JUL AYUDA PARA TRATAMIENTO DE DISTEMPER. Public. · Hosted by Valeria Pizarro Rojas. Interested. clock. Tuesday, July 17, at AM UTC [ 7 ] SINTOMAS DEL MOQUILLO EN PERROS y [ 1 ] FORMA MUY GRAVE MOQUILLO NERVIOSO. Sintomas del Moquillo en Perros – Tratamiento. Canine distemper is a contagious and serious disease caused by a virus that attacks the respiratory, gastrointestinal and nervous systems of puppies and dogs .
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Propolis is a resin that honey bees Apis mellifera produce by mixing wax, exudates distem;er from dostemper shoots, pollen, and enzymes. It has been used for its biological properties against pathogenic microorganisms including those of viral origin. In the present study, we demonstrate the antiviral effect of Mexican propolis, tratamiennto well as of the three commercial flavonoids quercetin, naringenin, and pinocembrin tratamento in its composition, in cell cultures infected with Canine Distemper Virus.
The treatments were carried out with propolis, flavonoids individually, and a mixture of the three flavonoids at three different times. Antiviral activity was evaluated by the inhibition of the relative tratammiento of the virus nucleoprotein gene Real-Time qPCR and by the determination of cellular viability MTT assay. Propolis applied before infection decreased viral expression 0. The administration of a flavonoid mixture containing the three commercial flavonoids before infection ttratamiento a slight decrease in viral expression 0.
Quercetin administrated at the same time of infection decreases viral expression 0. Pinocembrin and naringenin individually did not show any antiviral activity at the administration times evaluated in this tratamifnto. The present work is the first in vitro study of the effect of propolis in Canine Distemper Virus and demonstrated the antiviral tratamjento of Mexican propolis, in addition to the synergy that exists between the three flavonoids on cell viability and the expression of the nucleoprotein virus gene.
Propolis is a resinous mixture that honey bees Apis mellifera produce by mixing beeswax, exudates gathered from tree buds, pollen, and enzymes secreted by the same bees [ 1 — 3 ]. Among the therapeutic properties of propolis, several have been investigated: Among the organic compounds, it is possible to find phenolic compounds and esters as well as several forms of flavonoids, terpenes, steroids, aromatic beta-aldehydes, alcohols, sesquiterpenes, and stilbenes [ 1011 ].
The combination of these compounds results in a synergic effect that plays a central role in the biological activity of propolis [ 12 ]. Several studies have highlighted the beneficial effects of flavonoids, given their antioxidant, antitumor, and antimicrobial activities [ 1314 ]. Likewise, the antiviral activity of flavonoids of several viruses has been demonstrated, namely, against herpes simplex virus HSV-1 and HSV-2Sindbis virus, parainfluenza-3 virus, human cytomegalovirus, and dengue virus type 2 [ 15 — 18 ].
In general, it has been found that flavonoids are more active than flavones and that synergism is obtained when both compounds are combined, which explains the fact that propolis presents a better biological activity than either substance individually [ 19 ]. The antiviral activity of propolis against some pathogenic human viruses, such as the HSV-1 [ 1920 ], HSV-2 [ 21 ], and human immunodeficiency virus HIV [ 22 ], has been evaluated.
Propolis has also been tested for its activity against several animal viruses: Pinocembrin 5,7-dihydroxyflavone is an disfemper propolis flavonoid [ 2930 ] that is found in pine trees, dry fruits, eucalyptus leaves, and acacia gum.
It has been demonstrated that this flavonoid possesses extensive pharmacologic effects, including antimicrobial [ 3132 ], antioxidant [ 33 ], antimutagenic, and anti-inflammatory properties [ 34 ]. On the other hand, it has been demonstrated that the flavonoid naringenin presents both antioxidant and antiviral activities against dengue virus and herpes simplex viruses 1 and 2 [ 16 — 1835 ]. Finally, quercetin is a polyphenolic flavonoid [ 36 ] that can be found in several plants and is considered one of the most potent antioxidants of vegetable origin; it also possesses a wide range of antiviral [ 37 ], antidiabetic [ 38 ], anti-inflammatory [ 39 ], and neuroprotective effects [ 40 ].
Canine Distemper is a severe multisystemic viral disease that affects dogs and other carnivores. The etiologic agent is known as Canine Distemper Virus CDVwhich is closely related to the measles virus, bovine pestivirus, small ruminant pestivirus, and phocine distemper virus [ 364142 ].
It belongs to the family Paramyxoviridaesubfamily Paramyxovirinae, and the genus Morbillivirus [ 37 ].
This work aimed to evaluate the antiviral activity of the Mexican propolis and three commercial flavonoids and the mixture thereof quercetin, naringenin, and pinocembrin against the Canine Distemper Virus. The collected material was cleansed of ditemper contaminants like splints, plastic, and bee’s body parts, among other things. After this time had elapsed, the solution was filtered using a Whatman No. The constituents were identified based on a comparison of the retention time and UV spectrum with those of the standards.
The concentration used for each extract was of 0. Commercial flavonoids, quercetin Qpinocembrin Pand naringenin Nwere acquired from Sigma Laboratories.
The results of the trials were reported in absorbance units AU. The antiviral capacity of the EEP was evaluated in vitro and compared to that exhibited by trataiento of the previously mentioned commercial flavonoids, as well as that of a mixture of them.
Treatment effectiveness eistemper determined using two methods: Six experimental conditions were established with four treatment administration times: Four experimental conditions were used with three times of administration of EEP or flavonoids: The incubation time for all experimental conditions was 48 h.
Primers were designed by using the Primer3 software, version 4. All assays were carried out three times for each experiment. Thermocycling conditions used were as follows: Reactions were standardized at the same temperature and number of cycles for both genes.
In this way, the efficacy of the system was evaluated and the type of quantification to be used for this study was determined. Next, the threshold cycle CT was identified with the purpose of determining the cistemper expression present in each treatment.
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The viability of the cell cultures was evaluated according to the average absorbance units produced by the cytolytic effect of the CDV in each treatment. To determine the relative expression of the CDV-NP, concerning untreated control cultures, a Livak method was used, implementing the following formula: The results of the cytotoxic effect of the EEP and the three commercial flavonoids, to obtain the mean cytotoxic concentration CC 50are shown in Table 2.
With these results, a cytotoxicity curve was chosen, to obtain the adequate concentration for the antiviral treatments that were used in the present study, which are shown in Table 2.
Canine Distemper Virus Buzzell strain was adapted to Vero cells and after 24 hours of infection cytopathic effects were noted, namely, syncytia formation, rounding of the cells, and vacuolization. After 48 hours lytic plaque formation was observed followed by total culture lysis data not showed. The capacity of Buzzell strain to produce cellular lysis allowed use of two methods for the evaluation of the antiviral treatments: With the purpose of determining the antiviral effect of the EEP and the commercial flavonoids in the cellular culture infected with CDV, cellular viability was evaluated by the MTT colorimetric assay [ 44 ].
When the EEP is applied to the cell culture at three different moments before, during, and after infectiona reduction in the lytic effect produced by the virus is observed.
Quercetin Figure 1 b showed a better effect when it was administered at the same time of infection Figure 1 b -5 ; there is a greater effect than that of propolis when they are compared in the same treatment time Figure 1 b -5 versus Figure 1 a Figure 1 c On the other hand, naringenin kept cellular viability only when it was administered at the same time of the infection, as shown by the statistically significant difference with the positive control cells infected with the virus and the other two treatments used Figure 1 d -5 versus Figures 1 d -4 and 1 d Additionally, a treatment where a flavonoid mixture pinocembrin, naringenin, and quercetin was used, at the already mentioned concentrations and times, was performed Figure 1 e.
A more pronounced decline in the relative expression of the CDV-NP gene was found when propolis was applied before viral infection Figure 2 a -2 ; this decline was not as evident with treatments applied after Figure 2 a -3 and at the same time of infection Figure 2 a When quercetin was administered at the same time of viral infection Figure 2 b -3a small reduction in the relative expression of the virus nucleoprotein gene was observed.
On the other hand, pinocembrin Figure 2 c and naringenin Figure 2 dwhen administered at any of the three established times, did not induce any change in the relative expression of the CDV-NP gene. Treatment with the flavonoids mixture Figure 2 eapplied before viral infection Figure 2 e -2resulted in a more marked reduction of the relative expression of the CDV-NP gene than that observed when the treatment was implemented at the same time and after viral infection.
For the Mexican propolis, by making use of an analytical method, we vistemper able to identify several phenolic compounds; among them, flavonoids represent a way by which to establish a quality index for propolis. The higher the percentage of these compounds in any given propolis, the better its purity and quality [ 47 ]. FESC propolis harvested during October presented a higher content of flavonoids and phenols than that of other propolis collected at the same location [ 43 ], indicating that variation of factors such as vegetation, flowering, and climatic conditions of the location affect the chemical composition of propolis directly and therefore its biological properties.
For this reason, it can be expected for the FESC-EEP to exhibit intense biological activity, given its high content of flavonoids and phenols. Mean cytotoxic concentration CC 50 and the employed concentrations of EEP, quercetin, and naringenin, used during the present study, are lower than those mentioned by other authors [ 37 ], while the used pinocembrin concentration is similar to that reported in other articles [ 29 ]. The variability in concentrations can be attributed to several factors: With the purpose of evaluating whether the times at which the treatments with propolis and the commercial flavonoids were administered had any influence on the development of viral infection, three different treatment administration moments were established two hours before, during, and two hours after viral infection.
In this way, it was observed that a higher efficacy of the EEP was achieved when it was administered before viral infection, which suggests that EEP directly interacts with host cells by interfering with proper recognition between cellular receptors [ 26 ] and virus proteins, thus preventing virus internalization and further replication.
Therefore, a minor cytopathic effect on culture cells was observed [ 2148 distempeg, 49 ]. Quercetin administered at the same time of infection increases cell viability and decreases viral gene expression. This flavonoid has been the subject of several studies because it is generally found in all propolis [ 1937 ]; many flavonoids, quercetin being among them, have demonstrated presenting antiviral activity against CDV [ 37 ].
Considering the fact that quercetin displays a higher antiviral effect when it is administered distejper the distempef time of infection, it is hypothesized that it acts by inhibiting the intracellular phase of the replication cycle of the CDV, thanks to its capacity to inhibit viral polymerase and interfere with viral nucleic acid synthesis [ 2249 ].
Additionally, its antiviral activity has been associated with its capacity to bind viral proteins and to block cellular protein synthesis [ 49 — 51 ]. It is suggested that if the concentration of this flavonoid increases, its biological activity can disstemper improved; however, the range of cytotoxicity of quercetin in cell culture should be considered to avoid cell damage, causing loss of cell viability and therefore alterations in the results.
Pinocembrin is the most abundant flavonoid in the FESC propolis, providing its important biological properties, such as antiviral, are ascribed to it, as demonstrated by its activity against herpes simplex virus [ 19 ]. It has been determined that this flavonoid acts by inhibiting viral replication cycle through specific interference with viral DNA polymerase [ 21 ]. Its antiviral activity against dengue virus, when there has been a previous direct interaction between this substance and the virus followed by culture inoculation, has also been reported; the mentioned concentration is very similar to that used in this study [ 29 ].
Nonetheless, in the present study, when pinocembrin and naringenin were used individually, regardless of the time at which they were administered, viral gene expression reduction was not observed, indicating trayamiento both of these compounds do not affect CDV replication.
Flavonoids mixture pinocembrin, naringenin, and quercetin treatment application before viral infection showed a distemler pattern to that observed for the EEP, that is, maintaining cellular viability and diminishing CDV-NP gene expression, thus demonstrating the existence of a synergistic effect between the three compounds, given the fact that, when used individually, the obtained results for each flavonoid were different.
Several authors have mentioned the ditsemper effect between some flavonoids, even when they interact with another kind of antiviral agents [ 49 ], which explains the fact that both, honey and propolis, exhibit a higher antiviral activity than their components [ 48 ].
Within research on the use of propolis in vitro, it is worth noting that distempfr is a proven model in which continuing with the specific analysis of the fratamiento activity of each of the compounds and the synergy between them is suggested. It is thought that in the future it is possible to implement an in vivo model, but not before standardizing specific concentrations of each of the components. Since we must consider that each propolis presents some changes in its composition rtatamiento effectiveness, based on our results we can point out that the propolis administered as a compliment or preventive of the CDV disease could be favorable.
These results indicated that the antiviral activity of the ethanolic extract of Mexican propolis was demonstrated, as well as the synergistic effect that exists between the studied flavonoids, as shown by a reduction in the cytopathic effect, represented by cell viability, and CDV gene expression. These results offer new perspectives that may be useful for generating even more specific knowledge about the biological effects of propolis and its components in cell cultures and during viral infection in vitro.
Evidence-Based Complementary and Alternative Medicine. Indexed in Science Citation Index Expanded. Subscribe to Table of Contents Alerts. Table of Contents Alerts. Abstract Propolis is a resin that honey bees Apis mellifera produce by mixing wax, exudates collected from tree shoots, pollen, and enzymes. Introduction Propolis is a resinous mixture that honey bees Apis mellifera produce by mixing beeswax, exudates gathered from tree buds, pollen, and enzymes secreted by the same bees [ 1 — 3 ].
Materials and Methods 2. Antiviral Treatments The antiviral capacity of the EEP was evaluated in vitro and compared to that exhibited by each of the previously mentioned commercial flavonoids, as well as that of a mixture of them. Statistical Analysis The viability of the cell cultures was evaluated according to the average absorbance units produced by the cytolytic effect of the CDV in each treatment.
Mean cytotoxic concentration of EEP and commercial flavonoids. The different times at which the treatments were implemented are compared: