identication. 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 signicance 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 aected 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
rd
(Day 3), and 5
th
days (Day 5) of HOCl treatment.
Shape (***) indicates the statistical dierence 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:
3
rd
day of HOCl treatment, Day 5: 5
th
day of HOCl treatment. Statistical analyses
were conducted using SPSS followed by the McNemar test. Dierent numbers
of shapes (*) between columns indicate statistical dierences 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
rd
day
of the HOCl treatment (before the 6
th
milking), the percentage reduced
to 45.95%. On the 5
th
day of the treatment (before the 10
th
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
rd
(before 6
th
milking) and 5
th
(before 10
th
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 signicantly 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
rd
day of treatment,
this increased to 54.05% (40 samples), and by the 5
th
day, 77.03%
FIGURE 4. Bacteriological isolation results before the intramammary Hypochlorous Acid (HOCl) treatment (A), 3
rd
(B), and 5
th
(C) day of
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
rd
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
rd
day of the HOCl
treatment are presented in FIG. 4B. On the 5
th
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
th
day of the HOCl treatment are presented in FIG. 4C.
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In milk–producing operations, the most critical factor determining
the milk quality is the animal’s and udder’s health. Inammation 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 signicant 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 signicant 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 signicant 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 signicant 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
rd
and 5
th
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 inammatory 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
rd
and 5
th
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 signicant issue in the
dairy farming industry, particularly in cases of bovine SCM. These
bacteria can produce staphylococcal toxins and form biolms, 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 signicant
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
rd
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
th
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 identied 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).
Conict of interest
The authors declare that they have no conict of interest.
ACKNOWLEDGEMENT
The authors thanks to Prof. Dr. Süheyla Turkyilmaz for providing
support in microbiological analysis.
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