Ozono revew 2020

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Ozone therapy in veterinary medicine: A review
R.L. Sciorsci, E. Lillo, L. Occhiogrosso, A. Rizzo
PII:
S0034-5288(20)30056-4
DOI:
https://doi.org/10.1016/j.rvsc.2020.03.026
Reference:
YRVSC 4011
To appear in:
Research in Veterinary Science
Received date:
12 January 2020
Revised date:
18 March 2020
Accepted date:
25 March 2020
Please cite this article as: R.L. Sciorsci, E. Lillo, L. Occhiogrosso, et al., Ozone therapy
in veterinary medicine: A review, Research in Veterinary Science (2019), https://doi.org/
10.1016/j.rvsc.2020.03.026
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Ozone therapy in Veterinary Medicine: a review
Sciorsci R.L., Lillo E., Occhiogrosso L., Rizzo A. *
Department of Veterinary Medicine, University of Bari Aldo Moro, S.P. per Casamassima km. 3
70010 Valenzano (BA), Italy
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*Corresponding author:
Sciorsci R.L.
70010 Valenzano (BA), Italy.
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Tel.: +39 0805443882; fax: +39 0805443883.
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Department of Veterinary Medicine, University of Bari Aldo Moro, S.P. per Casamassima km. 3
ABSTRACT
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E-mail address: [email protected]
Ozone (O 3 ) is a triatomic form of oxygen. As O3 rapidly dissociates into water and releases a reactive
form of oxygen that may oxidize cells, the gas mixture of O 3 /O 2 is used in medicine. ATP is widely
available for cellular activity. O 3 can be administered via the systemic and local routes. Although O3
is known as one of the most powerful oxidants, it also promotes antioxidant enzymes. Additionally,
it stimulates some of the cells of the immune system and inactivates pathogens, including bacteria,
fungi, yeasts, protozoa, and viruses. Owing to these activities, O 3 is used to improve several diseases,
both in human and in veterinary medicine. Considering the wide scope of O3 application, the aim of
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this review was to reiterate the mechanisms of action of O3 and its utilization in different mammalian
species (bovine, ovine-caprine, equine, canine, porcine).
Keywords: Ozone; Oxidative Mechanism; Immune Response; Antimicrobial; Mammals.
1 INTRODUCTION
Ozone (O3 ), a gas discovered in the mid-nineteenth century, is a triatomic form of oxygen with a
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dynamically unstable structure due to the presence of mesomeric states. The word “Ozone” is
derived from “Ozein”, a Greek word for smell (Srikanth et al., 2013). It is colourless, acrid in odour,
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and explosive in its liquid or solid form (Elvis and Ekta, 2011).
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O3 is an atmospheric natural component; however, its distribution varies between the upper
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atmosphere (stratosphere) and lower atmosphere (troposphere). In the stratosphere, O3 forms an
indispensable layer that filters ultraviolet radiations. This layer is necessary for O3 generation via a
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chemical combination of atomic oxygen (O) with molecular oxygen (O2) (Hänninen, 2019). The
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process of O3 formation is in dynamic equilibrium with its natural destruction, which is caused by
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different reactions including the collision with ultraviolet and infrared radiations (Mustafa, 1990). In
the troposphere, O3 is considered a pollutant obtained from the reaction between nitrogen oxides
(released from sources such as automobile exhaust, power plant emissions, and wildfires) and
sunlight (Lange et al., 2018).
Despite its role as a pollutant, O3 is used in human and veterinary medicine. The medical use of a gas
mixture of O 3 /O 2 , called ozone therapy, is based on the assumption that O3 rapidly dissociates into
water and releases a reactive form of oxygen that may oxidize cells (Case et al., 2012). This
dissociation increases the availability of oxygen and ATP for cellular activity (Bhatt et al., 2016).
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O3 can be considered a pro-drug as it can induce the activation of a second messenger in a cascade
with multiple systemic actions and consequent rearrangement of the biochemical pathways (Re et al.,
2008).
The use of O 3 began over 150 years ago (Elvis and Ekta, 2011). It was first used as a microbicidal
molecule in 1856 and subsequently in 1860 to disinfect operating rooms and water treatment,
respectively (Merhi et al., 2019). During the last century, several studies revealed other important
activities, such as its antioxidant and anti-inflammatory functions and its immuno-stimulatory effect.
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Today, the effects of O 3 therapy have been proven. Additionally, these have been consistent and
possess minimal side effects, thereby enabling its wide use (Elvis and Ekta, 2011).
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O3 can be administered via two main routes, the systemic route and local route. For the systemic
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route, Ozone autohemotherapy (O3 -AHT) (Sagai and Bocci, 2011), which consists of instilling a
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precise concentration of O2 -O3 in a predetermined amount of blood, ex vivo, is employed.
2006).
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Thereafter, this oxygenated-ozonated blood is administered to the patient (Bocci, 1994a; Bocci,
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However, three main forms of O3 are used for local administration, namely ozonated water, ozonated
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oil, and O 2 -O 3 gas mixture (Saini, 2011), which are available on the market in different
pharmaceutical preparations (creams, gas, injections, paillettes, foams, pearls, boluses, ozonized oil)
(Ðuričić et al., 2012a). Local administration routes of O 3 include intramuscular, intradiscal, and
paravertebral route; however, the rectal, nasal, tubal, oral, vaginal, vesical, pleural, and peritoneal
cavities are prudent routes of administration (Smith et al., 2017).
The only impractical route for O 3 medical application is inhalation owing to its pulmonary toxicity.
Epiphora, rhinitis, cough, headache, and sometimes, nausea, and vomiting are the typical side-effects
of O 3 therapy. O 3 should not be administered under some conditions, such as pregnancy, favism,
hyperthyroidism, severe myasthenia, and anaemia (Nogales et al., 2008).
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Several techniques are applied in O 3 production (Seung et al., 2006). The most common device
currently available is a plasma reactor. This generator employs a corona discharge or dielectric
barrier discharge (DBD) (Park et al., 2006; Kogelschatz, 2003). Issues with these systems include
high electrical consumption and a relatively low efficiency (~1%–15%) for converting oxygen to O3
(Kogelschatz, 2000). Moreover, high-energy methods (UV light, beta rays, and lasers) exist;
however, they found minimum large-scale commercial applications (Pelster, 1995).
Because of the unstable structure of O 3 , obtaining high concentrations tends to be difficult (Smith et
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al., 2017). O 3 decomposes into molecular and atomic oxygen (Stockburger, 2002) with a half-life of
40 min at 20 °C and ~140 min at 0 °C (Elvis and Ekta, 2011).
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O3, for applications regarding human medicine, is used to improve several diseases such as
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abscesses, acne, eczema, psoriasis, human immunodeficiency virus and acquired immune deficiency
uveitis,
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syndromes, fibromyalgia, arthritis, asthma, cancers, inflammation, cardiac disease, liver disorders,
cystitis, chronic wounds, dyslipidaemia, osteomyelitis, Raynaud’s disease, Parkinson’s
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disease, sepsis, sinusitis, dental caries, infections of the oral cavity, and diabetic foot (Baysan and
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Lynch, 2005; Re et al., 2008; Zhang et al., 2014; Merhi et al., 2019).
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O3 has also been widely used in the treatment of herniated vertebral discs owing to its activity on
glycosaminoglycans, with consequent dehydration of the nucleus pulposus (Murphy et al., 2016).
Considering the wide scope of O3 application in medicine, the purpose of this review was to
summarise the mechanisms of action of O3 and its utilization, with particular attention to the
veterinary field with respect to reproduction in different mammalian species (Table 1).
1.1 Ozone: oxidant-antioxidant paradox
O3 is one of the most powerful known oxidants and has a standard redox potential of +2.07 V
(Weast, 1970). As a result, there has been uncertainties regarding its use as it can generate free
radicals and correlated pathologies. Today, O3 is known to present a paradoxical action as despite its
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role as an oxidizing molecule, it increases the antioxidant properties of structures affected by its
pathologies (Munoz, 1993).
Owing to its capacity to oxidize organic compounds, O3 has toxic effects on tissue cells, especially
on the respiratory tract, owing to its low antioxidant system.
O3
reacts with all macromolecules of the cellular membranes,
including lipids,
proteins,
carbohydrates, and DNA (Clavo et al., 2019).
Owing to the oxidant properties, the interaction between lipid and O3 almost exclusively appeals to
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the carbon-carbon double bonds in unsaturated fatty acids (UFA). UFA, which is found on the
cellular membrane, reacts with O 3 to generate hydrogen peroxide (H2 O2 ) (Bocci et al., 2009; Bocci et
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al., 2011; Viebahn-Hansler et al., 2012). Through the cellular membrane and cytosol, H2 O2 promotes
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the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway and the synthesis of proteins, which
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favour cell survival (Re et al., 2014; Galie et al., 2018; Siniscalco et al., 2018; Wang et al., 2018). 4HNE degradation sends a quick signal of a transient oxidative stress, activating the synthesis of
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several substances such as γ-glutamyl transpeptidase (a glycosylated enzyme that catalyses the
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transfer of the γ-glutamyl moiety from peptide donors to different acceptors, including amino acids,
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dipeptides, and H2 O), heat shock protein (HSP-70, a protein induced by the physiological response
of cells to stress), haem oxygenase-1 (a generalized response to oxidative stress), and antioxidant
enzymes, such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase, and
glucose-6-phosphate dehydrogenase (G6PDH) and the Nrf2 pathway (Figure 1).
This process represents the basis of the paradoxical phenomenon, for which an oxidizing molecule,
such as O3 , triggers a potent antioxidant reaction.
4-HNE has different actions depending on its concentration: at lower concentrations, it regulates the
proliferation, differentiation, and enhancement of Nrf2 and the antioxidant systems, and at high
concentrations, it causes oxidative stress, apoptosis, and necrosis (Clavo et al., 2019).
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The therapeutic effects of O3 are dose-dependent. Further, obtaining an appropriate concentration of
the ozonation products is crucial to avoid toxicity (Bocci, 1994b; Bocci, 1996). Adverse effects
occur when ozonation products overwhelm the antioxidant system, ultimately resulting in a toxic
effect that leads to tissue damage. Its mechanisms of toxicity can be summarised into the following
categories (Mustafa, 1990):
• formation of free radicals and reactive intermediates;
• initiation of lipid peroxidation chain reactions;
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• oxidative loss of functional groups and activities of biomolecules, including enzymes;
• alteration of membrane permeability and functions;
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• initiation of secondary processes.
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O3 regulates the generation of nitric oxide (NO), a powerful chemical mediator that can control
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several biological functions, such as that of the vascular endothelium. The exposure of rat
macrophages and type II cells to O3 causes NO production by inducible NO synthase induction (Re
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et al., 2008).
1.2 Ozone immuno-response
Many studies have revealed that O3 stimulates a certain number of cells in the immune
system (Sánchez et al., 2012). This effect is a consequence of the O3 reaction with polyunsaturated
fatty acids (PUFA) and other antioxidants. When peroxidation compounds are formed, H2 O 2 diffuses
into immune cells and regulates signal transduction, facilitating immune responses (Caliskan et al.,
2011).
Generally, when the T-cell antigen receptor (TCR) recognizes any foreign antigens, phospholipase C
is activated. Thereafter, the last phospholipase hydrolyses phosphatidylinositol-4,5-bisphosphate
(PIP2), a membrane lipid, to produce the secondary messengers, inositol triphosphate (IP3) and
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diacylglycerol (DG). IP3 induces the release of Ca2+ from the endoplasmic reticulum into the
cytosol, which dephosphorylates nuclear factor activated T-cells (NFAT) for transport into the
nucleus. NFAT then induces the transcription of cytokines, such as interleukin (IL)-2, tumour
necrosis factor (TNF)a, IL-6, and interferon (IFN)g, and immune response elements on DNA, which
are translated into their respective proteins. Mild oxidative stress induced by O 3 therapy may activate
NFAT followed by immune functions (Sagai and Bocci, 2011).
An increase in IL-2, IFN, and TNF was observed post-treatment with O3 concentrations within the
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“therapeutic window” (Elvis and Ekta, 2011). H2 O2 enhances the activity of tyrosine kinase, which
phosphorylates IκB, a subunit of the transcription factor, nuclear factor kappa B (NF-κB). NF-κB
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and transforming growth factor beta (TGF-β) activation increase the release of cytokines, such as
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TNF, INF γ, IL-8, IL-1β, IL6, and IL8 (Orakdogen et al., 2016; Clavo et al., 2019). However, as
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mentioned above, O 3 should be used at a low concentration; a higher dosage could promote
inflammation by activating the NF-κB pathway (Kafoury et al., 2007).
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O3 also has an effect on other immune mediators, such as vascular endothelial growth factor
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(VEGF), platelet-derived growth factor (PDGF), and TGF-β, ultimately inducing their action (Zhang
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et al., 2014). Low O3 doses also increase the secretion of macrophages and leukocytes and inhibits
prostaglandin synthesis and bradykinin release, as demonstrated in a study conducted with rats
(Orakdogen et al., 2016).
The change in O 3 -induced NO is also an important step as NO plays a fundamental role in the
physiological control of immune response (Re et al., 2008).
1.3 Ozone inactivates pathogens
The oxidant activity of O 3 is useful for destroying bacterial walls and fungi cytoplasmic membranes;
however, it still acts against yeasts, protozoa, and viruses.
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For bacteria, O 3 reacts with bacterial amines, amino acids, activated aromatic compounds, and
reduced sulphur residues (von Gunten, 2003). Additionally, it reacts with unsaturated bonds
of phospholipids, proteins, peptidoglycans, and liposaccharides on the bacterial cell surface (McNair
Scott and Lesher, 1963). After the membrane is damaged by oxidation, its permeability increases,
and the O3 molecules enter the cells (Bünning and Hempel, 1996). Through electron microscopic
analysis, Thanomsub et al. (2002) revealed the destruction of the bacterial membrane and the
consequent cellular lysis.
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O3 also acts on bacterial nucleic acids. First, it preferentially destroys guanine residues. Ozonolysis
of the supercoiled DNA has also been demonstrated (Sechi et al., 2001).
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water (Yamayoshi and Tatsumi, 1993).
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According to the structure of the cell wall, microorganisms exhibit different sensitivities to ozonated
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In 2001, the U.S. Food and Drug Administration approved O3 as a sanitizer for food contact surfaces
and direct application for food products (Food and Drug Administration, 2001) and water (Lezcano
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et al., 2001). However, in the alimentary field, an excessive concentration of O3 may damage food
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surfaces with consequent discoloration and deterioration of flavour (Kim et al., 1999).
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In addition to its use on food products and water, the antimicrobial activity of O 3 has been exploited
as a support to antibiotic therapy, thereby providing benefits in the treatment of infectious disorders.
O3 antimicrobial potential is useful for reducing bacterial charge on wounds and accelerate healing.
When combined with chlorhexidine, O3 improves anti-bacterial and yeast activity (Borges et al.,
2017). O3 treatment effectively reduces the colonization of drug-resistant Staphylococcus aureus on
plate culture (Dyas et al., 1983; Yamayoshi and Tatsumi, 1993). Silva et al. (2009) evaluated the
intraperitoneal application of O3 in the gaseous form of rats and reported that it potently inhibits
bacterial growth.
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O3 exhibits a synergic action with antibiotics. In fact, Bacillus subtilis spores treated with O 3 became
more sensitive to thermal and osmotic stresses due to inner membrane damage (Cortezzo et al.,
2004).
Owing to its antimicrobial activity, O 3 is useful for preventing the spread of antibiotic-resistant
bacteria (ARB) and antibiotic-resistance genes (ARGs). Ozonisation might be an effective method
for reducing this problem. A study conducted by Stange et al. (2019) demonstrated that, even at a
low concentration of 1 mg/L, O3 removed more than 99% of ARB and ARGs. As a result, only a
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minor utilization of antibiotics without residues is obtained in animal food derivates.
O3 is applied for virus inactivation (Roy et al., 1981; Bolton et al., 1982). O3 inactivation of viruses
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primarily occurs via two approaches: lipid peroxidation and protein peroxidation, ultimately
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enhancing the sensibility of the enveloped viruses (Wells et al., 1991; Murray et al., 2008). Envelop
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phospholipids contain many unsaturation points along their hydrocarbon chains; O 3 oxidizes these
bonds, leading to structural damage. Human Immunodeficiency virus type-1 has been inactivated by
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O3 treatment, even at non-cytotoxic concentrations (Wells et al., 1991; Carpendale and Freeberg,
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1991).
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However, protein peroxidation plays a key role in the inactivation of non-enveloped viruses. H2 O2 ,
the O3 -mediated ROS, can interact with proteins and cause damage and destruction of the capsid
structure (Murray et al., 2008). Regarding naked virus inactivation, a study conducted by ThurstonEnriquez et al. (2005) revealed that ozonated water could inactivate feline calicivirus and adenovirus
type 40 using 0.30 and 0.06 mg/l, respectively. However, in addition to damaging the capsid protein
to inactivate the virus, ozonisation also damages the viral nucleic acid. Jiang et al. (2019) reported
that Poliovirus 1 inactivation by O3 is associated with damage to the viral genome instead of the
capsid proteins.
O3 acts on fungi via the same oxidative mechanism used in cell membranes. O 3 , in its gaseous and
oiled
forms,
has
been
used
in
three
common
genera
of
dermatophytes
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(Epidermophyton, Microsporum, and Trichophyton),
revealing its fungicidal and inhibitory effects
on sporulation (Ouf et al., 2016). O3 also acts by reducing the production of the enzymes required for
fungus-host and
fungus-fungus interactions,
which are normally produced to facilitate their
multiplication within the host (Ouf et al., 2016).
Regarding yeasts, a study conducted by Zargaran et al. (2017) evaluated the fungicidal effects of O3
on different forms of Candida albicans. Furthermore, in a study on recurrent vulvovaginal
candidiasis, 85% of patients recovered using ozonized water, 10% remained asymptomatic, and only
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5% did not respond to O3 therapy (Schwartz, 2015).
O3 action against protozoa has been demonstrated in vitro with different parasites, such as
2 Use in veterinary medicine
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2.1 Bovine species
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(Rajabi et al., 2015; Khalifa et al., 2019).
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Leishmania Giardia, Cryptosporidia, and Microsporidia, using ozonated oil and ozonated water
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The therapeutic use of O 3 has been demonstrated for resolving different pathologies in the bovine
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species. Ogata and Nagata (2000) compared the use of O3 and antibiotics (Ampicillin, Kanamycin,
Benzylpenicillin procain, Cefazolin) in acute clinical mastitis. Infusion of O3 (6 mg/L) was
performed in an inflamed quarter of cows via a teat canal. As a result, cows did not require any
antibiotics for recovery. This innovative therapy was proven effective, safe, and cost effective,
thereby constituting no risk of drug residues in milk. O3 was also used for subclinical mastitis. In a
study conducted on Jersey cattle affected by subclinical mastitis caused by Staphylococcus Aureus
(SA), after topical application of ozonated sunflower oil at a concentration of 600 eq-kg (600 units of
peroxide) on the affected quarter, the bacteriological test of milk SA revealed a negative result
(Quintana et al., 2019).
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O3 is also used in reproduction, especially in urovagina. Ozonated spray foam application,
immediately after the removal of the excess fluid (urine), resulted in an improvement in reproductive
performances, such as the lowest number of inseminations up to pregnancy, the lowest number of
open days, and the reduction of the reduced culling rate. O 3 flush coupled with intracornual
insemination served as an effective treatment option for urovagina that can lead to successful
conceptions and pregnancies in dairy cows (Zobel et al., 2011). Moreover, O3 use in the vagina
following urovagina surgery proved useful for improved healing (author’s note; data not published),
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potentially by increasing fibroblast migration (Xiao et al., 2017).
O3 is used to prevent metritis (Ðuričić et al., 2015a), with intrauterine ovules or foam spray. By
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comparing two formulations, Ðuričić et al. (2012a) found that the use of O 3 in the foam form was
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more effective during the first puerperium and improved reproductive performance in dairy cow. O 3
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foam is also recommended as a non-antibiotic intrauterine treatment for cattle with retention of foetal
membranes (RFM) (Ðuričić et al., 2012b; Samardžija et al., 2017). A study compared O3 foam to
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intrauterine tetracycline bolus. The efficacy of O3 foam treatment in RFM was evaluated by
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shortening the days open until pregnancy and the intercalving period (Imhof et al., 2019).
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Preventive intrauterine O 3 is used to improve reproductive efficiency through foam spray or paraffin
pearls administered 24-48 h after calving (Ðuričić et al., 2012a).
2.2 Ovine-Caprine species
Recent studies evaluated both in sheep and goats, the use of intrauterine therapy with O3 compared to
the classic therapy with antibiotics for the treatment of RFM, dystocic, and assisted delivery. In the
caprine species, O3 treatment achieved similar results to standard antibiotic therapy (oxytetracycline
hydrochloride), indicating that it could be a new potential therapeutic alternative for RFM in dairy
goats. In sheep, O3 foam had a better performance than antibiotic therapy (Ðuričić et al 2015b;
Ðuričić et al., 2016; Samardžija et al., 2017).
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O3 therapy is also used to treat acute putrefaction of the sheep's foot. This disease is widespread and
contagious, and it causes severe lameness combined with reduced production of wool and meat and
reduced fertility (Ansari et al., 2014). The most widespread therapeutic strategy involves the use of
systemic antibiotics associated with antibiotic footbaths and topical antibiotic spray. Szponder et al.
(2017) evaluated the therapeutic association of O 3 (70 mg/ml) and platelet-rich plasma (PRP) on
sheep for 20 min, three times at weekly intervals. All sheep recovered without experiencing any side
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effects.
2.3 Equine species
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In horses, O 3 therapy was reportedly used to improve antioxidant capacity before exercise. Tsuzuki
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et al. (2015) infused 20 μg/kg of O 3 gas in autologous blood (400 ml). This ozonated blood reinfused
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into horses (3.2 ml/kg/h; defined as ozonated autohemotherapy) before exercise, improved the
antioxidant capacity of horses, with effects that lasted 7 to 14 days.
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Mastitis can adversely affect the health and productivity of mares, and is mainly caused by gram-
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negative (Klebsiella and E. coli) microbes (McCue and Wilson, 1989). Shinozuka et al. (2008)
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demonstrated that O3 therapy is better than antibiotic therapy (aminobenzylpenicillin, kanamycin,
oxytetracycline, sulphadimethoxine, and enrofloxacin). O3 aids in the reversal of the local and
systemic signs of acute clinical mastitis. The researchers hypothesized that O 3 increases leukocyte
function and respiratory burst.
O3 therapy has been used in equine orthopaedics to cure osteoarthritis (OA). OA is considered as a
progressive disease, with cartilage degeneration, accompanied by mild to moderate synovial
inflammation and changes in the subchondral bone structure (Rousseau et al., 2012). Vendruscolo et
al. (2018) analysed the effects of intra-articular (IA) O 3 at two concentrations (20 and 40 μg/mL) to
evaluate the changes in inflammation and cartilage catabolism biomarkers. As there were no
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significant changes in the concentrations of these biomarkers, they concluded that IA administration
of O 3 might serve as a therapeutic option for horses with OA.
Coelho et al. (2015) employed O3 for the treatment of chronic laminitis Obel grade IV in a 10-year
mare. The therapeutic regimen was comprised of hoof trimming followed by intramuscular,
peritendinous, and intrarectal administration of medical O3 (19 mg/ml) twice per week for 10 weeks.
Owing to the therapy, after six months, the mare recovered up to the Obel grade II lameness.
O3 therapy could be considered an alternative to anti-inflammatory treatment for lumbar pain, which
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is often developed by thoroughbred horses (Vigliani et al., 2005). In a study conducted by Ballardini
(2005), 30 horses suffering from back pain were treated with a local infiltration of 15 ml of oxygen-
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ozone mixture at an O3 concentration of 30 μg/mL into the affected muscle at the interspinous and
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paravertebral regions. O3 was found to produce an analgesic effect, as the animals did not suffer from
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pain upon palpation at the later stage. As a result, this study proved the efficacy of O3 therapy for
pain management.
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A new formulation of ozonized ophthalmic oil is used to promote wound healing and treat common
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eye disorders, not only in humans but also in horses. A study used this new O3 formulation to treat a
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horse with conjunctivitis resistant to treatment with topical antibiotic (tobramycin) and topical and
systemic non-steroidal anti-inflammatory drugs (piroxicam plus flunixin meglumine). This ocular
preparation, which was administered 3–4 times per day, was demonstrated to exhibit antiinflammatory effect, bactericidal activity, tissue repair and patient recovery potential (Spadea et al.,
2018).
2.4 Canine species
In canine,
O3
was used
for different ocular pathologies given the increasing incidence;
endophthalmitis associated with intraocular procedures, cataract extraction, and intravitreal injection
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(Vaziri et al., 2015). Marchegiani et al. (2019) assessed the effectiveness of preoperative liposomal
O3 dispersion at reducing bacterial colonization from the conjunctival sac and periocular skin in dogs
compared to povidone-iodine 5% and fluoroquinolone. The results of the present study, although
preliminary, demonstrated that liposomal O3 formulation could reduce, in a statistically significant
manner, ocular surface bacterial load without causing any corneal damage or side effect in ocular
tissues.
Liposomal O 3 is also used as a therapeutic strategy in pharmacodermia, which is rare in dogs and can
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be caused by β-lactam antibiotics. Silva Júnior et al. (2019) reported the use of ozonized sunflower
oil to treat a dog that developed pharmacodermia instead of one of the classical therapies expected
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after ovary-hysterectomy (omeprazole, cephalexin, tramadol hydrochloride, carprofen). However,
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ozonized sunflower oil is often applied to wounds (4 drops, BID). Further, cleaning with ozonized
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(47 μg/mL) saline solution (0.9%) was found to cure pharmacodermia at 30 days of treatment.
A study evaluated the analgesic effects of O3 administered intrarectally or into the acupoints of
undergoing
ovariohysterectomy
compared
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bitches
to
commonly
used
non-steroidal
anti-
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inflammatory drug therapy (meloxicam). No statistically significant differences in the pain scales
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were found among the three analgesic protocols. O3 is an alternative option for promoting pain relief
in bitches undergoing ovariohysterectomy (Teixeira et al., 2013).
Han et al. (2007) evaluated fluoroscopic-guided intradiscal oxygen-ozone injection therapy for
thoracolumbar intervertebral disc herniations in dogs. Briefly, five dogs were administered a
percutaneous injection of an O 2 -O3 gas mixture, with an O 3 concentration of 32 μg/μL applied
intradiscally (1.5–2 μL) under fluoroscopy guidance. Five weeks after treatment, the mean size of the
herniated discs was measured by computed tomography. Based on their findings, a significant
reduction in the disc volumes of all animals (8.8%±3.82%) was observed, including a recovery of
their gait function intradiscally. The researchers concluded that the O2 -O3 technique can decompress
disc herniation with minimum invasiveness.
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In addition to its application in humans and even dogs, O 3 is applied in dentistry. A prior study
evaluated the response of the periradicular tissues to the endodontic treatment of infected roots using
ozonized oil or calcium hydroxide in camphorated paramonochlorophenol (CMCP) dressings. Based
on the histological and histobacteriological analysis results of treated dogs, the researchers
demonstrated that the ozonized oil conferred a success rate greater (77%) than CMCP (74%)
(Silveira et al., 2007).
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2.5. Porcine species
There are few reports on the use of therapeutic O 3 in porcine species. Generally, pigs are involved in
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scientific studies as they are a valid model for studying O3 application in other species. For example,
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the porcine species was previously used to elucidate the mechanism of action of intradiscal O2 -O3
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therapy for herniated intervertebral disc therapy. Yucatan miniature pigs were administered different
concentrations of percutaneous intradiscal O2 -O3 . As a result, the administered O3 reacted with
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proteoglycans, reducing disc volume through dehydration. This might thus serve as the primary
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(Murphy et al., 2016).
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mechanism whereby O3 relieves nerve root compression and alleviates herniated disc–related pain.
In another study, Eroglu et al. (2019) evaluated the histological and immunohistochemical effects of
O3 therapy on secondary wound healing on the palatal gingiva. Routine histological analysis and
immuno-histochemical staining were performed to investigate the expression of TGF-β. The topical
application of O 3 gas was deemed effective in the early stages of wound healing as it increased the
amount of VEGF expression.
Conclusion
The use of O3 has been intensively studied in human medicine for several years; however, in
veterinary medicine, it remains in the development stage.
Journal Pre-proof
The medical use of O3 is based on its antioxidant, immunostimulant, and antimicrobial activities. In
particular, when O3 is administered to elicit its antibacterial, antiviral, antifungal, anti-yeast, and
antiprotozoal effects, no residues are found in tissues and biological fluids after its administration. As
a result, O3 could be a potential solution for preventing antibiotic resistance.
Many aspects regarding the mechanism of action of O3 during local and systemic administration
remain unknown. A complete understanding of the mechanisms involved in O 3 therapy should be
understood before some of the O3 applications used in humans or examined in vitro and in rat models
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f
are transferred to veterinary medicine.
Moreover, a study of the optimal pharmaceutical forms, according to the clinical case, should be
pr
conducted. The O3 concentrations applied locally or in O3 -AHT should be defined early in humans
e-
and mainly animals as high O3 concentrations in the O2 /O3 gas mixture can be deleterious for tissues.
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rn
References
al
Pr
Owing to the above, studies in veterinary medicine should be implemented.
Ansari, M.M., Dar, K.H., Tantray, H.A., Bhat, M.M., Dar, S.H., Naikoo, M., 2014. Efficacy of
different regimens for acute foot rot in adult sheep. J. Adv. Vet. Anim. Res. 1, 114–118.
Ballardini, E., 2005. Oxygen-Ozone Therapy for spinal muscle disorders in the horse. Rivista Italiana
di Ossigeno-Ozonoterapia 4, 70-73.
Baysan. A., Lynch, E., 2005. The use of ozone in dentistry and medicine. Prim. Dent. Care 12, 47–
52.
Bhatt, J., Bhat, A.R., Kuldeep, D., Amarpal, A., 2016. An overview of ozone therapy in equine-an
emerging healthcare solution J.E.B.A.S. 4, 203-210.
Journal Pre-proof
Bocci, V., 1994a. Autohaemotherapy after treatment of blood with ozone. A reappraisal. J. Int. Med.
Res. 22, 131-144.
Bocci, V., 1994b. A reasonable approach for the treatment of HIV infection in the early phase with
ozone therapy (autohemotherapy).
How inflammatory cytokines may have therapeutic role.
Mediators Inflamm. 3, 315-321.
Bocci, V., 1996. Ozone: a mixed blessing. Res. Complementary Med. 3, 25-33.Bocci, V., 2006.
Scientific and medical aspects of ozone therapy. State of the art. Arch Med Res. 37, 425-435.
as well as a medical drug. Med. Res. Rev. 29, 646–682.
oo
f
Bocci, V., Borrelli, E., Travagli, V., Zanardi, I., 2009. The ozone paradox: ozone is a strong oxidant
e-
response relationship. J. Transl. Med. 9, 66.
pr
Bocci, V., Zanardi, I., Travagli, V. 2011. Ozone acting on human blood yields a hormetic dose-
Pr
Bolton, D.C., Zee, Y.C., Osebold, J.W., 1982. The biological effects of ozone on representative
members of five groups of animal viruses. Environ. Res. 27: 476.
al
Borges, G.A., Elias, S.T., Mazutti da Silva, S.M., Magalhães, P.O., Macedo, S.B., Ribeiro, A.P.D.,
rn
Guerra, E.N.S., 2017. In vitro evaluation of wound healing and antimicrobial potential of ozone
Bünning,
G.,
Jo
u
therapy. J. Craniomaxillofac. Surg. 45, 364-370.
Hempel,
D.C.,
1996.
Vital‐fluorochromization of microorganisms using‐3′, 6′‐
diacetylfluorescein to determine damages of cell membranes and loss of metabolic activity by
ozonation. Ozone Sci. Eng. 18, 173–181.
Caliskan, B., Guven, A., Ozler, M., Cayci, T., Ozcan, A., Bedir, O., Surer, I., Korkmaz, A., 2011.
Ozone therapy prevents renal inflammation and fibrosis in a rat model of acute pyelonephritis.
Scand. J. Clin. Lab. Invest. 71, 473-480.
Carpendale, M.T., Freeberg, J.K., 1991. Ozone inactivates HIV at noncytotoxic concentrations.
Antiviral. Res. 16, 281–292.
Journal Pre-proof
Case, P.D., Bird, P.S., Kahler, W.A., George, R., Walsh, L.J., 2012. Treatment of root canal biofilms
of Enterococcus faecalis with ozone gas and passive ultrasound activation. J. Endod. 38, 523–526.
Clavo, B., Rodríguez-Esparragón, F., Rodríguez-Abreu, D., Martínez-Sánchez, G., Llontop, P.,
Aguiar-Bujanda, D., Fernández-Pérez L. Santana-Rodríguez, N., 2019. Modulation of oxidative
stress by ozone therapy in the prevention and treatment of chemotherapy-induced toxicity: review
and prospects. Antioxidants 8, 1-20.
Coelho, C.S., Abreu-Bernadi, W., Ginelli, A.M., Spagnol, T., Gardel, L.S., Souza, V.R.C., 2015. Use
oo
f
of ozone therapy in chronic laminitis in a horse. J.O3.T. 1, 30-35.
Cortezzo, C.E., Koziol-Dube, K., Setlow, B., Setlow, P., 2004. Treatment with oxidizing agents
pr
damages the inner membrane of spores of Bacillus subtilis and sensitizes spores to subsequent stress.
e-
J. Appl. Microbiol. 97, 838-852.
Pr
Ðuričić, D., Dobranić, T., Vince, S., Getz, I., Gračner, D., Grizelj, J., Prvanović, N., Folnožić, I.,
Smolec, O., Samardžiia, M., 2011. Shortening days open using intrauterine ozone therapy in
al
Simmental cows. Conference paper: Veterinarska Stanica, 42, 149-152.
rn
Ðuričić, D., Valpotic, H., Samardzija M., 2015b. The intrauterine treatment of the retained foetal
236-239.
Jo
u
membrane in dairy goats by ozone: novel alternative to antibiotic therapy. Reprod. Dom. Anim. 50,
Ðuričić, D., Valpotić, H., Samardžija, M., 2015a. Prophylaxis and therapeutic potential of ozone in
buiatrics: Current knowledge. Anim. Reprod. Sci. 159, 1-7.
Ðuričić, D., Valpotic, H., Zura Zaja, I., Samardzija M., 2016. Comparison of intrauterine antibiotics
versus ozone medical use in sheep with retained placenta and following obstetric assistance. Reprod.
Dom. Anim. 51, 538–540.
Ðuričić, D., Vince, S., Ablondi, M., Dobranić, T., Samardžiia, M., 2012a. Effect of preventive
intrauterine ozone application on reproductive efficency in Holstain cows. Reprod. Dom. Anim. 47,
87-91.
Journal Pre-proof
Ðuričić, D., Vince, S., Ablondi, M., Dobranić, T., Samardzija, M., 2012b. Intrauterine ozone
treatment of retained fetal membrane in Simmental cows. Anim. Reprod. Sci. 134, 119-124.
Dyas, A., Boughton, B.J., Das, B.C., 1983. Ozone killing action against bacterial and fungal species;
microbiological testing of a domestic ozone generator. J. Clin. Pathol. 36,1102-1104.
Elvis, A.M., Ekta, J.S., 2011. Ozone therapy: A clinical review. J. Nat. Sci. Biol. Med. 2, 66-70.
Eroglu, Z.T., Kurtis, B., Altug, H.A., Sahin, S., Tuter, G., Baris, E., 2019. Effect of topical
ozonetherapy on gingival wound healing in pigs: histological and immuno-histochemical analysis. J.
oo
f
Appl. Oral Sci. 27, 1-11.
Galie, M., Costanzo, M., Nodari, A., Boschi, F., Calderan, L., Mannucci, S., Covi, V., Tabaracci, G.,
pr
Malatesta, M., 2018. Mild ozonisation activates antioxidant cell response by the Keap1/Nrf2
e-
dependent pathway. Free Radic. Biol. Med. 124, 114–121.
Pr
Han, H.J., Kim, J.Y., Jang, H.Y., Lee, B., Yoon, J.H., Jang, S.K., Choi, S.H., Jeong, S.W., 2007.
Fluoroscopic-guided intradiscal oxygen-ozone injection therapy for thoracolumbar intervertebral disc
al
herniations in dogs. In Vivo 21, 609-614.
rn
Hänninen, K., 2019. Contribution of excited ozone and oxygen molecules to the formation of the
Jo
u
stratospheric ozone layer. Environ. Ecol. Res. 7, 121-134.
Imhof, S., Luternauer, M., Hüsler, J., Steiner, A., Hirsbrunner, G., 2019. Therapy of retained fetal
membranes in cattle: Comparison of two treatment protocols. Anim. Reprod. Sci. 206, 11–16.
Jiang, H.J., Chen, N., Shen, Z.Q., Yin, J., Qiu, Z.G., Miao, J.,, Yang, Z.W., Shi, D.Y., Wang, H.R.,
Wang, X.W., Li, J.W., Yang, D., Jin, M., 2019. Inactivation of Poliovirus by ozone and the impact of
ozone on the viral genome. Biomed Environ Sci. 32, 324-333.
Kafoury, R.M., Hernandez, J.M., Lasky, J.A., Toscano, W.A.Jr., Friedman, M., 2007. Activation of
transcription factor IL-6 (NF-IL-6) and nuclear factor-kappaB (NF-kappaB) by lipid ozonation
products is crucial to interleukin-8 gene expression in human airway epithelial cells. Environ
Toxicol. 22, 159-168.
Journal Pre-proof
Khalifa, A.M., El Temsahy, M.M., Abou El Naga, I.F., 2001. Effect of ozone on the viability of
some protozoa in drinking water. J Egypt Soc Parasitol. 31, 603-16.
Kim, J.-G., Ahmed, E.Y., Sandhya, D., 1999. Application of Ozone for Enhancing the
Microbiological Safety and Quality of Foods: A Review. J. Food Prot. 62, 1071–1087.
Kogelschatz, U., 2000. Ozone generation and dust collection, in van Veldhuizen, E.M., (Ed.),
Electrical Discharge for Environmental Purposes: Fundamentals and Applications. Nova Science
Publishers, Inc., New York.
oo
f
Kogelschatz, U., 2003. Dielectric-barrier discharges: their history, discharge physics, and industrial
applications. Plasma Chem. Plasma P. 23, 1–46.
pr
Lange, S.S., Mulholland, S.E., Honeycutt, M.E., 2018. What are the net benefits of reducing the
e-
ozone standard to 65 ppb? An alternative analysis. Int. J. Environ. Res. Public. Healt 15, 1586.
Pr
Lezcano, I., Rey, R.P., Gutiérrez, M.S., Baluja, C., Sánchez, E., 2001. Ozone inactivation of
microorganisms in water. Gram positive Bacteria and yeast. Ozone Sci. Eng. 23, 183–7.
al
Marchegiani, A., Magagnini, M., Cerquetella, M., Troiano, P., Franchini, I., Franchini, A.,
rn
Scapagnini, G., Spaterna, A., 2019. Preoperative topical liposomal ozone dispersion to reduce
Res. 189, 107848.
Jo
u
bacterial colonization in conjunctival sac and periocular skin: Preliminary study in dogs. Exp. Eye
McCue, P.M., Wilson, W.D., 1989. Equine mastitis a review of 28 cases. Equine Vet. J. 21, 351-353.
McNair Scott, D.B., Lesher, E.C., 1963. Effect of ozone on survival and permeability of Escherichia
coli. J. Bacteriol. 85, 567-576.
Merhi, Z., Garg, B., Moseley-LaRue, R., Moseley, A.R., Smith, A.H., Zhang, J., 2019. Ozone
therapy: a potential therapeutic adjunct for improving female reproductive health. Med. Gas. Res. 9,
101-105.
Muñoz, A., 1993. Design and analysis of studies of the health effects of ozone. Environ Health
Perspect Suppl, 101, 231.
Journal Pre-proof
Murphy, K., Elias, G., Steppan, J., Boxley, C., Balagurunathan, K., Victor, X., Meaders, T., Muto,
M., 2016. Percutaneous treatment of herniated lumbar discs with ozone: Investigation of the
mechanisms of action. J. Vasc. Interv. Radiol. 27, 1242-1250.e3.
Murray, B.K., Ohmineb, S., Tomera, D.P., Jensena, K.J., Johnsona, F.B., Kirsi, J.J., Robisona, R.A.,
O’Neill, K.L., 2008. Virion disruption by ozone-mediated reactive oxygen species. J. Virol. Methods
153, 74–77.
Mustafa, M.G., 1990. Biochemical basis of Ozone toxicity. Free Radic. Biol. Med. 9, 245-265.
medicine and dentistry. J Contemp Dent Pract. 9, 75-84.
oo
f
Nogales, C.G, Ferrari, P.H, Kantorovich, E.O., Lage-Marques, J.L., 2008. Ozone therapy in
e-
dairy cows. J. Vet. Med. Sci. 62, 681-686.
pr
Ogata, A., Naghata H., 2000. Intramammary application of ozone therapy to acute clinical mastitis in
Pr
Orakdogen, M., Uslu, S., Emon, S.T., Somay, H., Meric, Z.C., Hakan, T., 2016. The effect of ozone
therapy in experimental vasospasm in the rat femoral artery. Turk Neurosurg. 26, 860-865.
al
Ouf, S.A., Moussa, T.A., Abd-Elmegeed, A.M., Eltahlawy, S.R., 2016. Anti-fungal potential of
rn
ozone against some dermatophytes. Braz. J. Microbiol. 47, 1-13.
Jo
u
Park, S.L., Moon, J.D., Lee, S.H., Shin, S.Y., 2006. Effective ozone generation utilizing a meshedplate electrode in a dielectric-barrier discharge type ozone generator. J. Electrostat. 64, 275–282.
Pelster, D.E., 1995. Apparatus and method for water purification using ozone generated by UV
radiation with a continuous filament bulb. United States Patent No. 5, 387-400.
Quintana, M.C.F., Domingues, I.M., Ribeiro, A.R., 2019. Uso de óleo ozonizado no tratamento de
mastite subclínica em vaca Jersey: Relato de caso. Pubvet 13, 1-4.
Rajabi, O., Sazgarnia, A., Abbasi, F., Layegh, P., 2015. The activity of ozonated olive oil
against Leishmania major promastigotes. Iran J. Basic Med. Sci. 18, 915–919.
Journal Pre-proof
Re, L., Martinez-Sanchez, G., Bordicchia, M., Malcangi, G., Pocognoli, A., Morales-Segura, M.A.,
Rothchild, J., Rojas, A., 2014. Is ozone pre-conditioning effect linked to Nrf2/EpRE activation
pathway in vivo? A preliminary result. Eur. J. Pharmacol. 742, 158-162.
Re, L., Mawsouf, M.N., Menéndez, S., León, O.S., Sánchez, G.M., Hernández, F., 2008. Ozone
Therapy: Clinical and Basic Evidence of Its Therapeutic Potential. Arch. Med. Res. 39, 17–26.
Rousseau, J.C., Garnero, P., 2012. Biological markers in osteoarthritis. Bone. 51, 265-277.
oo
inactivation by ozone. Appl. Environ. Microbiol. 41, 718-723.
f
Roy, D., Wong, P.K.Y., Engelbrecht, R.S., Chian, E.S.K., 1981. Mechanism of enteroviral
Sagai, M., Bocci, V., 2011. Mechanism of action involved in ozone therapy: is healing induced via a
pr
mild oxidative stress? Med. Gas. Res. 1, 1-18.
e-
Saini, R., 2011. Ozone therapy in dentistry: a strategic review. J. Nat. Sci. Biol. Med. 2, 151-153.
Pr
Samardžija, M., Turk, R., Sobiech, P., Valpotic, H., Harapin, I., Gračner, D., Đuričić, D., 2017.
Arhiv 87, 363-375.
al
Intrauterine ozone treatment of puerperal disorders in domestic ruminants: A review. Veterinarski
rn
Sánchez, G.M., Re, L., Perez-Davison, G., Delaporte, R.H., 2012. Las aplicaciones médicas de los
Jo
u
aceites ozonizados, actualización. Rev. Esp. Ozonoter. 2, 121–139.
Schwartz, A., 2015. Ozone therapy in the treatment of recurrent vulvo-vaginitis by Candida
albicans. Rev. Esp. Ozonoter. 5, 99–107.
Sechi, L.A., Leczano, I., Nunez, N., Espim, M., Duprè, I., Pinna, A., Molicotti, P., Fadda, G.,
Zanetti, S., 2001. Antibacterial activity of ozonized sunflower oil (Oleozon). J. Appl. Microbiol., 99,
279-274.
Seung, L.P., Jae, D.M., Seug, H.L., Soo, Y.S., 2006. Effective ozone generation utilizing a meshedplate electrode in a dielectric-barrier discharge type ozone generator. J. Electrostat. 64, 275-282.
Journal Pre-proof
Shinozuka, Y., Uematsu, K., Takagi, M., Taura, Y., 2008. Comparison of the amounts of endotoxin
released from Escherichia coli after exposure to antibiotics and ozone: an in vitro evaluation. J. Vet.
Med. Sci. 70, 419-422.
Silva Júnior, J.I.S, Santos, C.S.F., Silva, B.M., Santos, I.F.C, Ferro, B.S., Barros, T.I.S.,
Tomacheuski, R.M., Simões-Mattos, L., 2019. Topical ozone therapy in the treatment of
pharmacodermia in a dog (Canis lupus familiaris). Acta Sci. Vet. 47, 425.
Silva, R.A., Garotti, J.E.R., Silva, R.S.B., Navarini, A., Adhemar, M.P., 2009. Analysis of the
oo
f
bactericida effect of ozone pneumoperitoneum. Acta Cir. Bras. 24, 124-127.
Silveira, A.M., Lopes, H.P., Siqueira, J.F. Jr., Macedo, S.B., Consolaro, A., 2007. Periradicular
pr
repair after two-visit endodontic treatment using two different intracanalmedications compared to
e-
single visit endodontic treatment. Braz. Dent. J. 18, 299-304.
Pr
Siniscalco, D., Trotta, M.C., Brigida, A.L., Maisto, R., Luongo, M., Ferraraccio, F., D’Amico, M.,
Di Filippo, C.,2018. Intraperitoneal administration of oxygen/ozone to rats reduces the pancreatic
al
damage induced by streptozotocin. Biology (Basel) 7, 1-13.
rn
Smith, N.L., Wilson, A.L., Gandhi, J., Vatsia, S., Khan, S.A., 2017. Ozone therapy: an overview of
Jo
u
pharmacodynamics, current research, and clinical utility. Med. Gas. Res. 7, 212-219.
Spadea, L., Tonti, E., Spaterna, A., Marchegiani, A., 2018. Use of ozone-based eye drops: a series of
cases in veterinary and human spontaneous ocular pathologies. Case Rep. Ophthalmol. 9, 287–298.
Srikanth, A., Sathish, M., Sri Harsha, A,V., 2013. Application of ozone in the treatment of
periodontal disease. J. Pharm. Bioallied Sci. 5, S89–S94.
Stange, C., Sidhu, J.P.S., Toze, S., Tiehm, A., 2019. Comparative removal of antibiotic resistance
genes during chlorination, ozonation, and UV treatment. Int. J. Hyg. Environ. Health 222, 541-548.
Szponder, T., Wessely-Szponder, J., Smolira, A., 2017. Evaluation of platelet-rich plasma and
neutrophil antimicrobial extract as two autologous blood-derived agents. Tissue Eng. Regen. Med.
14, 287–296.
Journal Pre-proof
Teixeira, L.R., Luna, S.P.L., Taffarel, M.O., Lima, A.F.M., Sousa, N.R., Joaquim, J.G.F., Freitas,
P.M.C., 2013. Comparison of intrarectal ozone, ozone administered in acupoints and meloxicam for
postoperative analgesia in bitches undergoing ovariohysterectomy. Vet. J. 197, 794–799.
Thanomsub, B., Anupunpisit, V., Chanphetch, S., Watcharachaipong, T., Poonkhum, R., Srisukonth,
C., 2002. Effects of ozone treatment on cell growth and ultrastructural changes in bacteria. J Gen
Appl Microbiol. 48, 193-199.
Thurston-Enriquez, J.A., Haas, C.N., Jacangelo, J., Gerba, C.P., 2005. Inactivation of enteric
oo
f
adenovirus and feline calicivirus by ozone. Water Res. 39, 3650-3656.
Tsuzuki, N., Endo, Y., Kikkawa, L., Korosue, K., Kaneko, Y., Kitauchi, A.,Torisu, S., 2015. Effects
pr
of ozonated autohemotherapy on the antioxidant capacity of Thoroughbred horses. J Vet. Med. Sci.
e-
77, 1647-1650.
Pr
U.S. Food and Drug Administration. 2001. Secondary direct food additives permitted in food for
human consumption. Fed Regist. 66:33829–33830.
rn
Ophthalmol. 9, 95–108.
al
Vaziri, K., Schwartz, S.G., Kishor, K., Flynn Jr., H.W., 2015. Endophthalmitis: state of the art. Clin.
Jo
u
Vendruscolo, C.D.P., Moreira, J.J., Seidel, S.R.T., Fülber, J., Neuenschwander, H.M., Bonagura, G.,
Agreste, F.R., Baccarin, R.Y.A., 2018. Effects of medical ozone upon healthy equine joints: Clinical
and laboratorial aspects. PLoS ONE 13, 1-27.
Viebahn-Hansler, R., Leon Fernandez, O.S., Fahmy, Z., 2012. Ozone in medicine: The low-dose
ozone concept—Guidelines and treatment strategies. Ozone-Sci. Eng., 34, 408–424.
Vigliani, A., Boniperti, E., Scudo, E., 2005. Paravertebral O2-O3 treatment in mechanical lumbar
pain in riding horses. Rivista Italiana di Ossigeno-Ozonoterapia 4, 64-69.
von Gunten, U., 2003. Ozonation of drinking water: Part I. Oxidation kinetics and product formation.
Water Res. 37, 1443-1467.
Journal Pre-proof
Wang, L., Chen, Z., Liu, Y., Du, Y., Liu, X.,2018. Ozone oxidative postconditioning inhibits
oxidative stress and apoptosis in renal ischemia and reperfusion injury through inhibition of MAPK
signaling pathway. Drug Des. Devel. Ther. 12, 1293–1301.
Weast, R.C., 1970. Handbook of chemistry and physics, 51st ed. Chemical Rubber Co., Cleveland.
Wells, K.H., Latino, J., Gavalchin, J., Poiesz, B.J., 1991. Inactivation of human immunodeficiency
bvirus type 1 by ozone in vitro. Blood 78, 1882–1890.
W., Tang,
H., Wu,
M., Liao,
Y., Li,
fibroblasts
L., Xu,
via
X.,
2017.
PI3K/Akt/mTOR
oo
Ozone oil promotes wound healing by increasing the migration of
K., Li,
f
Xiao,
signaling pathway. Biosci Rep. 9, 37-42.
pr
Yamayoshi, T., Tatsumi, N., 1993. Microbicidal effects of ozone solution on methicillin-resistant
e-
Staphylococcus aureus. Drugs Exp. Clin. Res. 19, 59-64.
Pr
Zargaran, M., Fatahinia, M., Zarei Mahmoudabadi, A., 2017. The efficacy of gaseous ozone against
different forms of Candida albicans. Curr. Med. Mycol. 3, 26–32.
al
Zhang, J., Guan, M., Xie, C., Luo, X., Zhang, Q., Xue, Y., 2014. Increased growth factors play a role
rn
in wound healing promoted by noninvasive oxygen-ozone therapy in diabetic patients with foot
Jo
u
ulcers. Oxid. Med. Cell Longev. 2014,1-9.
Zobel, R., Tkalčić, S., Štoković, I., Pipal, I., & Buić, V., 2011. Efficacy of ozone as a novel treatment
option for urovagina in dairy cows. Reprod. Domest. Anim. 47, 293–298.
Legend
Figure 1. The oxidative mechanism of action of Ozone. Ozone and the unsaturated fatty acids (UFA)
present in the cellular membrane react and generate hydrogen peroxide (H2 O2 ) and aldehyde 4hydroxynonenal (4-HNE). 4-HNE can activate the synthesis of several substances, such as γglutamyl transferase, γ-glutamyl transpeptidase, heat shock protein (HSP-70), haem oxygenase-1
(HO-1), and antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase
(GSH-Px), catalase, and glucose-6-phosphate dehydrogenase (G6PDH) and the nuclear factor
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erythroid 2-related factor 2 (Nrf2); the latter is responsible for activating the synthesis of the
antioxidant enzymes. Through the cellular membranes and in the cytosol, H2 O2 promotes Nrf2.
Application
Clinical mastitis
Formulation
Route
O2 -O3 mixture Intranipple
gas
Concentrations
6 mg/L
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f
Species
Table 1. Application of Ozone Therapy in different species.
References
Ogata
and
Nagata, 2000
Journal Pre-proof
Sunflower oil
Udder
600 eq/Kg
Urovagina
Spray foam
Intravaginal
Equine
Chronic laminitis
mixture Intra-articular
mixture Intrascapular
mixture Interspinous
muscle and
paravertebral
Conjunctivitis
Ophtalmic oil
Intraocular
No concentration
avaible
Ocular pathologies Liposomal
Topical
No concentration
dispersion
avaible
Pharmacodermia
Sunflower oil
Local
No concentration
avaible
Disc herniations
O2 -O3 mixture Intradiscally
32 µg/mL
gas
Postoperative
O2 -O3 mixture Intrarectally
30 µg/mL
analgesia
gas
and acupoints
Dentistry
Ozonized oil
Root canal
No concentration
avaible
Herniated
O2 -O3 mixture Percutaneous 30 μg/mL
intervertebral disc gas
intradiscal
Secondary wound O2 -O3 mixture Topical
60 μg/µL
healing
gas
application
Porcine
Tab. 1
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u
rn
Canine
al
Pr
Lumbar pain
O2 -O3
gas
O2 -O3
gas
O2 -O3
gas
pr
Osteartritis
e-
Ovinecaprine
Ovules or spray Intrauterine
foam
Retention of fetal Spray foam
Intrauterine
membranes
Reproductive
Spray foam or Intrauterine
efficiency
pearls
Retention of fetal Spray foam
Intrauterine
membranes
Acute putrefaction O2 -O3 mixture Local wraps
of foot
gas
Antioxidant
O2 -O3 mixture Autohemothe
capacity
gas
rapy
oo
Metritis
Quintana et al.,
2019
No concentration Zobel et al., 2011
avaible
No concentration Ðuričić
et al.,
avaible
2012
No concentration Imhof et al., 2019
avaible
No concentration Ðuričić
et al.,
avaible
2011, 2012
No concentration Duricic et al.,
avaible
2016
70 mg/ml
Szpondera et al.,
2017
20 µg/Kg in 400 Tsukuki et al.,
mL
autologus 2015
blood
20-40 µg/mL
Vendruscolo
et
al., 2018
19 mg/L
Coelho et al.,
2015
30 µg/mL
Ballardini, 2005
f
Bovine
Suclinical mastitis
Spadea et al.,
2018
Marchegiani
et
al., 2019
Silva Junior et al.,
2019
Han et al., 2007
Teixeira et
2013
Silveira et
2007
Murphy et
2016
Eroglu et
2019
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Highlights
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Ozone (O 3 ) is a triatomic form of oxygen normally present in athmosphere
O 3 is widely used in human and veterinary medicine through several pharmaceutical forms
O 3 has antioxidant, anti-inflammatory, immuno-stimulant and microbicidal effect
This review focuses on O 3 mechanisms of action and on its main application in Veterinary
Medicine
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Figure 1