Nanosilver against acute hepatopancreatic necrosis AHPND/EMS
In this study, the scientists successfully synthesized silver nanoparticles and studied the antibacterial effect of silver nanoparticles against Vibrio parahaemolyticus strain. These are bacteria species isolated from white shrimp samples infected with acute hepatopancreatic necrosis disease (AHPND). The study was carried out for 7 days to feed shrimp with silver NPs solution at different concentrations from 10 – 10,000ppm. It was then studied on the survival rate, antimicrobial ability, growth stimulation, and nutritional absorption efficiency of shrimp in the presence of nanosilver particles.
The biosynthetic Ag / AgCl NPs showed important in vitro antimicrobial effects against three Vibrio parahaemolyticus strains isolated from cultured shrimp affected with acute liver necrosis disease (AHPND) in northwestern Mexico. Litopenaeus vannamei shrimp were exposed for 7 days with NP Ag / AgCl supplementation at 10, 100, 1000 or 10,000 ppm (nanosilver concentration fed). Supplementing with NP in the diet did not affect shrimp survival, growth or feed conversion rates, but high concentrations (1000 and 10,000 ppm) significantly reduced liver index.
Short-term consumption of NP Ag / AgCl produced a significant bioaccumulation of Ag in the hepatopancreas and to a lesser extent in the epidermis, while no significant muscle bioaccumulation. Studies of purification have confirmed the rapid digestion of nanosilver in shrimp hepatopancreas and showed a rapid reduction in hepatopancreas as well.
Materials and synthesis methods nanosilver
Chemical reagent, sterile filtered water (W3500), silver nitrate (209139), sodium salt resazurine (R7017) and sodium chloride (V000106) from Sigma-Aldrich (USA). The cultures of Tryptic Soy Agar (TSA), Tryptic Soy Broth (TSB), Mueller Hinton Agar (MHA), Mueller Hinton Broth (MHB) and Agar Agar (AA) were obtained from Difco (USA), Ulva seaweed. clathrata (Roth)
Bacterial strains Vibrio parahaemolyticus strains were obtained from collections cultured in a laboratory at CIAD Mazatlan, Sinaloa, Mexico. Samples were isolated from the stomach and hepatopancreas of cultured shrimp affected with AHPND in northwestern Mexico. The strains were maintained in cryopreservation (- 80 ° C) until activated in TSB supplemented with 2% NaCl.
Short-term preparation and analysis of acute toxicity
- Supplemented feed preparation
A commercial pellet feed with 44% protein and 8% lipid content (742 UANL, Nutrimar, Sinaloa, Mexico) was added by soaking with Ag / AgCl nanosilver solution to obtain Ag 0 concentration, 10, 100, 1000 and 10,000 mg Ag kg-1. Different Ag / AgCl NP stock solutions are diluted or concentrated in the same final volume of distilled water (40 mL) and sprayed onto 100 g of previously weighed feed and placed in aluminum trays. The diet was mixed for 5 minutes to ensure complete homogenization. Finally, the feed was dried at 50 ° C for 1 hour. Final Ag concentration in the NP / AgCl supplement was quantified using AAS as described above.
- Content of short-term acute toxicity assessment and tissue distribution of nanosilver in vivo test
The first in vivo trial evaluated acute toxicity by behavioral performance parameters, survival and growth and distributed analysis of nano-silver Ag / AgCl after 7 days of low feed consumption ( 0, 10 and 100 mg Ag kg-1) and high (1000, 10,000 mg Ag kg-1) diet levels. In the experiment, 150 juveniles [Litopenaeus vannamei (Boone, 1931) supplied by FITMAR, Sinaloa, Mexico (initial weight 0.400 ± 0.02)] were randomly distributed in 15 rectangular fiberglass tanks. 10L (10 shrimp per tank, five treatments and three replicates). The experimental tanks had their own continuously maintained and daily aeration with 100% manual artificial seawater exchange (Pro Aquatics, Fritz Industrial Inc., Mesquite TX, USA) and photoperiod natural conditions. Water temperature, salinity and pH, ammonia, nitrite and dissolved oxygen were monitored daily and maintained within limits favorable for shrimp growth. The daily feed diet was 5% of the total biomass per tank.
- In vivo experiment content on the elimination of nanosilver
A second in vivo trial was performed to evaluate NP Ag / AgCl depletion using the supplementary at 0 and 100 mg Ag kg-1 nominal concentrations. Litopaneis vannnamei juvenile shrimp supplied by FITMAR, Sinaloa, Mexico (initial weight 1.9 ± 0.20 g) were randomly distributed in nine rectangular 10 L fiberglass tanks (19 shrimp per each tank, two treatments and three repeats). The organisms were maintained under the same conditions as the first test. During the absorption phase (first 7 days), the organisms were fed a low-dose test diet, while the degenerative phase (the next 14 days) began on day 8 of the test by switching to NP-free diet (0 mg Ag / kg). The trial period is 21 days.
- Disc diffusion test
Antimicrobial tests performed by disc diffusion showed significant differences in growth inhibition zone diameter between the three strains of V. parahaemolyticus (p = 0.001), for products (p = 0.001) and in their interaction (p = 0.001) with a two-dimensional ANOVA (Figure 6). Plates impregnated with NP Ag / AgCl produce an IZ of 9.2 ± 0.2, 5.3 ± 0.1 and 5.8 ± 0.2 mm for strains M5-28, M5-06, respectively. M9-04 V.parahaemolyticus. AgNO3 solution is tested at the same concentration that produces a lower IZ than NP Ag / AgCl, with values of 6.5 ± 0.6, 4.6 ± 0.1 and 4.5 ± 0.2, respectively. mm for three strains. In contrast, seaweed extract (AEU) is not inhibitory.
- Microfiltration test
An antibacterial test performed by a microfiltration assay is performed only on products obtained with IZ on a disc diffusion assay. The Ag / AgCl silver NPs solution showed a minimum inhibitory concentration (MIC) of 3.2 mg mL-1 for all strains tested, while the AgNO3 solution showed a MIC of 1.6 μg / mL. for strains V. parahaemolyticus M5-28, M6-05 and 3.2 µg / mL for V. parahaemolyticus M9-04 .. The NP Ag / AgCl solution showed a minimum bactericidal concentration (MBC) of 6. , 3 μg / mL for the three strains, while the AgNO3 solution presented the MIC was 3.2 μg / mL for the strains M5-28 and M6-05 and 6.3 mg / mL for the M9-04 (Table 1).
- Short-term evaluation in vivo trials
The stability of the nanoparticles in the Ag fortified feed in the test feed is shown in Table 2. The dehydrated feed had higher Ag content than the original feed and the silver nanoparticles were lost from the substrate. surface of food into sea water after 1 hour is less than 5%.
Nano silver test results
- Short-term acute toxicity in vivo and the ability to eliminate nano-silver in tissues
In the first short-term food exposure trial, the organisms showed no change in behavior and a 100% survival rate for all treatments. Complementary feed consumption for 7 days did not cause a significant change in feed conversion but had an effect on weight gain (%), slightly improved in feed consuming 10-100 mg of shrimp. Ag kg-1 feed, and slightly (but not significantly) were depressed in animals consuming a feed containing 10,000 mg Ag kg-1 (Table 3). In contrast, the liver index (HPI) showed a significant decrease with consumption of the highly fortified feed (1000 and 10,000 mg Ag kg -1; Figure 7a).
Mean concentration values of Ag that were reported for the different organ groups were analyzed at the end of 7 days of supplement consumption (Fig. 1; hepatopancreas 7b, abdominal epidermis 7c and muscle 7d).
A greater bio-accumulation of Ag was observed in hepatopancreas showing a dose-dependent relationship up to 1500 mg kg-1 dry weight (dw), while concentrations in the epidermis and muscle less than 8 mg kg-1 dw. The levels of hepatopancreas and epidermal Ag became significantly different when the diet contained 1000 and 10,000 ppm Ag. In contrast, the muscle Ag content did not change significantly with any treatment.
Attenuation parameters (second test) Attenuation rate (k2), anabolic efficiency (α), half-life (t1 / 2) and bio-condensation coefficient (BMF) are only possible identified for liver and gill only (Figure 8). The model used to calculate attenuation parameters is not applicable in muscle tissue due to the low Ag concentration.
However, results indicated that hepatopancreas had higher k2 and α values (0.125 day-1 and 33.053%, respectively) compared with gills (0.072 d-1 and 0.169%). The reduction time of 50% (or (t1 / 2) was shorter in the gills than in the hepatopancreas, at 4.4 and 9.5 days, respectively. Hepatopancreas showed food biological factor (BMF) value. The high is 10,553, while the gill has a value of 0.125. Finally, the reduction of Ag accumulated in the hepatopancreas and gill is 89 and 99%, respectively, after only 14 days of reduction (i.e. with NP Ag / AgCl).
In the first in vivo trial, consuming the supplement at a low concentration (10 and 100 ppm) had a positive effect on growth. This result coincides with survival and growth results reported by Sivaramasamy and Zhiwei (2016), who raised L. vannamei shrimp (6.82 ± 2.16 g) with a synthetic AgNP concentration of 10,000 ppm Bacillus. subtilis for 65 days.
In contrast, high concentrations of dietary supplements (1000 and 10,000 ppm) can lead to moderate (invisible) liver damage, as suggested by a significant reduction in HPI. This negative effect on HPI, followed by histological damage, has been reported in salmon after 8 weeks of exposure to water with NPs at 3300 and 1000 mg Ag L-1 (Monfared and Soltani 2013). .
However, no similar studies in shrimp compared with our results on the reduction of HPI and toxicity of nano-silver in the diet. In terms of bioaccumulation and biological distribution, the highest Ag concentration found in the hepatopancreas is not surprising, since the crustacean hepatopancreas can be considered a target organ for accumulation of many types of substances.
On the contrary, muscle shows practically no bioaccumulation, which is expected to represent the most edible portion of the shrimp. However, the bioaccumulation at medium intensity in the cephalothorax cuticle suggests that the tail may contain certain levels of Ag residual. In this regard, several studies have found that nanoparticle biological distribution and toxicity are strongly influenced by particle size, shape, surface charge, surface coating, and solubility ( Sivaramasamy and Zhiwei 2016).
Nanosilver half-lives of 4 to 9 days were found in the tissues sampled in the degradation study (hepatopancreas, gills, and muscle).
This means waiting for half the half-life can remove the remaining 50% of Ag. These values are lower (but consistent with) that previously reported by Metian et al. (2010), who showed a t½ value of 10.8 ± 2 days for degeneration of Litopenaeus stylirostris after exposure to the labeled Ag + diet.
Silver nanoparticles against acute hepatopancreatic necrosis disease (AHPND) in shrimp and their depuration kinetics
Maribel Maldonado-Muñiz 1 & Carlos Luna2 & Raquel Mendoza-Reséndez 2 & Enrique Díaz Barriga-Castro3 & Sonia Soto-Rodriguez4 & Denis Ricque-Marie1 & Lucia Elizabeth Cruz-Suarez1