Propolis is a resinous ma­terial produced by bees (Apis mellifera) using wax and plant exudates. The word propolis is believed to have been given by Aristotle to indicate that propolis is used to pro­tect and defend the hive (Alves et al., 2013). Propolis means ‘before the city’ or ‘defender of the city’ (Greek pro: in de­fense, and polis: city). In the beginnings of Greek civilization, Aristotle observed that propolis had the ability to defend a city with thousands of residents, the hive.

Propolis was useful for both structural repair and for maintenance of the species through the preparation of aseptic places for the deposit of eggs of the queen bee (Kuropatnicki et al., 2013). Propolis is important for the bees, since it is an ad­hesive material used to seal openings and cracks in the hive (Bankova et al., 2012), and is also used to smooth internal walls (Burdock, 1998) and to protect the colo­ny from diseases and cover the corpses of intruders that have died in the hive, preventing their decomposition (Bankova et al., 2000). It has been observed that bees collect the protective resin from flowers, leaves and buds with their jaws and then take them to the hive on its hind legs, its color varying from green to reddish brown depending on its botanical source (Kuropatnicki et al., 2013).

Propolis has been wide­ly used since antiquity. The Egyptians took advantage from the anti-putrefaction properties of propolis to embalm their dead and the Greek and Roman physi­cians used it as an antiseptic and healing medicine (Sforcin and Bankova, 2011). In the last decades numerous articles have been published describing different as­pects of the biological properties of prop­olis (Rocha et al. 2013. However, in the majority of them, the information is lim­ited (Sforcin and Bankova, 2011). Currently, propolis is used as a folk rem­edy and is available in different pharma­ceuticals forms (capsules, gel, powder, mouthwash, cream and powder) and com­binations thereoff (Castaldo and Capasso, 2002). Propolis has become popular con­stituent that complements health care products, as a natural ingredient in the pharmaceutical industry and in food preparation. Antimicrobial, antioxidant, antiviral, antifungal, anti-inflammatory, immunomodulatory and anti-tumoral ac­tivities are among the therapeutic proper­ties that have been studied (Monzote et al., 2012; Chan, 2013).

The most common methods for propolis extraction uses ethanol as solvent in different mixtures of water and etanol (Rocha et al., 2013). The precise composition of prop­olis can be determined through chemi­cal analysis by HPLC (Castro, 2001). As with honey, propolis composition varies with a variety of factors, such as the source of the exudates, the climate and environmental conditions (Chen and Wong, 1996). The presence of at least 300 compoundsof has been identi­fied, out of which approximately 50% correspond to resins, 30% to wax, 10 % to essential oils, 5% to pollen and 5% to other organic compounds (Gómez et al., 2006). Among these organic compounds, it is possible to find phe­nolic compounds, esters and flavonoids (flavonols, flavones, flavonones, dihy­droflavonols and chalcones), terpenes, steroids, aromatic beta-aldehydes, alco­hols, sesquiterpenes and stilbene (Aga et al., 1994;Russo et al., 2002).

Propolis has been used in veterinary medicine in different fields and pharmaceutical forms and in differ­ent animal species, among which have been reported: solutions for the preven­tion and control of foot diseases in sheep (Bogdanov, 2012), mammary infu­sion for treating mastitis, antidiarrheal powders, boluses and injectable solu­tions in genitourinary diseases such as endometritis (Bogdanov, 2012; Martos et al., 2008), eyedrops and ointments for keratitis and keratoconjunctivitis infec­tious; tinctures and pomades for wounds, disinfectant solutions and repel­lents as therapy in calf omphalitis (Burdock, 1998), and it has also been used against rabbit infections by Pasteurella multocida (Gutiérrez, 2011).

Antiviral activity

Propolis is capable to inhibit viral propagation. Most of the studies published describe activities against viruses that affect humans. In vi­tro studies demonstrate the effects of propolis over both DNA and RNA virus­es. The observed effects include reduc­tion in viral multiplication and even a virucidal action (Amoros et al., 1992), but the mechanism of the antiviral action of propolis is still unclear. The first hints about the mechanism of antiviral action of propolis was given by Selway (1986) by mentioning that the flavonoids com­pounds can be related to the inhibition of viral polymerase and the binding of viral nucleic acid or viral capsid proteins (Selway, 1986). The mechanism of action could suggest a receptor cell membrane blockage by the propolis, or propolis in­teractions that induce internal changes in the host cells, which in turn affects the virus replication cycle (Amoros et al., 1992). Other authors state that the effect is due to the flavonoids and other pheno­lic compounds that are present in propo­lis and interact with viral proteins, form­ing complexes unstable compounds and therefore altering the stages of adsorption and penetration (Selway, 1986; Amoros et al., 1992).

Different components of propolis appear to act synergistically, which would explain the fact that honey and propolis posses a greater antiviral activity than their individual components (Kujumgiev et al., 1999). The antiviral activity of propolis has been tested, with promising results, against some patho­genic viruses to humans (Gutiérrez, 2011) and animals (Amoros et al., 1992), such as the herpes simplex virus type 1 (HSV- 1; Hegazi et al., 2001) and type 2 (HSV- 2), human immunodeficiency virus (HIV) (Gekker et al., 2005) and avian influenza (Bogdanov, 2012). However, few studies have been published regard­ing the use of propolis in viral diseases of veterinary interest. This review de­scribes the biological properties of propo­lis antiviral mentioned specifically in veterinary medicine widening the pros­pect of use.

Uses and Perspectives of Propolis in Animal Viral Diseases

Poultry

Hegazi et al. (2001) used propolis of four different origins (Egypt, Austria, France and Germany) to prove the antiviral activity of propolis on Infectious Bursal Disease Virus (IBDV) and Avian Reovirus (ARV). The active components of propolis were identified by mass spectrometry. To assess the in­fectivity of the virus and the antiviral ef­fect of propolis, primary cultures of chicken embryo fibroblasts (CEF) were used. All the propolis varieties used caused a reduction in the titer of IBDV and ARV, as determined by its TCID50. Further, when the cytopathic effect (CPE) was evaluated, they found that the degree of reduction was different in each of the samples, but all of them reduced viral in­fectivity at different degrees. In this study the Egyptian propolis showed the greatest activity against ARV and IBDV. Authors suggest that propolis show quali­tative similarities between them and the quantitative differences obtained could be due to the participation of different pop­lar species, therefore the reduction of in­fectivity is completely dependent on the chemical composition of each propolis (Hegazi et al., 2001; Bankova et al., 2002; Sforcin and Bnkova, 2011).

The components of three propolis originating from different regions of Egypt (Dakalia, Ismailia and Sharkia) were determined by mass spectrometry and gas chromatography. The antiviral ac­tivity against IBDV and ARV was also evaluated by determining the viral titer (TCID50) in cultures of chicken fibro­blasts. The sample from Dakalia showed the presence of aliphatic acids, aromatic acids, esters of aromatic acids, flavonoids and some triterpenoids; a total of 65 com­pounds were identified. The composition of propolis from Sharkia does not vary significantly fron the Dakalia propolis, while propolis from Ismailia does not con­tain aromatic acids, aromatic esters (ex­cept phthalate esters) and flavonoids (ex­cept hexamethoxyflavone). The propolis that produced the largest reduction in vi­ral titer were Dakahlia against IBDV and Ismailia versus ARV. On the other hand, propolis from Sharkia showed moderate activity against both viruses. Reduced in­fectivity depends on the chemical compo­sition presented by the propolis samples from the three provinces. A reduction in cytopathic effect was observed, but this changed with the chemical composition of each of the samples (Abd El Hadya and Hegazi, 2001).

The activity of propolis has also been tested in conjunction with other components, as is the case of the studies carried out using the Newcastle disease virus (NDV) and IBDV (Kong et al., 2006). They analyzed eight extracts of propolis from Egypt in which 42 poly­phenol components were determined by HPLC. Thirteen aromatic acids were found, as well as esters and alcohols, and 29 flavonoids, 6 of which had not been previously reported. Viral titration was performed in cultures of fibroblasts. The determination of antiviral activity was made by mixing an equal volume of serial dilutions of each virus with stock solutions of propolis. and the antiviral activity of the samples was determined by the de­crease in the TCID50 in chicken embryo fibroblasts. All propolis samples led to a reduction in TCID50 of IBDV and NDV, and the reduction varied according to the origin of propolis. Similar results were re­ported with herpes simplex virus (Abd El Hadya et al., 2007), avian influenza virus (Amoros et al., 1992) and infectious bursal disease virus and reovirus (Abd El Hadya et al., 2007).

On the other hand, (Kong et al., 2006), determined the effec­tiveness of four components of a prepared Chinese herbal product (CHI) in chick­ens, both in vitro and in vivo. The prod­uct used included astragalus polysaccha­ride (ASP), isatis root polysaccharide (IRP), propolis polysaccharide (PP) and epimedium flavone (EF). Animals distrib­uted in 10 groups were used, with a total of 200 animals. Chickens were inoculated with the IV strain of NDV by intranasal and intraocular administration. Other groups were administered for 3 consecu­tive days with 0.5ml of CHI subcutane­ously, corresponding to different doses/kg body weight of APS and IRP. Control groups (CHI-free control an a vaccination control and CHI-free) of chickens were injected with physiological saline. The re­sults indicate that most of the CHI used in appropriate concentrations were effec­tive. The administration of CHI to the vaccinated chickens increased the concen­tration of antibodies anti-Newcastle in se­rum, as determined by hemagglutination inhibition, compared to single administra­tion of Newcastle. The effect on the pro­duction of antibodies was observed 21 days after the start of vaccinations.

Bovines

There are studies per­formed on cattle about the antiviral activ­ity of propolis experimentally used to counteract the causal agents of diarrhea syndromes, as are the presence of entero­toxigenic bacteria Escherichia coli, Cryptosporidiumsp. and rotavirus in groups of calves. Two schemes of treat­ment were applied: group A was treated orally with a propolis solution, while the group B was subjected to standard thera­py with oximicine. The results revealed that animals treated with propolis showed a better recovery at 24, 48, and 72h. When comparing the percentage of reco-vered animals with those obtained in group B it was determined that these dif­ferences were statistically significant (Bernal, 1991).

Porcines

An in vitro evaluation was carried out of the antiviral effect of a Mexican propolis on pseudorabies virus (PRV) by infecting cultures of MDBK cells (González, 2015). In order to infect these cultures with the Shope strain of PRV, an infective dose was determined and, subsequently, an ethanol extract of propolis (EEP) was allowed to interact with the virus (2h before, during and 2h after infection). Also, in order to deter­mine the effects of EEP on virus, sam­ples from cell cultures subjected to the different previously mentioned treatments were processed for transmission electron microscopy (TEM). It was observed that administration of EEP two hours before infection resulted in a reduction in the number of plaque forming units com­pared to the other treatments or with the infected culture without treatment. The difference found was statistically significant. Under TEM, viral particles with al­tered structure were observed, suggesting the ocurrence of damage to proteins of the viral envelope, causing structural de­stabilization, and an electron-dense layer was also observed around the membrane of cells in which viral particles were found, so that propolis seems to affect both penetration and the viral replication cycle. In another work, the extract aque­ous of Israel propolis was tested on cul­tured Vero cells infected with Herpes Simplex Virus type 1 (HSV-1), treating the cultures with propolis at various con­centrations for 2h before and 2h after completion of the infection. The cultures were maintained for 10 days and resulted in a lower CPE with the more concentrat­ed extract of propolis, showing that the treatment applied prior to infection result­ed in less penetration of the virus into the host cell. In this same study, the ef­fect of the virus was evaluated when ap­plied after the treatment with propolis, without changes in the cytopathic effect. On the other hand, when virus and prop­olis were applied simultaneously to the culture, similar results were obtained as in the pre-treatment, in which a lower cy­topathic effect was shown to take place (Mahmoud y Isanu, 2002).

Similar results were ob­tained by Schnitzler et al. (2010), who studied the antiviral effect in cell culture of aqueous and ethanolic extracts of propolis, as well as that of caffeic, p-cou­maric and benzoic acids, galangin, pino-cembrin and chrysin, against Herpes Simplex Virus type 1 (HSV-1). The two extracts of propolis showed high levels of antiviral activity against HSV-1, as plaque formation was significantly re­duced when applied before viral infec­tion. The compounds galangin and chry­sin showed significant antiviral activity when compared to the rest. The study shows that extracts differ quantitatively and qualitatively, indicating that the etha­nolic extract has more flavonoids and therefore its greater antiviral capacity. The activity of each propolis is reflected in the decrease in the cytopathic effect in relation to the exposure time and the con­centration at which they are used in the tests (Schnitzler et al. 2010).

Dog and cat

The antiviral activity of two ethanol extracts of propolis (EP1 and EP2) from two different sources against feline calicivirus (FCV), canine adenovi­rus type 2 (CAV-2) and Bovine Viral Diarrhea Virus (BVDV) was determined by Cueto et al. (2011). The choice of these viruses is justified because the CAV-2 and FCV are etiologic agents of respiratory diseases in the respective spe­cies, while BVDV has been widely used as a model to study the antiviral activity against hepatitis C. In the analysis of propolis extract by high pressure liquid chromatography, the presence of flavo­noids such as rutin, quercetin and gallic acid was detected. In the chromatogram, it is possible to visualize the presence of different peaks between samples of the commercial propolis (EP2) and the propo­lis extract obtained in the laboratory (EP1). There were quantitative differences in the chromatographic peaks, especially in the rutin fraction. For evaluation of antiviral activity of EP1 and EP2 diverse cells (MDBK, MDCK and CRFK) were infected with the viruses (25 μl/well of viral dilution), incubated for 2h and the inoculum was removed. A negative con­trol (cells and medium) and a positive control (viruses, cells and medium) were included. The extracts were added: I) be­fore viral inoculation (interaction of cells-extract for 1 h); II) after the viral inoculation (the extracts were added im­mediately after inoculation) and III) be­fore and after viral inoculation. After three days of incubation, the culture medium was removed and cell viability was quantified by determining the degree of metabolization of 1-(4,5-dimethyl-thiazol-2-yl)-3,5-diphenylformazan. Under these conditions the median effective concentration (EC50), which is the con­centration of propolis extract that can inhibit the cytopathic effect of the virus in the 50% of the cultures, was deter­mined. The results (Cueto et al., 2011) showed that when the extracts are added before infection an increased antiviral ef­fect is observed. The activity of the ex­tract obtained in the laboratory (EP1) was superior to the commercial one (EP2). Both propolis extracts showed higher ac­tivity against BVDV that to the other two viruses, although the differences between BVDV and CAV-2 were small. BVDV has a lipid envelope surrounding the nucleo­capsid and this could constitute a struc­tural difference with the other two virus­es and could explain the greater sensitivi­ty of this virus to the antiviral action of the extracts.

Conclusions

Propolis appears to be a promising alternative for the prevention and possible treatment of some viral dis­eases in several animal species. It is not­ed that all the explored antiviral activities of propolis have been performed in vitro (Table I). It is important to translate this knowledge into clinical practice and ex­plore their properties and their mecha­nism of action. On the other hand, the adjuvant properties of propolis have been explored in vivo with promising results, but it is essential to conduct further stud­ies in order to know which components are responsible for the different biological activities

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Notes