Nanosilver treats black spot disease NHPB caused by Rickettsia bacteria
Antibacterial effects of biosynthesized nanosilver particles on Pacific white shrimp Litopenaeus vannamei (Boone) infected with necrotic hepatitis pancreatitis (NHPB), also known as black spot disease
Abstract
Necrotizing fasciitis in shrimp is caused by polymorphic Gram-negative Rickettsialike bacteria also known as black spot disease. It is well known that silver is an effective disinfectant, but experiments with aquatic organisms are scarce, at the nanoparticle level even more rare. The objective of this study was to determine the antibacterial effect of nanosilver particles (AgNP) on black spot disease in Litopenaeus vannamei in the Pacific. Therefore, AgNO3 was used as a source of silver, and dried leaf extracts of green tea plants Camellia sinensis and neem Azadirachta indica were used as reducing agents. Various infected shrimp batches were treated with 0.5 and 35 μg AgNP by forced feeding. The difference between the number of bacterial nodules in the shrimp hepatopancreas and the mortality compared to the amount of AgNP has been shown to be effective against this pathogen.
(Copyright by NanoCMM Technology)
INTRODUCE SOME APPLICATIONS OF NANOSILVER
Certain diseases caused by many types of pathogens such as viruses, fungi, protozoa and bacteria, have negatively affected shrimp production in large producing countries such as China, Thailand, Taiwan and Ecuador (Lightner & Pantoja, 2003). Intensive culture conditions, stocking densities, or the use of feed with low and unbalanced nutritional quality can lead to stress, leading to the entry of pathogenic microorganisms. Therefore, research to develop effective techniques and methods that allow for early detection and control of diseases is very important. Disease control in broodstock and larvae culture is more effective than in the nursery stage, where it is much more difficult to do. Necrotizing fasciitis (black spot) bacteria, an intracellular bacterium like the Gram-negative Rickettsia, has emerged as one of the major diseases affecting shrimp farming (Krol et al., 1991). During the acute phase of the disease, the hepatopancreas of shrimp is atrophy while in the chronic stage, the hepatopancreas is deformed, the tubes are necrotic, and many other defects. The disease causes mortality rates up to 40% and its diagnosis is based on the use of histological and molecular tools (Morales-Covarrubias, 2004; Avila-Villa et al., 2012; Nunan et al. , 2013; Varela-Mejías & Peña-Navarro, 2016).
Nanotechnology, the technique of functional systems at the molecular scale, has been focused on the manufacturing industry, but it has also been used in medicine as in vitro detection and diagnostic agents. in vivo, multimodal imagery, chemotherapy, phototherapy, and immunotherapy (Giasuddin et al., 2013; Lin, 2015). Furthermore, in agricultural operations, it has been shown that biopesticides of nanometer sizes offer higher efficiency and lower cost than conventional pesticides, providing a reliable choice ( Huang et al., 2015). On the other hand, in the application of nanoscience in aquaculture production, Khalafalla et al. (2011) reported that selenium nanoparticles were able to improve the reproduction, growth and survival of Oreochromis niloticus (Tilapia). In addition, nanotechnology can be applied on food to prevent microbial contamination or degradation. Some other applications involve nano media as transporters for nutrients, drugs, enzymes or foods and antimicrobial additives (Can et al., 2011; Handy, 2012; Giasuddin et al. the, 2013). Certain nanoparticles may have antimicrobial activity and affect many species of bacteria (Lu et al., 2013; Huang et al., 2015; Bakare et al., 2016). For example, copper nanoparticles have been shown to have antimicrobial potential against the intestinal bacteria Aeromonas hydrophila, Vibrio parahaemolyticus, and Pseudomonas fluorescens in silver and grass carp (Huang et al., 2015), and Kim et al. (2007) reported that the use of nanosilver particles (AgNPs) inhibited the growth of yeasts, Escherichia coli (Escherich), and Staphylococcus aureus (Rosenbach).
Several studies have demonstrated antimicrobial effects of nano silver against pathogenic bacteria that are common to infecting aquaculture species. For example, the antibacterial effects of AgNPs synthesized using the Prosopis chilensis extract were tested on the fungus Penaeus monodon infected with Vibrio cholerae, V. harveyi and V. parahaemolyticus (Kandasamy et al., 2013). Furthermore, the effects of AgNPs, synthesized using green tea extracts were tested on Fenneropenaeus indicus infected with V. harveyi (Vaseeharan et al., 2010). Recently, Juárez-Moreno et al. (2017) demonstrated the antiviral properties of AgNPs against the white spot syndrome virus infecting Litopenaeus vannamei juveniles. Therefore, the use of nanoparticles against pathogens is a promising area in aquaculture (Can et al., 2011; Rather et al., 2011; Selvaraj et al., 2014). The above mechanisms of the bactericidal action of silver are mainly related to the sulfhydryl groups of enzymes and proteins such as glucose-6-phosphate dehydrogenase and glutathione reductase (Shukla & Chandra, 1977), by interfering with the flask functions. usually of protein. Nanosilver also inhibits DNA replication, and in bacteria it induces oxidative stress in the cell wall where important cell functions occur, affecting the maintenance of internal ionic balance. In this way, bacteria exposed to silver showed inhibition of growth, loss of potassium and inhibition of chemical transport from the cell wall (Hwang et al., 2007; Luoma, 2008). It is well recognized that certain bacteria that live in vacuoles or phagosomes can synthesize certain proteins and toxins to form holes in the host’s vacuole membrane to escape from these organelles (Van der Meer-Janssen et al., 2010). Broken cell membranes can facilitate the penetration of nanoparticles into the host cell and some reports have demonstrated that Gram-negative bacteria showed increased sensitivity to nanosilver particles (Sondi). & Salopek-Sondi, 2004; Radzig et al., 2013), this makes the candidate vehicle suitable as an antibacterial agent against black spot disease – NHPB. The application of nanoparticles to inhibit the invasion of bacteria in human cells has been discovered, but no progress has been reported against black spot disease – NHPB in aquaculture species. Despite the fact that the effective antimicrobial activity of nanosilver has been proven, there are very few experiments conducted on aquatic organisms. Therefore, the objective of this study was to determine the antibacterial effects of AgNPs against NHPB in Pacific white shrimp, Litopenaeus vannamei.
MATERIALS AND METHODS – EFFICIENCY OF NANO SILVER
- Synthetic nanosilver
The dried leaves of green tea (Camellia sinensis, Kuntze) and the fresh leaves of the neem plant (Azadirachta indica, Jussieu) were used as reducing agents to determine which of the two species preferred to produce single-dispersed nanosilver. Neem leaves were dried in the dark at room temperature and stored in paper bags for three days. Nanoparticle synthesis was performed based on the method described by Vasseharan et al. (2010) with some modifications in quantity and timing as follows: For both plants, 6 g of dried leaves were added to 60 mL of boiling deionized water for 2 minutes, stirring occasionally. Then, after 5 minutes, the infusion is filtered through a 4 μm filter three times to separate suspended solids. The infusion was performed just before starting to synthesize the nanoparticles. Reaction was conducted in a 50 mL test tube with 1.0 mL of 0.01 M silver nitrate, 3.0 mL of deionized water, and 30 mL of phytonutrients. The absorbance of the reaction was evaluated in Shimadzu UV-2450 spectrophotometer every 5 minutes for 1 hour. To assess the form and dispersion of the nanoparticles, a sample of 3 μL of reactive solution was placed and analyzed in an electron microscope transmitted through JEOL 2000 using a 200 mesh copper mesh that was dried at room temperature. 6-12 hours. Images were analyzed using ImageJ 1.44 p (NIH) software.
- Black spot bacteria sample (NHPB)
To store shrimp tissue infected with black spot disease – NHPB, 60 live specimens were collected from two shrimp farms (30 samples per farm). Farms were anonymized at the request of the farmer, and identified as Farm A and Farm B. The entire hepatopancreas of each shrimp was dissected using flame-sterilized surgical material in the laboratory. The test, and hepatopancreas were divided into three parts distributed as follows: one piece was placed in a 1.5 mL tube with absolute ethanol for further PCR analysis to confirm the presence of bacteria and WSSV. Another piece was soaked in Davidson solution for histological analysis. According to Gracia-Valenzuela et al., To transplant bacteria into healthy shrimp if the PCR diagnosis is positive, the third piece is transferred to a cryoprotectant (50% glycerol). (2011). Samples in alcohol and glycerol were stored at -20 ° C, and the Davidson samples were converted to 70% ethanol for histological analysis. To confirm bacterial contamination, DNA was isolated from the hepatopancreas previously dissected using the QIAamp DNA Mini Kit (QIAGEN), according to the manufacturer’s instructions. DNA quantification and A260 / A280 ratio verification were performed in a NanoDrop 1000 spectrophotometer. PCR reaction was performed using Illustra PuRe Taq’s PCR Ready-To-Go Particle (GE Healthcare). According to DNA quantification results, PCR reaction is performed using 1 µL of DNA if the concentration is greater than 100 ng µL-1 and 2 µL if the DNA concentration is less than 100 ng µL-1. One µL of oligonucleotides was described by Loy et al. (1996) for the diagnosis of black spot – NHPB (pf-1: 5′-ACGTTGGAGGT TCGTCCTTCAG-3 ‘and pr-1: 5′-TCACCCCCTTGC TTCTCATTGT-3’) was added to the reaction mixture. Thermal cycle conditions are described by Loy et al. (1996).
Since the white spot syndrome virus (WSSV) has shown a high incidence of disease on shrimp farms in northwestern Mexico, a PCR analysis was also conducted to exclude samples tested positive for the virus. withdraw. WSSVVP28-F1 molecules: 5’-CTCGCTTGCCAATTGTCCTGT TA-3 ‘and WSSVVP28-R1: 5’-ATTTCCACCGGC GGTAGCTGC-3’ were reported by Ramos-Paredes et al. (2012) were used, as well as thermal cycle conditions recommended by these authors. A DNA-free negative control was used in all cases. Since the white spot syndrome virus (WSSV) has shown a high incidence of disease on shrimp farms in northwestern Mexico, a PCR analysis was also conducted to exclude samples tested positive for the virus. withdraw. WSSVVP28-F1 molecules: 5’-CTCGCTTGCCAATTGTCCTGT TA-3 ‘and WSSVVP28-R1: 5’-ATTTCCACCGGC GGTAGCTGC-3’ were reported by Ramos-Paredes et al. (2012) were used, as well as thermal cycle conditions recommended by these authors. A DNA-free negative control was used in all cases.
- Activate the inoculum of the black spot – NHPB again
Ten live shrimp from the experimental culture at the Universidad de Sonora facilities in Bahía de Kino, Sonora were transported to our domestic laboratory.
The specimens were placed in a 15 L tank of filtered seawater and fed with commercial shrimp feed twice daily for 5 days. Thereafter, no feed was subsequently provided for 24 hours for intestinal cleansing and then the hepatopancreas pressured organism was harvested from NHPB / WSSV negative shrimp from the stored tissue bank. used a catheter adapted to a 20-100 µL microipette, and the procedure was repeated every third day for three times. After 30 days, the entire hepatopancreas was dissected, most of it preserved in glycerol and a small fraction was used for DNA extraction and PCR analysis for the presence of NHPB according to the method described above. Positive NHPB assay organisms participate in infected tissue banks to conduct infection treatment and bioassay treatment with nano silver.
- Biological test for NHPB infection
One week after the NHPB strain was reactivated, one hundred and ninety six g live shrimp from our facilities in Bahía de Kino, Sonora, were shipped to our domestic facilities. . These shrimp numbers are considered for the possibility of mortality from handling, stress, cannibalism or disease, and therefore, to ensure that there are sufficient numbers of shrimp that survive for a biological test. To assess that these shrimp were not infected with both NHPB and WSSV, 11 shrimp were randomly selected and analyzed by PCR using DNA extracted from one white shrimp, with the method described previously. All shrimp were transferred to 16 culture tanks (10 to 12 each) containing 40 L of seawater that was filtered and sterilized with UV light and maintained at 28-29 ° C. Shrimps were monitored for three months with continuous aeration and pellet industrial feed, supplied twice a day at a dosage of 5% of their biomass. At that time, some shrimp died from cannibalism or because they jumped out of the tank, but none of them got sick. 80% water changes are done every 3-4 days with UV filtered and disinfected sea water. One hundred and twenty shrimp mentioned in the preceding paragraph, weighing 16 g, were infected with NHPB from forced feeding using previously obtained contaminants. To ensure infection and disease progression, shrimp were maintained for 50 days. This is done because, in previous breeding trials, clinical signs of the disease in shrimp were observed after 50 days. Control treatment included uninfected shrimp. To detect NHPB infection in shrimp, feces were collected daily. To do so, the shrimp of each culture tank were placed in a cylindrical mesh and left for about 30-40 minutes to allow time for manure deposition. Feces are stored in 1.5 mL tubes with> 95% ethanol. After NHPB infection in all shrimp, we proceeded to apply nano silver-based treatments.
- Nanosilver treatment methods
From the infected shrimp, 45 shrimp were selected and three groups were established, with three tanks per group (n = 5), for the AgNPs treatments were explained as follows. nanosilver synthesized with A. indica were used in two different treatments, 5 µg and 35 µg AgNPs in aqueous solution, and were administered forced feed to 15 shrimp in each treatment, allotted to 5 shrimp in each aquarium. This was determined by the extreme concentrations used by Vaseeharan et al. (2010) against Vibrio harveyi. A third group of infected shrimp were cultured with aqueous solution of no nanoparticles. The fourth group of 15 shrimp without infection without the nanoparticles was still the control group. One shrimp in each tank (three per treatment) on days 12 and 24 was collected, and sacrificed by hypothermia to reduce its metabolism, and treated immediately. Cephalothorax fractions (3-5 mm thick) from the sampled organisms were placed in a cassette for histological examination in the Davidson solution. A small portion of hepatopancreas was collected and placed in 1.5 mL tubes with> 95% ethanol for PCR analysis as explained above. Hepatopancreatic tissue infection was evaluated histologically, based on Lightner & Pantoja (2003) and Del Río-Rodríguez et al. (2006).
Determination of hepatopancreatic metabolites Hepatopancreatic glucose, total protein, glycogen, total lipid, acylglyceride and extracted sterols and measured according to Sánchez-Paz et al. (2007) in the Synergy micro-plate reader (BioTek Instrumets), using a commercial kit (Randox Laboratory). Briefly, a weighed hepatopancreas is marinated with a buffer volume A (100 mM buffer of potassium phosphate, pH 7.2 and 1.0 mM EDTA, 10 µM PMSF) and one volume of chloroform-methanol- water (2: 2: 1) and centrifuge at 15,000 × g for 15 minutes. Aqueous phase is used to quantify glucose, total protein and glycogen levels. Glycogen determination was performed by hydrolysis and was measured as glucose according to Passonneau & Lauderdale (1974). The chloroform-containing solvent phase was collected and dried in air for 12 hours in complete darkness at room temperature (25 ° C), then homogenized with distilled water and used for total quantification. lipids, acylglycerides and sterols. The metabolite data, expressed in mg g-1 of the hepatopancreas, were statistically analyzed by KruskalWallis analyzing one-way variance with Dunn’s multiple comparison test in post-production analysis by SigmaPlot v .12.0.
FINDINGS ON EFFICIENCY STUDIES OF NANOSILVER
- Properties of AgNPs nanoparticles
The reactive solution of Azadirachta indica on exposure to AgNO3 showed a single absorption peak at 440 nm, while the reaction solution of Camelia sinensis showed a small absorption peak at 390 nm and a more pronounced peak at 440 nm. The reference solutions of AgNO3 do not have an absorption peak in either case. Nanosilver particles synthesized by A. indica showed both hemispherical and flattened after 120 min of reaction time. The size of nanosilver ranged from 5 to 45 nm with an average of 14.84 ± 5.81 nm. 84.65% of nanoparticles were found in the 5 to 21 nm range with an average of 13.01 ± 3.71 nm. AgNPs obtained from A. indica are monolithic due to the high proportion of nanoparticles of the same size type (Figure 1a), and this is the criterion for its use in bio-treatment assay. On the other hand, for C. sinensis, after 120 minutes of reaction time, polyhedra, hemispherical, flattened and several nanowire shapes were observed. The size range is between 7.4 and 68.8 nm with an average of 29.67 ± 14.17 nm, 29% from 13.1 to 20.8 nm with an average of 16.60 ± 2, 02 nm and 20% are between 37.05 and 44.03 nm with the mean of 40.56 ± 1.88 nm (Figure 1b). This is reason enough to remove these nanoparticles from biological tests.
- Detect pathogens
The organisms from Farm A indicated that 17% positive for NHPB, 23% positive for WSSV, 3% positive for both infections and 57% without the pathogen.
Furthermore, samples from farm B showed that 63% were infected with WSSV, 3% had a double infection and in 33% had no detectable presence of these pathogens. NHPB chain shows 100% identity with NHPB chain U65509 of GenBank and is additionally registered with joining number KM305771.
Trial Biological testing for NHPB infection Experimentally, shrimp infected with NHPB have the typical clinical signs of the disease such as soft exoskeleton, decreased hepatopancreas, erratic swimming and lethargy (Vincent et al., 2004 ). In addition, this result showed that each shrimp exposed to material infection was successfully infested because 100% of diagnostic tests yielded positive (Figure 2).
It is noteworthy that the mortality of shrimp treated with NHPB treated with 35 μg AgNPs was 0%, while shrimp treated with 5 μg AgNPs had a mortality rate of 40% at the end of the experiment. Organisms without nanoparticles, but being infected showed a mortality rate of 41%. After 12 days of treatment, multiple bacterial nodules were observed in shrimp hepatopancreatic tissue with 5 µg AgNPs and these tubes slightly contracted and had bacterial nodules (Figure 3a). After 24 days, severely enlarged hepatopancreas and bacterial nodules were observed in the epithelial tissue of the hepatopancreatic duct (Figure 3b). During the 35 µg AgNPs treatment, and after 12 and 24 days of treatment, shrimp hepatopancreas showed signs of infection with damaged hepatopancreas and severely vacuumed epithelium (Fig. 3c-3d). Infected shrimp hepatopancreatic tissue without nanosilver showed a markedly severe infection since the first sample on day 12, with many bacterial nodules and severely atrophic hepatopancreas (Figure 3e).
- Metabolites through the hepatopancreas
Statistical analysis of the biochemical components analyzed at 12 and 24 days of bacterial challenge showed no significant difference in total protein (P = 0.225), acylglyceride (P = 0.174), total number of lipids (P = 0.166), glucose (P = 0.225
) and glycogen (P = 0.270). However, there was a difference in sterol concentrations (P = 0.017), with higher values in infected shrimp that did not receive AgNPs (Figure 4).
DISCUSS
Nanosilver particles from green tea extract and neem leaf have been successfully synthesized quickly, practically and economically compared to other chemical and physical techniques (Poole & Owens, 2007; Guzmán et al. , 2009). However, the difference in shape and dispersion of the nanoparticles was compared with Vaseeharan et al. (2010) and Gavhane et al. (2012). Green tea extract, as a reducing agent, showed two peaks in the absorption spectrum, showing an increase in polydispersite (Hsu & Wu, 2010; Kamal et al., 2010), confirmed in transmission electron microscopy. through, where the size of nanosilver is widely dispersed. In this study, different shapes of nanoparticles were observed, and although it is not clear whether the antibacterial activity depends on the nanoparticle shape, it seems certain. It is reasonable to assume that all forms found may lead to antimicrobial effects. However, more studies are needed to confirm that neither the form nor the size of the nanoparticles are suitable for therapeutic use.
Histological results of NHPB-infected shrimp were consistent with those reported by Del RíoRodríguez et al. (2006). Shrimp treated with 5 µg AgNPs showed similar tissue damage to infected shrimp without nanoparticles, also with tissue necrosis as well as presence of bacterial nodules and blood cell infiltrates. Furthermore, while shrimp were treated with 35 µg AgNPs, some tissue damage was observed, no bacteria or blood cell infiltration was found as evidence of NHPB. In addition, the epithelium of the tubes is slightly thinner and tissue integrity is altered. This may indicate that treatment with AgNPs removes or significantly reduces NHPB in shrimp cells. In this case, a positive PCR analysis indicates the presence of bacteria, perhaps in small numbers, but not as clear as microscopic analysis, or residual bacterial DNA from dead bacteria remains. still in shrimp. The probability that nano-silver remains in the tissue is high. Bianchini et al. (2007) reported that in Penaeus duorarum (Burkenroad), silver accumulation mainly occurs in hepatopancreas, followed by hemolymph, however, in gills, muscles and eyes there is very little silver accumulation. Perhaps, there is a great benefit that silver accumulates in the hepatopancreas as it is the target organ of the NHPB (Lightner et al., 1992), so most of the silver will be available as an antimicrobial agent. In addition, due to the great concern about the effects of nanosilver on human health (Korani et al., 2015), and because the consumer market mainly considers the abdomen rather than the cephalothorax, it is in fact the muscle of the shrimp. Less nanoparticle accumulation is an advantage.
According to Vincent et al. (2004), the course of NHPB disease indicated that during the first 20 days the disease was in an advanced stage. After that time, the disease turns to an acute phase and begins to die, and from 35 days after infection, the chronic phase begins. In the current study, normal behavior and clinical signs were observed in shrimp up to 20 days, while mortality occurred during the trial. However, the chronic phase began at 56 days of challenge, after the end of our trial. This difference is with Vincent et al. (2004) research needs to be evaluated, looking for genetic variants NHPB or immune health status of shrimp. The slight difference in mortality between the 0 µg and 5 µg AgNPs treatments could indicate that the concentration of silver nanoparticles was insufficient to resist the pathogen, which is consistent with the findings in the analysis. histology. Furthermore, the organisms used 35 µg AgNPs had a 100% survival rate, and although the molecular analysis results were positive at the end of the experiment, the tissue was almost anatomically restored when compared compared with the first samples. Morales Covarrubias et al. (2012) tested florfenicol and oxytetracycline on NHPB-infected shrimp, and observed a survival rate of 56 to 83%, with antibiotic residues up to 10 days in shrimp muscle. It is important to note that the use of antibiotics in aquaculture is associated with the production of resistant bacteria. Bacteria can tolerate certain silver concentrations and reduce them to form nanocrystals in the extracellular space as a protective measure (Klaus-Joerger et al., 2001). However, most pathogenic bacteria are not resistant to nanosilver (Parameswari et al., 2010; Saxena et al., 2010; Vaseeharan et al., 2010). Furthermore, it was found that antibiotic-resistant organisms, compared to non-resistant, were equally sensitive to nanosilver. For example, Lara et al. (2010) found that antibiotic resistant strains of Staphylococcus aureus and Pseudomonas aeruginosa (Schroeter) were sensitive to nanosilver.
The application of nanosilver particles is synthesized using tea extracts that include inexpensive and readily available ingredients compared to other reducing agents. This means a significant cost reduction as well as the application of simple methodologies. In addition, the benefits of applying nanoparticles synthesized in an environmentally friendly manner and in low dosage volumes could be an excellent candidate as an antimicrobial agent in a number of shrimp farming industries, for example. for example, in maintaining broodstock. Although it was found that the nanoparticles had antibacterial effects against the intracellular bacteria that cause NHPB, it is necessary to understand the mechanism of action of these nanoparticles against these bacteria. The absence of statistical differences in the five biochemical components analyzed showed insufficient evidence to prove that the levels of those components were affected by the effects of the infection and the different concentrations of the nanoparticles in infected and healthy shrimp. Future studies should take into account a larger sample size to avoid large dispersion values such as observed in the analysis of metabolites, as high dispersion values mask any possible differences. exist. It is also important to test a wider range of nanoparticle concentrations and check the feasibility of the application of nanoparticles in large aquaculture systems, possibly adding nanoparticles as well. Food Additives. Recently indicated an adverse effect of nanosilver on coastal biota (Burić et al., 2015; Degger et al., 2015; Gambardella et al., 2015; Rocha et al., 2015), but fortunately Juárez-Moreno et al. (2017) demonstrated that nano-silver at therapeutic doses was nontoxic and did not affect metabolic rate or total blood cell counts in juvenile Litopenaeus vannamei. However, given the abundance of metals and metals used as nanoparticles, it is important to study the impact of their use on aquaculture species, human health and the coastal environment. , to implement future strategies for sustainable and responsible aquaculture development.
Martín Rodrigo Acedo-Valdez, José Manuel Grijalva-Chon, Eduardo Larios-Rodríguez, Amir Darío Maldonado-Arce, Fernando Mendoza-Cano, José Arturo Sánchez-Paz & Reina Castro-Longoria, Departamento de Investigaciones Científicas y Tecnológicas, Unidad Regional Centro Universidad de Sonora, Hermosillo, Sonora, México, Departamento de Ingeniería Química y Metalurgia, Unidad Regional Centro Universidad de Sonora, Hermosillo, Sonora, México, Departamento de Física, Unidad Regional Centro Universidad de Sonora, Hermosillo, Sonora, México, Laboratorio de Referencia, Análisis y Diagnóstico en Sanidad Acuícola, Centro de Investigaciones Biológicas del Noroeste S.C., Hermosillo, Sonora, México