Nanosilver against African Swine Fever virus – a safe solution
African swine fever, also known as ASF, from the time it was discovered, scientists have yet to find a treatment vaccine. Viruses have the ability to exist for a long time in the environment, with diverse transmission routes and difficult to control. Causing great economic damage to the livestock industry. Nanosilver particles have been studied and shown to be effective as a potential antiviral agent against the African Swine Fever virus.
OVERVIEW
The African Swine Fever Virus (ASFV) is the cause of a highly contagious and fatal disease in farmed pigs, but so far there is no effective vaccine or drug, so the finding a new and effective anti-ASFV is an urgent task. Nanosilver particles (SNPs) have recently emerged as a new antiviral agent against many viruses, but their antiviral activity against ASFV has not been studied. In this study, the antiviral ability of SNP to ASFV was reported. Microbiological contamination in pig pens was significantly reduced by spraying with 25 ppm SNP. SNP solution with a concentration of 0.78 ppm did not have any toxicity to alveolar macrophage cells of pigs; while completely inhibiting ASFV at 103 HAD50 (RBC adsorption dose). This study confirms that nano-silver is highly resistant to ASFV and is a promising disinfectant that can be used to prevent ASFV transmission.
(Copyright by NanoCMM Technology)
INTRODUCE
The African Swine Fever Virus (ASFV) is a member of the genus Asfivirus in the family Asfarviridae, a double-stranded DNA virus with a complex tetrahedral morphology [1]. ASFV causes a viral illness in pigs with very high mortality in domestic pigs, while the disease is asymptomatic in natural fat hosts. The virus can be transmitted through direct contact with infected animals, by bites of infected arthropods, especially soft mites of the genus Ornithodoros, and by contact with materials or materials. infected with a virus such as undercooked meat, blood or fluid from an infected pig [2 -4]. This virus persists for a long time in the blood and tissues after death, so it is easily transmitted through the transport of pork products. Viruses can be found in all secretions, especially in nasal fluids. Airborne transmission also occurs in pig pens [3]. Outbreaks of the disease can have a significant economic impact on the affected area, vaccination is the best control measure, but unfortunately no vaccines have been sold so far. school. Vaccine development has been hampered by large gaps in understanding ASFV infection, immunity, and the mechanism by which the virus modulates host responses to infection [5]. Without a vaccine against ASFV, early diagnosis and effective hygiene measures are very important strategies for eliminating the disease in the affected area.
Since there is no vaccine yet, implementing biosafety measures is still the key to disease control and prevention. In addition to strict regulations on the transport of animals and animal products, management of pork-related waste, and monitoring of infected areas, effective disinfection practices play a very important role. disease control spread [6-8].
ASFV is inactivated by pH <3.9 or> 11.5 and some traditional disinfectants such as calcium hydroxide, hypochlorite, formalin, ortho-phenylphenol, Glutaraldehyde and iodine compounds [6, 9]. Traditional disinfectants often cause a number of disadvantages such as a bad odor, rapidly losing their antiviral activity and carcinogenicity; therefore, only certain disinfectants are recommended by the US Environmental Protection Agency for use in the fight against ASFV [9].
Significant efforts have recently been made to find more effective antiviral agents, especially those of natural origin, in the hope of developing an effective drug to treat the disease. or an antiviral agent for disinfection [5, 10, 11]. Plant-made Resveratrol and Oxyresveratrol (found in grape skins, blueberries, raspberries, and peanuts) to deal with biotic and abiotic stress can inhibit viral infection by disrupting functions. of cells [1].
Genistein exhibited the strongest anti-ASFV-Ba71V activity with a 3.8 log decrease in viral titer at concentrations of 50 mM and 8 hpi [12]. Genistain’s antiviral activity on the ASFV Ba71V strain has been determined by a number of ways including impaired viral DNA synthesis, inhibition of the post-invasive stages of the ASFV life cycle, and inhibition of enzyme activity. ASFV type II topoisomerase. Sulfated polysaccharides may inhibit viral adsorption on host cells [13].
Apigenin, a natural flavonoid found in many plants can inhibit ASFV-specific protein synthesis and cause a reduction in ASFV virus formation 99.99% by adding 1 hour after inoculation [14] . Fluoroquinolones were found to influence viral DNA replication and ASFV protein synthesis disrupted viral infection [15]. One group of HDAC inhibitors were able to eliminate ASFV-Ba71V replication and late protein synthesis [16]. Despite great efforts, there are no effective drugs on the market to treat the disease; Therefore, it remains a challenge to find an effective disinfectant with high antiviral resistance.
Silver nanoparticles (SNP) have been widely used as an antiseptic in applications ranging from daily hygiene to medicine [17-21]. Recently, nanomaterials including nanosilver particles have emerged as a new type of therapeutic agent that can inhibit viral replication [22-24]. It is believed that SNPs, particularly particles less than 10 nm in diameter, can interact with active sulfur groups in glycoprotein gp120 nodes causing inhibition of human immunodeficiency virus infection ( HIV-1) [25-27] and similar interactions can be found in herpes simplex virus type 1 (HVS-1) [28], influenza A virus [29-31], and influenza virus. H3N2 [32].
SNPs were stronger than gold nanoparticles, but did not show acute cytotoxicity to Hut / CCR5 and human peripheral blood mononuclear cells, while inhibiting HIV-1 replication [33] . The stabilizers used for SNP synthesis also influence their antiviral activity; may get higher antiviral activity against RSV when the SNP is stabilized by curcumin compared with citric acid [34]. After HIV-1, it has been shown that SNP can interact and inhibit many other viruses that cause viral illnesses in humans such as HVS-1 and 2 [28, 35], hepatitis B [36 ], respiratory syncytial virus (RSV) [37], influenza A H1N1 [29] [31], and influenza H3N2 [32], human parainfluenza type 3 [38], poliovirus [39], and adenovirus type 3 [40].
Several studies have investigated the antiviral activity of silver nanoparticles in animal viral diseases and indicated that SNP can effectively inhibit Tacaribe virus in bats [41], which Infectious bursal causes chicken gumboro disease [42] and the infectious gastroenteritis virus (TGEV) can cause severe diarrhea in pigs [43]. The grapheme oxide-attached SNP has also shown high antiviral resistance against a virus that causes respiratory and reproductive syndrome in pigs (PRRSV) called blue ear pig disease and swine diarrhea virus (PEDV) [ 44].
The spread of ASFV in Europe, China and several other countries causes great economic losses and threatens food security around the world [45-52], however, the Disease control and prevention are still based on biosafety measures due to lack of vaccines and medications. Therefore, the need for an effective antiviral agent to fight the disease that is spreading is high and urgent. SNP has exerted high antiviral activity against many viruses, but their antiviral ability against ASFV has not been studied. Therefore, in this study, the antiviral activity of nanosilver against ASFV will be examined to evaluate their use in controlling ASFV transmission.
EXPERIENCES
- Nanosilver particle synthesis
SNPs were synthesized by chemical reduction methods as described elsewhere [53]. In a typical experiment, 20 mL of 10000 ppm chitosan as stabilizer was first added to a 1000 mL beaker containing 180 mL of distilled water under stirring at 300 rpm. After the solution becomes homogeneous; Approximately 10 minutes of continuous stirring, 250 mL of 1 000 ppm AgNO3 solution is added and the mixture is stirred at the same rate for 1 h. The experiment was then continued by increasing the stirring rate to 500 rpm and adding 200 ppm of NaBH4 solution (20 mL) as the reducing agent by the drip method. Immediately after instillation of NaBH4 solution, the solution in the beaker turns yellow and darkens when the reducing agent is added. The experiment ends when the addition of NaBH4 is completed. The obtained silver NPs were transferred to brown sample bottles and kept at room conditions for further investigation.
- Study on sterilization properties
The sterilization capacity of nano silver was investigated in a laboratory using Salmonella bacteria and using the microbiological air pollution index in pig pens as the corresponding index. For the laboratory antibacterial test, Salmonella was first developed by adding 100 µL of stock to the BHI medium and incubating for 6 hours at 37 ° C then by centrifugation to separate the supernatant. Bacterial cell pellets are then dispersed in sterile distilled water to obtain a Salmonella suspension for later use. Salmonella (0.5 mL) suspension was then mixed with 0.5 mL of SNPs solution of varying concentrations from 0.025 to 250 ppm in a 1.5 mL tube.
After incubation at 37 ° C for 1 hour, a suspension (100 µL) is taken and diluted with sterile distilled water with ten successive dilutions and then 100 L of each dilution is extracted and Arrange on NA plate, incubate for 24 h at 37 ° C for colonies. count. Control experiment was performed according to the same method but sterilized distilled water was added instead of SNPs solution.
Index of microbiological contamination in pig pens is investigated by a passive method called sedimentation plate method. Accordingly, the PCA medium containing the Petri dishes (diameter 9 cm) was placed at the four corners and the center of the pigpen, 40 cm from the floor and 1 m from the wall or obstacle and exposed to the air for 10 minutes. The experiment begins with a site preparation, in which the floor is cleaned with water and allowed to dry until the settling plate is tested. When the floor is dry, the test of the reference sedimentation plate is carried out by opening the Petri plates for 10 min thereafter; nanosilver particles with a concentration of 25 ppm were sprayed all over the floors and walls. When the floor dries again, another sedimentation test is carried out by opening the plates for 10 min. Once collected, the plates were sealed to prevent further contamination and incubated for 24 hours at 37 ° C to check for colony counts.
- Cell culture and virus preparation
Primary pig alveolar macrophages (PAM) were collected from the lungs of pigs 6–8 weeks, weighing 20–40 kg, and healthy Big White pigs. Alveolar cells were cultured in RPMI medium with 10% pig serum and 1% antibiotic. For monolayer cultures, alveolar cells were inoculated into tissue culture plastic plates at approximately 4 105 cells / cm2. After 24 hours at 37 ° C in a humid air with 5% CO2, non-adhesive cells are removed by rinsing with medium.
VNUA / HY-ASF1, a genotype II p72 virus derived from infected pigs on a farm in Hung Yen province, Vietnam, was extracted and cultured to 106 HAD50 (Red blood cell adsorption dose). ) as reported in a previous study, which was used for this investigation [51].
- Cell toxicity test
Silver nanoparticles (500 ppm) are diluted to different concentrations from 0.024 ppm to 50 ppm to perform cytotoxicity test. PAM cells were seeded previously in 96-well plates which were decanted to remove the medium, then added 50 µL of diluted SNP and 50 µL of RPMI broth to each well. The final silver NP concentration in the cell homogenizer was calculated by dividing the dilute SNP concentration added to the well by 2 due to the dilution (1: 1). The plates were then incubated for about 1 hour at 37 ° C in 5% CO2 medium and then 200 µL of fresh medium was added to monitor cell viability. Dead and alive cells were identified by determining their morphology after treatment with nano silver. The live ones appear spherical and clear like the ones in the control, while the dead ones are shrunk and sometimes have an aspherical or crumbling form.
- Test for virus suppression
To test the inhibitory activity of SNP against viruses, the nanosilver particle with the highest non-toxic concentration was selected and mixed with the virus supernatant in a 1: 1 volume ratio. In a typical experiment , titrant 106 HAD50 virus was diluted by medium using ten series dilutions before mixing with SNP solution. The supernatant of the virus, after mixing with SNPs, was kept at room temperature for 1 hour for further experiments.
Experiments were then followed by adding 100 µL solutions containing both SNP and viruses at different titres to cell wells after removal of the medium. Controls were prepared by culturing only cells in the medium and adding only virus. The plate was incubated at 37 ° C for 5% CO2 for about 8 hours for the surviving virus to replicate. After incubation, the virus and SNPs mixture were removed from the cell and washed with PBS 1X solution and then a 200 µL medium supplemented with 1% pig red blood cells was added.
Finally, cells were incubated at 37 ° C for 7 days and examined on a microscope to evaluate their growth based on their change in shape and characteristic asteroid formation representing autoclaving. Red blood cell receptors around infected cells. The antiviral activity of SNP was determined based on the viability of cells at the research level in the absence of a cytosol (CPE) effect.
- Results and Discussion
AgNO3 solution turns yellow immediately after addition of NaBH4 solution demonstrates the possibility of forming SNP. This phenomenon may be related to the characteristic surface Plasmon resonance of silver NPs. The UV-Vis spectroscopy properties showed that the absorption peaked at 401 nm (Figure 1 A), coinciding with the Plasmon resonance surface of SNP reported in many previous works [25, 54, 55]. The dark solution observed when NaBH4 solution is added is due to the increased SNP concentration, proportional to the intensity of absorption. This has been demonstrated in previous studies [56, 57] in which evolution of SNP was monitored using UV-Vis spectra collected at different reaction times.
The position of the absorption peak may vary slightly in different studies due to the influence of the nanoparticle size and shape; however, it is an easy method for a quick initial assessment of SNP formation. To further confirm the presence of SNP, high resolution TEM images (Fig. 1 B) were performed to analyze their size, shape and crystallinity. TEM image analysis showed that the particles obtained were spherical with an average diameter of approximately 14 nm. These particles are highly crystalline with a distance d of 0.23 nm (inner figure 1B), corresponding to the plane (111) of metallic silver. This result proves that metal SNP has been successfully synthesized and can be used for further experiments.
The antibacterial properties of the synthetic nanosilver particles were tested against Salmonella at different concentrations from 0.025 to 250 ppm and the results were shown in Figures 2 and 3. Control samples without supplemented SNPs contained 1.23×10^8. CFU / mL, meanwhile, no bacteria were found in the SNP-treated sample at concentrations above 25 ppm (Figure 2). As seen in Figure 3, SNP can inhibit more than 95% of salmonella at an SNP concentration of 2.5 ppm and reach 100% at a SNP concentration of 25 ppm. The antibacterial effect seems to be concentration dependent, decreasing from 100% to 44.7% when the SNP concentration decreases from 25 to 0.025 ppm, respectively. The mechanism for explaining the antibacterial properties of SNP has not been fully elucidated, but there is broad consensus that SNP can inhibit bacteria through three mechanisms: (1) Ag + ions disrupt ATP production and DNA replication, (2) SNP and Ag + ions generate excess oxygen free radicals such as O, OH (ROS) that disrupt membranes and mitochondrial function or cause DNA damage , and (3) SNP interacts and disrupts bacterial cell membranes [18]. Results showed that SNP was an effective antimicrobial agent and possibly a potential disinfectant against ASFV.
STUDY RESULTS OF NANOSILVER
AgNO3 solution turns yellow immediately after addition of NaBH4 solution demonstrates the possibility of forming SNP. This phenomenon may be related to the characteristic surface Plasmon resonance of silver NPs. The UV-Vis spectroscopy properties showed that the absorption peaked at 401 nm (Figure 1 A), coinciding with the Plasmon resonance surface of SNP reported in many previous works.
The dark solution observed when NaBH4 solution is added is due to the increased SNP concentration, proportional to the intensity of absorption. This has been demonstrated in previous studies [56, 57] in which evolution of SNP was monitored using UV-Vis spectra collected at different reaction times. The position of the absorption peak may vary slightly in different studies due to the influence of the nanoparticle size and shape; however, it is an easy method for a quick initial assessment of SNP formation.
To further confirm the presence of SNP, high resolution TEM images (Fig. 1 B) were performed to analyze their size, shape and crystallinity. TEM image analysis showed that the particles obtained were spherical with an average diameter of approximately 14 nm. These particles are highly crystalline with a distance d of 0.23 nm (inner figure 1B), corresponding to the plane (111) of metallic silver. This result proves that metal SNP has been successfully synthesized and can be used for further experiments.
The antibacterial properties of the synthetic silver nanoparticles were tested against Salmonella at different concentrations from 0.025 to 250 ppm and the results were shown in Figures 2 and 3. Control samples without supplemented SNPs contained 1.23×108. CFU / mL, meanwhile, no bacteria were found in the SNP-treated sample at concentrations above 25 ppm (Figure 2). As seen in Figure 3, SNP can inhibit more than 95% of salmonella at an SNP concentration of 2.5 ppm and reach 100% at a SNP concentration of 25 ppm.
The antibacterial effect seems to be concentration dependent, decreasing from 100% to 44.7% when the SNP concentration decreases from 25 to 0.025 ppm, respectively. The mechanism for explaining the antibacterial properties of SNP has not been fully elucidated, but there is broad consensus that SNP can inhibit bacteria through three mechanisms: (1) Ag + ions disrupt the production of ATP and replication of DNA, SNP and Ag + ions produces excess oxygen free radicals such as O, OH (ROS) that disrupt membranes and mitochondrial function or cause DNA damage, and ( 3) SNP interacts and disrupts the bacterial cell membrane. Results showed that nano silver was an effective antimicrobial agent and possibly a potential disinfectant against ASFV.
Figure 2. Antimicrobial ability of SNPs against Salmonella; colonies from the control sample diluted at 106 times (A) and the sample treated with 25 ppm SNPs diluted at 10 times (B), and microbiological contamination checked by the sedimentation plate method; bacterial colonies grow on agar plates exposed to air 10 minutes before (C) and after (D) sprayed SNPs 25 ppm
Figure 3. Antimicrobial effect of SNPs against Salmonella
- Cell toxicity
To evaluate the toxicity of nano-silver on PAM cells, SNPs at different concentrations were added to cell wells to observe their development. The layout of the cytotoxicity test in a 96-well plate is depicted in Figure 4. Each column has 7 wells for each SNP concentration (rows A to G) and the 8th well (row H) is an empty cell. have SNP to control. Toxic effects were observed at a SNP concentration of 1.56 ppm with approximately 70% of cells deactivated. Above 1.56 ppm, SNPs showed high toxicity with 100% deactivated cells, while at more dilute concentrations (≤0.78 ppm), toxic effects were not observed with more than 80% cells grow normally. A microscopic observation (Fig. 5) shows that the cells in the control have a clear spherical shape, similar to the samples treated with SNP concentrations of 0.78 ppm or less, while the cells in the control are spherical. In samples treated with nano-silver of 1.56 ppm or more, it was clearly divided. This confirmed that SNP at concentrations of 0.78 ppm or less was not toxic to PAM cells.
Ag +-induced toxicity has been studied for over 50 years and its mechanism has been known with the general consensus that mitochondria are Ag + ‘primary targets. Mitochondria are susceptible to damage by the “permeability transition pathway”, characterized by the formation of protein pores in the mitochondrial membrane. The lowest levels for Ag + possibly causing adverse effects in mammalian cells were observed to be between 222 and 362 mg Ag / Kg * day [58]. Toxicity caused by SNPs is not similar to that of Ag +, there are a number of factors that allow SNP to exert toxic effects on cells and organisms. SNPs can penetrate cell walls and membranes and then release intracellular Ag +. Ag + here can interact directly with DNA to induce cytotoxic effects and genotoxicity due to the disruption of cell transport and depletion of glutathione and other antioxidants [59, 60]. Nanosilver can stimulate ROS production and reduce ATP production, inducing oxidative stress and genotoxic effects. Since most of the toxic effects occur in cells, it is believed that the toxicity of nano-silver is size dependent; may be more toxic in smaller particles, especially particles with size ≤5nm [61].
The toxicity of nanosilver is concentration dependent and its effects can be different on cell type [59]. RAW cell survival 246.7 decreased by 20% and 40% at SNPs concentrations of 0.2 and 1.6 ppm, respectively [62]. Toxicity was also observed in mouse liver cell lines (BRL 3A) at SNPs concentrations between 1 and 25 ppm [63]. The threshold of toxicity of SNPs measured in HeLa and U937 cells was 2 ppm after 4 hours of treatment [64]. However, no toxicity was found on HepG2 cells at the SNPs concentration from 0.01 to 5ppm [65]. Microscopic observation on the skin of pigs treated with SNP for 14 days showed that their toxicity in pig skin was a concentration-dependent reaction [66]. A slight intracellular and intercellular edema was found in skin treated with SNPs of 0.34 ppm (size 20 nm) and severe focal dermatitis was observed when the SNP level increased to 34. ppm. Apparently, the toxicity of SNPs varies significantly with different cell lines, therefore, depending on the purpose of their application; The threshold for toxicity on the relevant cell lines should be investigated. In this study, the observed toxicity threshold (0.78 ppm) allowed us to further study the antiviral activity of silver nanoparticles on ASF virus while avoiding destruction of PAM cells ( Alveolar macrophage cells).
Figure 4. Layout of the cytotoxicity test. Each column has 7 wells from rows A to G for each concentration of SNP and the 8th well in row H is for the non-nano silver control. Red shows high toxicity, causes cell deactivation, yellow shows toxicity with a high percentage of deactivated cells, and green shows a safe SNP concentration with more than 80 percent of the remaining cells. living
Figure 5. Representative image of control sample with normal cells (a) and dead cells in sample treated with SNPs at 3,125 ppm (b). Image taken on Leica DM IL LED microscope at 200x magnification.
- Anti-virus activity
Antiviral activity of silver nanoparticles on ASFV was investigated at a SNPs concentration of 0.78 ppm, the threshold for toxicity of SNPs. An antiviral experiment layout is shown in Figure 6.
Virus was detected in all dilutions when the original homogeneous virus was not treated with SNP (green circle), in contrast, no virus was found in the virus-free control and without SNP (red circle). Differences can be seen in samples treated with SNPs of 0.78 ppm. No virus was detected when the stock homogeneous was diluted 3 log or more (Red Circle), while it was observed at all dilutions below 2 log.
These results indicate that ASFV at ≤103 HAD50 levels can be completely inhibited by SNPs at a concentration of 0.78 ppm. Previous studies have shown that the antiviral effect of SNP is concentration dependent [29, 33, 36, 43, 44], so their antiviral effect may be higher when the SNP concentration increases. up; however, it may be more cytotoxic at higher concentrations. Pictures of infected cells and normal PAM cells are shown in Figure 7.
Apparently, if not treated with nanosilver, its virulence would cause infection and death in PAM cells. As can be seen in Figure 7a, the cells in the sample, where the virus was not treated with 0.78 ppm SNPs, had an altered morphology and showed erythrocyte absorption around the infected cells. Red blood cell absorption is due to an interaction between erythrocytes and viral glycoproteins [67]. Meanwhile, PAM cells were found to live with clear round shape and no erythrocyte adsorption in blank samples and in virus homogeneous fluid diluted by ≥3 log after treatment with 0.78 ppm SNPs. (Figure 7b). The results confirmed the inhibitory ability of SNP against ASFV.
According to previous studies, silver nanoparticles can interact with the viral glycoprotein capsid gp120, especially the glycoprotein nodes, the more exposed and accessible parts on the viral capsid and thus preventing interbreeding and host cell and thus induce an effective inhibitory activity against the virus [29–31]. This mechanism may explain the viral inhibitory effect on some viruses with glycoprotein on the seed membrane including influenza A virus [29-31], influenza H3N2 virus [32], and parainfluenza virus. type 3.
SNPs may also inhibit the formation of intracellular RNA and extracellular virions by interacting with suspected DNA or viral particles of HBV [36]. ASFV is a DNA virus encapsulated with particles between 170 and 190 nm in diameter encapsulated with more than 50 proteins including glycoproteins [67]. Therefore, it is possible that SNPs may interact with these proteins, especially glycoproteins on the outer membrane, to prevent viral entry or replication and thereby induce viral suppression.
Since viral suppression is based on the interaction between nanosilver and viral proteins, the antiviral effect is significantly dependent on the exposure of the active sites on both the virus and SNP. Therefore, the size of the SNPs and the limiting substances used in the synthesis can make an important contribution to the antiviral effect of the resulting SNPs. Gaikwad et al. demonstrated that SNPs with an average diameter of 47 nm and 45 nm produced by fungi, Alternaria and Phoma, had a low viral suppression (between 0 and 40%), while SNP was produced by F. oxysporum 24 nm) and C. Indicum (45 nm) have a inhibitory effect of up to 80% (F. Oxysporium) and 90% (C. Indicum).
Higher antiviral activity against RSV may also be obtained when the SNP is stabilized by curcumin compared with citric acid [34]. In this study, SNPs with a mean diameter of approximately 14 nm were able to completely inhibit ASFV at the titer of 103 HAD50 in vitro. However, the actual antiviral effect can be greatly influenced by the pollutants that exist in the environment. Adverse effects can be more serious when SNPs are applied to the cleaning and disinfection of barns because many organic compounds from pig waste and excretions can interact strongly with SNPs.
To assess this effect on the disinfection ability of SNPs in pig pens, 25 ppm SNPs were sprayed onto the floors, walls and partitions of the pigpen and then microbial contamination in the air before and after. Spraying was monitored by a passive method called deposition [68]. Trials were conducted in 3 different pens for piglets, sows and adults.
Before spraying the SNPs solution, the bacteria growing on the plate were so numerous that it could not be counted as shown in Figures 2 C and D, however, after spraying the SNPs solution, the average number of bacteria on the plate was 34, 95 and 813 cfu / sheet corresponds to 2674, 5760 and 63914 cfu / m3 of air in sows, piglets, and adult pigs, respectively. The results showed that microbial pollution decreased significantly after spraying SNP 25 ppm solution. The higher bacterial count in piglets and adult pigs does not mean that the disinfection efficiency of nano silver is reduced in those cases. It is likely that the concentration of bacteria in the air varies with the level of activity of the pig indoors; Pig activity produces and pumps more aerosols containing bacteria into the atmosphere.
In the sow pen, each animal is separated in a septum with very limited activity; it spends more time lying on the floor and thus causes less aerosolization with airborne bacteria. As a result, less bacteria are deposited on agar plates. Meanwhile, piglets and adult pigs that are freely moved through the barn space cause more bacterial mist with bacteria supplied into the air and as a result, more bacteria are found on agar plates. . The results obtained confirm that the disinfection activity of SNPs is not significantly affected by contaminants remaining on the floor.
Figure 6. Layout of antiviral experiment. Rows A, B, C, D correspond to virus blanks and without SNPs (A) treatment, SNP-free and virus-free (B) controls, and virus samples treated with SNP 0, 78 ppm (C and D) respectively. Columns 1 to 7 correspond to a 10-serial dilution of a homogeneous virus at 106 HAD50; The minus number in the circle represents the dilution log. A green circle indicates that a live virus has been detected, while a red circle indicates no live virus has been detected.
Figure 7. Representative microscopic image of PAM cell cultured ASFV (a) and PAM cell cultured with ASFV treated in SNP solution 0.78 ppm for 1 h (b). Image was taken on Leica DM IL LED microscope at 200x magnification
SNPs have been widely used as an antimicrobial agent in many applications from the textile industry, water disinfection, food packaging to medicine [19]. SNPs have several advantages over conventional disinfectants. They have strong and long-lasting antibacterial activity against a wide variety of microorganisms including bacteria, fungi, and viruses. They can be easily incorporated with other substrates and materials; therefore, they can be used in liquid or solid form. Hence, SNP offers a wide range of applications for water disinfection, air purification, surface disinfection, lotion or ointment for skin, pads or fabrics for wound care. Thanks to its flexible application and long operation, SNP can create an effective barrier to disrupt ASFV transmission. This suggests that SNP could be a potential disinfectant and an effective tool for preventing the rapid spread of ASFV.
CONCLUSION
This study demonstrated that nano silver is an effective disinfectant against both Salmonella and ASFV. Complete inhibition of Salmonella and ASFV was observed at SNP concentrations of 25 ppm and 0.78 ppm and at bacterial concentrations of 108 CFU / mL and titer of the virus respectively 103 HAD50. SNPs did not show cytotoxicity to PAM cells at a concentration of 0.78 ppm. The results confirm that SNP has a strong antiviral ability against ASFV and could be a promising tool to fight the disease on a large scale. Controlling the spread of ASFV, in addition to the need for an effective vaccine, requires the implementation of multiple biosecurity measures to isolate the outbreak area, disinfect the site of infection and create a boundary. protection for the area not infected. SNP can make a major contribution to the implementation of biosafety measures; however, the use of SNPs should be combined with existing precautions to optimize their cost and effectiveness. Therefore, more in-depth studies, especially field studies, need to be performed in order to achieve an effective combination of SNP with existing preventive measures and to reduce preventive measures costs.
Reference:
Silver nanoparticles as potential antiviral agent against the African swine fever virus
Tran Thi Ngoc Dung a,*, Vu Nang Nam a, Tran Thi Nhan a, Trinh Thi Bich Ngocb, Luu Quang Minh c, Bui Thi To Nga b, Van Phan Le b,**, Dang Viet Quang d
a Institute of Environmental Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Hanoi, Vietnam
b College of Veterinary Medicine, University of Agriculture of Vietnam, Hanoi, Vietnam
c Ministry of Science and Technology (MOST), Vietnam
d Faculty of Biotechnology, Chemical and Environmental Engineering, Phenikaa University, Hanoi 12116, Vietnam