The Journal of Veterinary Science.
Photon 114 (2013) 240-244
https://sites.google.com/site/photonfoundationorganization/home/the-journal-of-veterinary-science
Original Research Article. ISJN: 1784-6372
The Journal of Veterinary Science
Innovative method for the treatment of mastitis in dairy animals
Hussein A. Kaouda*, Salah Yosseifb
a
Department of Veterinary Hygiene and Environmental Pollution, Faculty of Veterinary Medicine, Cairo
University, Egypt
b
Veterinary Medical Researcher, Austria
Article history:
Received: 16 December, 2012
Accepted: 19 January, 2013
Available online: 04 June, 2013
Keywords:
Mastitis, Innovation, Supperoxide water,
nanoparticles
Abbreviation:
AgNPs: Silvernanoparticles, ORP: Oxidation Reduction
Potentials
Corresponding Author:
Kaoud H.A.*
Perofessor
Email: ka-oud@link.net
Phone: 0201224207641
Yosseif S.
Researcher
Email: salah.youssef1950@Gmail.com
Phone: 0201270972146
Abstract
The study was conducted in a herd consists of 800
buffaloes, in which 120 out of 160 clinically mastitic
animals were selected to carry out the field study
and to apply a novel strategy to control microbial
1. Introduction
Economic losses were summarized by
Barmely et al.(1986) and Varsheny & Naresh,
(2004) including loss in milk production,
discarded abnormal milk, degrading milk
quality and price due to high bacterial or
somatic cell count, cost of drugs, veterinary
services, increased labor costs, increased risk
of subsequent mastitis, herd replacement and
problems related to antibiotics residues in milk
and its products. Good quality starts on the
farm by application of good hygienic measures
to minimize the population of organisms on
teats and udder (Fox et al., 2005).
The microorganisms responsible for most
episodes of mastitis in dairy herds are the
environmental pathogens (E. coli, Salmonella,
Mycoplasma and Staphylococcus aureus
(Cullor & Davis 2004).
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infections in animal production. The mastitic
buffaloes were having mixed infection with E. coli,
Staphylococcus aureus and Streptococcus
agalactae. When applying 6 lines of treatment, the
diseased animals were classified into 6 groups (20
Silver each). The first group received local treatment by
intramammary infusion of ceftiofur hydrochloride;
the second one received systemic treatment of both
enrofloxacin and carprofen;the third one received a
combination of both local (ceftiofur) and systemic
treatment (enrofloxacin and carprofen. The forth
group received local treatment by intramammary
infusion with 1\500 Envirolyte-Anolyte (2 mg of
active chlorine). The fifth group received local
treatment by intramammary infusion with AgNPs
suspension. The sixth group received both local
treatments with 1\500 Envirolyte-Anolyte (2 mg of
active chlorine) and systemic treatment
(enrofloxacin and carprofen. The cure rate was 60
% for the first group, 80 % for the second and third
group. The fourth and fifth group both were 60 %,
while 100% in the sixth group.
Citation:
Kaoud H.A., Yosseif S. 2013. Innovative method for the
treatment of mastitis in dairy animals. The Journal of
Veterinary Science. Photon 114, 240-244.
It is thought that contagious mastitis is
primarily caused by S. aureus and
Streptococcus agalactiae. Environmental
mastitis can be caused by a variety of different
bacteria, including, but not limited to, K.
pneumoniae, Escherichia coli, Klebsiella
oxytoca, Enterobacter aerogenes,
Streptococcus uberis, Streptococcus bovis,
and Streptococcus dysgalactia.
Mastitis caused by Staphylococcus aureus is
the most significant cause of economic losses
of dairy industry; once it is established in the
mammary gland, it is difficult to eradicate. To
establish an infection process, it must
overcome the different host defence
mechanisms (phagocytosis, elimination by
milking etc) (Aguilar et al., 2001).
240
Insufficient contact of the antibiotics with
pathogenic bacteria at the site of infection is a
major cause of mastitis treatment failure
(Sandholm et al., 1990). The route of of
administration, intramammary or parenteral of
medicinal products to treat mastitis is an
important issue. It determines the biological
barriers encountered by the active compound
and the routes by which it may make contact
with the causal microorganism (Serieys et al.,
2005).
Traditional prevention of bovine mastitis
involves a complex regimen of daily teat-
dipping with a disinfectant solution, and may
involve antibiotic-containing teat dips. When
infection does occur, intramammary infusion of
antibiotics is indicated; however this leads to
increasingly resistant strains of bacteria.
Ceftiofur is a new broad-spectrum third
generation cephalosporin antibiotics for
veterinary use. It inhibits bacterial cell wall
synthesis by interfering with enzymes
essential for peptidoglycan synthesis (Hornish
and Kotarski, 2002). Consequently, this new
antibiotic should be effective against a wide
variety of mastitis pathogens, including
environmental mastitis pathogens (Oliver et
al., 2004).
Therefore, it is necessary to discover novel
strategies and identify new antimicrobial
agents to develop the next generation of
agents to control microbial infections in animal
production.
2. Experimental
Traditional prevention of bovine mastitis
involves a complex regimen of daily teat-
dipping with a disinfectant solution, and may
involve antibiotic-containing teat dips. When
infection does occur, intramammary infusion of
antibiotics is indicated; however this leads to
increasingly resistant strains of bacteria.
2.1 Animal
This study was conducted in a farm at
Alexandria Desert Road (a herd consists of
800 buffaloes) in which 120 out of 160
clinically mastitic animals were selected to
carry out the field study.
2.2 Housing
Animals were housed in a separate yard
stands on straw bedding floor, automatically
milked twice daily and the milk collected in
milk tank until treatment. They receive their
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needs of water through a common water
trough.
2.3 Examination
A palpation evaluation was conducted for
animals after complete milk out at the milking
time according to Massart-Leen et al., (1988).
Animals scored 1. showed pliable udder, heat
pain, redness, and/or swelling were not
detected; animals exhibited no signs of
discomfort scored 2,where the udder was less
pliable with some firmness, redness, heat, and
pain were generally not detectable; animals
exhibited no signs of discomfort scored 3.
Animals exhibited moderate swelling udder,
definitely firm, reddened, and warm to the
touch and generally exhibited signs of
discomfort (irritable, performed stepping
motion with feet and/or kicked during the
preparing and milking procedures) were
scored.4. Udder showed sever swelling (the
udder was very hard, red, hot, and noticeably
larger than the other quarters) and of
extremely uncomfortable and very irritable
were scored 5. Udder was generally pliable
and a hardened lump was palpable and
present from milking to milking overtime
without change in size, no pain, heat, redness,
or swelling was associated with this condition
scored 6. Udder was swollen, reddened, hard,
and often extending forward toward the navel,
as well as posteriorly up the rear quarters
where the udder attaches to the body.
Appearance of foremilk was scored as follows:
1. Normal, 2. Flakes, 3. Small slugs, 4. Large
slugs, 5. Stringy (watery), 6. Bloody. The
mammary glands were considered to have
clinical mastitis when the udder or milk score
was 3 or higher.
2.4 Sampling
All samples were collected using standard
procedures described by Harmon et al.,
(1990). After discarding the first few milk
drops, milk samples were taken from all
clinically mastitc buffaloes. In clean
environment, thoroughly wiping the Teats and
teat orifices were thoroughly wiped with 70 %
ethyl alcohol. Each milk sample was collected
in a sterile screw capped bottle and sent
directly to the laboratory with minimum of
delay for routine cultural identification.
All samples were kept on melting ice (1° C)
during transport and at the laboratory until
analysis were performed or aliquots were
prepared for freezing (-20° C).
Milk samples were centrifuged at 300 rpm for
15 Minutes and a loopful from the sediment
241
and the rest of all samples were inoculated as the disappearance of clinical signs which
into a nutrient broth, brain heart infusion broth, were observed on day before treatment, on
then incubated aerobically overnight at 37° C other words, by the return to normal feed
for enrichment and enhancement of bacterial intake, good general condition, absence of
growth. Subcultures were streaked on 10 % udder edema, normal milk appearance and
sheep blood agar and MacConkey agar plates normal milk yield.
according to Carter and Cole (1990).
Suspected colonies were identified Samples were taken from clinical mastitic
morphologically, microscopically and quarters and examined bacteriologically to
biochemically according to Quinn et al. (1994) identify the causative agent.
and Waage et al. (1999). For Mycoplasma, a
loopful was cultured on modified Hay flicks 3. Results and Discussion
media, incubated at 37° C with 10 % CO2 for 7
- 10 days (Hogan et al., 1999). Specification of Clinically mastitic cases were detected by
culture material was performed by an enzyme- clinically infected quarters often showing
linked immunosorbent assay (ELISA). moderate swelling, firmness, visible signs of
chunks of milk, clots in milk and in some
2.5 Treatment of Mastitic Cases cases, the milk become viscous. 120 out of
The test was performed according to Sayed 160 clinically mastitic buffaloes of mixed
and Abdel-Rady (2008). All diseased animals infection (E. coli, S. aureus, and S. agalactae)
were classified into 4 groups (20 animals were selected to carry out the experiment.
each). The first group received local treatment
by intramammary infusion with 125 mg of Table 1: The efficiency of different lines of
ceftiofur hydrochloride (Pfizer Animal Health, treatment which applied to cases of clinically
Egypt); the second one received systemic mastitic buffaloes
treatment by I/M injection of both enrofloxacin
5 mg/kg body weight for 5 successive days
and I/V injection of 2.9 ml/kg body weight of
carprofen as an immunomodulator drug and
the third one received a combination of both
local (local intramammary infusion with
ceftiofur) and systemic treatment ( I /M
injection of both enrofloxacin 5mg/kg body
weight for 5 successive days and I /V injection
of 2.9 ml/kg body weight of carprofen as an
immunomodulator drug).. The forth group
received local treatment by intramammary 1: Local intramammary infusion with ceftiofur; 2:
infusion with 1\500 Envirolyte-Anolyte (It received systemic treatment by I/M in enrofloxacin
contains various mixed oxidants predominantly and I/V carprofen, 3: received a combination of both
local and systemic treatment, 4: Local
hypochlorous acid and sodium hypochlorite
intramammary infusion with Envirolyte-Anolyte
(HClO, ClO2, HClO3, HClO4, H2O2, O2, ClO-, 1\500), 5: Local intramammary infusion with AgNPs,
- - - - -
ClO2 , ClO3 , O , HO2 , OH - working 6: received both local treatment by intramammary
substances, (1\500) [pH 2.5-3.5, infusion with 1\500 Envirolyte-Anolyte (2 mg of
ORP>1150mV, Cactive ~500mg/l] , 1\500 = 2 active chlorine) and received systemic treatment.
mg /L active chlorine, 1\1000 = 1mg /L active
chlorine. Figure 1: Different lines of treatment applied to
cases of clinical mastitis and the cure percentages
The fifth group received local treatment by
intramammary infusion with AgNPs
suspension. The sixth group received both
local treatments by intramammary infusion
with 1\500 Envirolyte-Anolyte (2 mg of active
chlorine) and received systemic treatment (I
/M injection of both enrofloxacin 5mg/kg body
weight for 5 successive days and I /V injection
of 2.9 ml/kg body weight of carprofen as an
immunomodulator drug).

The treatment applied once daily and for 5

successive days. Clinical cure was defined

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The cured cases were 60 % for the first group,
80 % for the second and third one together
with stimulation of the innate immune
mechanisms of the animal. Improvement of
local clinical signs of the swelling and milk
appearance at the level of affected quarters by
carprofen was observed, we think that
carprofen may be able to relief most cases (80
%), these observations are in accordance with
Vangroenweghe et al., (2005).
The fourth and fifth group both were 60 %
while, it was 100% in the sixth group which
received both local treatment by
intramammary infusion with 1\500 Envirolyte-
Anolyte (2 mg of active chlorine) and received
systemic treatment by I/M injection of both
enrofloxacin 5mg/kg body weight for 5
successive days and I/V injection of 2.9ml/kg
body weight of carprofen.
The cure % of the first group may be due to
insufficient to induce cure for this group.
Results of the study by Oliver et al., (2004)
demonstrated that ceftiofur therapy was
effecting in eliminating naturally occurring
subclinical mastitis in lactating dairy cows
when the therapy is extended more than 5
days.
Envirolyte-Anolyte (1\500) and AgNPs were
induced similar curing rate (60%) for 5 days
local treatment.
The unique physiochemical properties of the
nanoparticles combined with the growth
inhibitory capacity against microbes has led to
the upsurge in the research on nanoparticles
and their potential application as
antimicrobials. The mechanism of antibacterial
effect of silver nanoparticles has been
reported in the literature (Sondi and Salopek-
Sondi, 2004), which suggests that the particles
are bactericidal.
Several possible modes of action are
discussed in the literature on nano-Ag effects
on bacteria and fungi. These are (1)
membrane disruption through direct
attachment of the nanoparticle to the bacterial
membrane, (2) cellular invasion and enzyme
disruption by nanoparticles, (3) changes in cell
membrane permeability (4) interference with
cellular S-containing compounds, and (5)
intracellular ROS accumulation (Hwang et al.,
2008; Kim et al., 2009; Lok et al., 2006;
Morones et al., 2005; Pal et al., 2007;
Panáček et al., 2006; Sondi and Salopek-
Sondi, 2004). That several of these events
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might act together to result in cell death is
probable, but the specific processes and
interactions required for toxicity have not been
fully confirmed.
In one hand, in spite that, the third line was a
combination of both second and third ones
(intramammary infusion of 125 mg of ceftiofur
hydrochloride plus systemic treatment by I/M
injection of enrofloxacin and I/V injection of
carprofen for 5 successive days), this line of
treatment induced 80% curing of cases. These
results are in accordance with those reported
by Grewal et al., (2005). On the other hand it
is interesting to say that, the combination of
Envirolyte-Anolyte (1\500) as intramammary
infusion of mastitic udder and I/M injection of
enrofloxacin and of carprofen for 5 successive
days were induced 100% curing.
Conclusion
The unique physiochemical properties of the
nanoparticles combined with the growth
inhibitory capacity against microbes has led to
the upsurge in the research on nanoparticles
and their potential application as
antimicrobials. Silver nanoparticles and
superoxide activated water are representing
new antimicrobial agents to develop the next
generation of agents to control microbial
infections in animal production.
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