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A vibriosis outbreak in the Pacific white shrimp, Litopenaeus vannamei reared in biofloc and clear seawater
Journal of Invertebrate Pathology 167 (2019) 107246
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A vibriosis outbreak in the Pacific white shrimp, Litopenaeus vannamei reared in biofloc and clear seawater
T
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Diana Aguilera-Riveraa, , Alejandra Prieto-Davóc, Gabriela Rodríguez-Fuentesc, Karla Susana Escalante-Herrerab, Gabriela Gaxiolab a
Escuela Nacional de Estudios Superiores, Unidad Mérida, Universidad Nacional Autónoma de México, Mérida, Yucatán 97205, Mexico Unidad Multidisciplinaria de Docencia e Investigación de Sisal, Facultad de Ciencias, Universidad Nacional Autónoma de México, Sisal, Yucatán 97356, Mexico c Unidad de Química-Sisal, Facultad de Química, Universidad Nacional Autónoma de México, Sisal, Yucatán 97356, Mexico b
A R T I C LE I N FO
A B S T R A C T
Keywords: Vibriosis Biofloc Pathogen Oxytetracycline Resistance
In May and June 2015, moderate and severe lesions were observed in Litopenaeus vannamei reared in clear seawater while, at the same time, lesions in shrimp reared in biofloc were considerably fewer. The signs of disease included anorexia, lethargy, melanization, expanded chromatophores, luminescence and necrotic areas in the uropods, suggesting a possible vibriosis. However, lesions observed in shrimp reared in biofloc disappeared after a certain time and without mortality in tanks, whereas mortality and severe signs continued to be observed in shrimp reared in clear seawater. To treat the possible vibriosis, oxytetracycline was administered only in clear seawater tanks, but the results were not successful. Bacterial cultures from hepatopancreas tissues of shrimp from both rearing systems confirmed a vibriosis outbreak only in the clear seawater system. Subsequently, Vibrio harveyi, Vibrio rotiferianus, Photobacterium sp. and Photobacterium damselae were identified from bacterial culture previously isolated for both rearing systems by molecular methods. Shewanella sp. was isolated and identified only in biofloc. To understand the possible pathogenicity and resistance mechanisms of the Vibiro strains for both rearing systems, pathogenicity (toxR) and oxytetracycline resistance-related genes (tet (B), tet(D), tet(G)) were determined. Although these genes were expressed for both rearing systems, biofloc proved to have the ability to control the development of the disease, in comparison to clear water, where the vibriosis was evident regardless of the administration of oxytetracycline as a treatment.
1. Introduction One of the major problems for aquaculture farms are diseases caused by infectious agents and non-infectious factors. Vibrio bacteria are particularly important due the relationship between the pathogens, the environment and the aquatic invertebrate hosts. Vibrio spp. are Gram-negative, mobile (with 1-2 flagella), mesophilic and chemoorganotrophic bacteria, with a heterotrophic facultative fermentative metabolism. They are oxidase positive and can grow in selective media (TCBS) (Thompson et al., 2004). Vibrios are part of the microbiome of estuarine environments (Heidelberg et al., 2000; Urakawa and Yoshida, 2000; Denner et al., 2002; Potasman et al., 2002; Baffone et al., 2003). However, are considered as opportunistic microorganisms for aquatic organisms (corals, fishes, molluscs, crustaceans, zooplankton) (Rosenberg and Ben‐Haim, 2002; Huys et al., 2001; Sawabe et al., 2003; Vandenberghe et al., 2003; Heidelberg et al., 2002) by the environmental disturbances produced by the salinity,
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temperature or dissolved oxygen (Baffone et al., 2003; Páez-Osuna et al., 2003). Several species of vibrios are well documented as pathogens in shrimp, and include Vibrio harveyi (Mirbakhsh et al., 2014; SotoRodríguez et al., 2012), Vibrio alginolyticus (Vandenberghe et al., 1999), Vibrio parahaemolyticus (Tran et al., 2013), Vibrio campbellii (SotoRodríguez et al., 2006), Vibrio nigripulchritudo (Goarant et al., 2006) and Vibrio penaecida (Goarant and Merien, 2006). Traditional shrimp rearing systems often use antibiotics, chemical substances produced by microorganisms that have the capacity to inhibit or remove pathogens (Santiago et al., 2009) to treat diseases, especially those caused by Vibrio strains. Unfortunately, the use of these antibiotics is inefficient because of inaccurate dosages and time of exposure, affecting the mechanism of action and producing resistance in bacteria. As a sustainable alternative disease control in aquaculture, biofloc rearing systems are used to maintain the water quality. The medium is
Corresponding author. E-mail addresses: [email protected], [email protected] (D. Aguilera-Rivera).
https://doi.org/10.1016/j.jip.2019.107246 Received 28 April 2019; Received in revised form 11 September 2019; Accepted 11 September 2019 Available online 12 September 2019 0022-2011/ © 2019 Published by Elsevier Inc.
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G2 = moderate lesions, severe; G3 = moderate to severe lesions, more severe; and G4 = severe lesions, lethal (Lightner, 1996).
rich in organic matter composed of particulate biomass and colonized by bacteria with a probiotic effect (Aguilera-Rivera et al., 2014). The ability of biofloc to prevent disease outbreaks has been attributed to its protective effect on intestinal epithelium by the presence of short chain fatty acids (Nhan et al., 2010), disruption of the quorum sensing of pathogenic V. harveyi (Avnimelech, 2015) and a contribution to shrimp immune system improvement before and during vibriosis outbreaks (Aguilera-Rivera et al., 2018). For this reason, biofloc is considered to be an alternative to disease reduction in aquaculture farms. Here we report a vibriosis outbreak in a biofloc rearing system and the recovery of shrimp without the administration of antibiotics. In addition, a similar outbreak was observed in shrimp reared in clear seawater and, in this case, the use of oxytetracycline as a treatment was inefficient. To understand the mechanisms related to the susceptibility or resistance to Vibrio of Litopenaeus vannamei juveniles reared in biofloc or clear seawater during a vibriosis outbreak, we isolated and identified Vibrio species through the CFU determination in hepatopancreas tissue, molecular identification and expression of pathogenicity and oxytetracycline resistance-related genes of Vibrio strains.
2.4. Bacterial isolation and molecular identification of strains Hepatopancreas tissues of the moribund shrimp previously analyzed were extracted (100 mg) and homogenized under aseptic conditions in sterile saline solution (0.85% NaCl). Sample solutions were serially diluted from 10−1 to 10−5. Three spread plates (100 μL) were made from each sample on thiosulfate-citrate-bile salts-sucrose (TCBS) and incubated at 30 °C for 24 h (Thompson et al., 2004). Subsequently, colonies were counted to estimate colony forming units (CFU) per mg of hepatopancreas tissue. The colony morphology of the bacteria was visually inspected after its growth on agar plates: colony size (measured in mm), color (green or yellow), translucency (opaque and translucent), and colony circumference (smooth or irregulate). Representative morphologies were selected for further analyses. Representative bacteria previously isolated were processed for its DNA extraction (Rojas-Herrera et al., 2008). PCR amplification of the 16S ribosomal RNA, purification and sequencing were conducted as previously described (Aguilera-Rivera et al., 2014). Sequences analyses were conducted using MEGA v5 interphase (Tamura et al., 2011), along with the 16S ribosomal gene sequences from Vibrio strains available in the GenBank data base, using nucleotide BLAST comparison software (Altschul et al., 1990) from the National Center for Biotechnology Information (NCBI). A phylogenetic tree was constructed on alignments using 590 bp. The phylogenetic tree was generated using the NeighborJoining statistical method and a Jukes–Cantor substitution model. The phylogenetic tree generated was validated by a bootstrap method as a support test of their phylogeny.
2. Materials and methods 2.1. Culture conditions Juvenile L. vannamei were reared at Unidad Multidisciplinaria de Docencia e Investigación, Facultad de Ciencias (Sisal, Yucatan, Mexico) in geomembrane circular lined tanks (20,000 L, deep 1 m). Shrimp were maintained 4 months under biofloc (13.98 ± 2.94 g) and clear seawater conditions (11.34 ± 1.54 g). The biofloc rearing system consisted of zero water exchange, heterotrophic medium (filamentous cyanobacteria, protozoa, nematodes, rotifer and copepods), constant aeration, ad libitum commercial feed with 35% of crude protein (Malta Cleyton, Culiacan, Sinaloa, Mexico) and organic fertilization with molasses (Emerenciano et al., 2013). The clear seawater rearing system consisted of daily water exchange of ~25% per day, and the same commercial feeding scheme as for the biofloc system. Water quality parameters were recorded before and during the appearance of lesions and mortality in tanks for both biofloc and clear seawater systems (temperature: 29.3 ± 1.34 and 29.8 ± 1.85 °C; dissolved oxygen: 6.32 ± 2.48 and 6.19 ± 1.89 mg L−1; salinity: 39%o; pH: 8.2 and 8.3, respectively). Total ammonia nitrogen (TAN) and nitrite (NO2) in biofloc also were recorded in the tank (TAN: 1.34 ± 0.43 mg L−1; NO2-N: 0.8 ± 1.09 mg L−1).
2.5. Identification of pathogenicity and oxytetracycline resistance-related genes In order to confirm the presence of the pathogenicity (Pang et al., 2006) and resistance oxytetracycline genes (Lyu et al., 2004) expressed in the strains previously identified by molecular methods, PCR analyzes was performed using primers shown in Table 1. The PCR mixture consisted of 25 µL reaction containing 2 µL of DNA sample, 5 µL of PCR buffer, 2 µL of 25 mmolL−1 MgCl2, 0.5 µL of 2.5 mmol L−1 dNTPs, 1 μL of each gene-specific forward and reverse primers, 0.2 µL of Taq polymerase (ThermoFisher Scientific, Waltham, MA, USA) and 13.3 µL of pyrogen free water. The PCR was carried out in a fluorometric thermocycler IQ5 (BIO-RAD®, Philadelphia, PA, USA). The amplification conditions for toxR were an initial denaturation at 94 °C for 5 min, followed by 30 cycles of amplification with a
2.2. History and gross signs In May and June 2015, mortalities were detected in shrimp reared in biofloc and clear seawater (25 and 80%, respectively). Moderate and severe signs observed in shrimp reared in clear seawater included anorexia, lethargy, melanization, expanded chromatophores, luminescence and necrotic areas in the uropods. Treatment with oxytetracycline (150 mg kg−1) (Gómez-Jiménez et al., 2008) was applied in the feed by repelletization with only occasionally positive results, possibly due to resistance that the bacteria developed against the antibiotic. Interestingly, shrimp reared in biofloc only were observed to have less multifocal melanization in the exoskeleton and redness in appendages. These signs then disappeared without the addition of antibiotics to treat the possible infection.
Table 1 Nucleotide sequences used to amplified toxR, tet(A), tet(B), tet(C), tet(D), tet(E) and tet(G) genes of strains isolated from diseased L. vannamei by conventional PCR. Gene
Sequence Forward/Reverse
Size (bp)
Reference
toxR
5′- GAAGCAGCACTCACCGAT – 3′ 5′- GGTGAAGACTCATCAGCA – 3′ 5′- GCGCTNTATGCGTTGATGCA – 3′ 5′- ACAGCCCGTCAGGAAATT – 3′ 5′- GCGCTNTATGCGTTGATGCA −3′ 5′-TGAAAGCAAACGGCCTAA-3′ 5′- GCGCTNTATGCGTTGATGCA – 3′ 5′ - CGTGCAAGATTCCGAATA – 3′ 5′ – GCGCTNTATGCGTTGATGCA – 3′ 5′- CCAGAGGTTTAAGCAGTGT – 3 5′- GCGCTNTATGCGTTGATGCA – 3′ 5′- ATGTGTCCTGGATTCCT – 3′ 5′- GCGCTNTATGCGTTGATGCA – 3′ 5′- ATGCCAACACCCCCGGCG −3′
382
Pang et al. (2006)
387
Lyu et al. (2004)
tet(A) tet(B) tet(C)
2.3. Signs in shrimp
tet(D)
Ten moribund shrimp with signs of disease were collected from each rearing system and were placed in an ice chest at 23 °C (~5 °C less than the temperature measured in the rearing tanks). A laboratory assay was conducted to determine the severity grade of lesions identified by the following scale: G0 = without lesions; G1 = few lesions, less severe;
tet(E) tet(G)
2
171 631 484 246 803
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denaturation at 94 °C for 1 min, alignment at 58 °C for 1 min and an extension at 72 °C for 1 min (Luan et al., 2006). For tet(A), tet(B), tet(C), tet(D), tet(E) and tet(G), amplification conditions were an initial denaturation at 94 °C for 3 min, followed by 30 cycles of amplification with a denaturation at 94 °C for 30 sec, alignment at 55 °C for 30 sec and an extension at 72 °C for 30 sec (Lyu et al., 2004).
Table 2 Signs identified in the shrimp L. vannamei reared in biofloc and clear seawater systems. Characterization of signs observed: G0 = without signs; G1 = few signs, less severe; G2 = moderate signs, severe; G3 = moderate to severe signs, more severe; and G4 = severe signs, lethal (Lightner, 1996).
2.6. Statistical analyses
Signs
Biofloc
Clear seawater
Lethargy
8 (G0) 2 (G1) 10 (G0)
7 (G2) 3 (G1) 4 (G2) 6 (G3) 3 (G0) 7 (G3) 2 (G0) 8 (G3) 1 (G2) 3 (G3) 6 (G4) 10 (G3)
Incomplete appendages
For CFU analyzes, data were checked for normality (KolmogorovSmirnov test) and homogeneity of variances (Cochran-C test). One-way ANOVA was used to determine significative differences between CFU counted in shrimp reared in clear seawater and biofloc. If the effect was significant, the ANOVA was followed by Tukeýs test. The significance level was set at 0.05. We used the software package STATISTICA v10 for Windows (StatSoft Inc., 2011) for statistical analyses.
Chromatophores expanded Melanization Necrosis in uropods
Luminescence in uropods
3. Results
7 3 6 4 9 1
(G0) (G1) (G0) (G1) (G0) (G1)
10 (G0)
damselae (Table 3, Fig. 2). The five most dominant morphological characteristics in bacteria grown on agar plates included yellow, green and transparent colonies with different sizes, translucency, and circumferences. The yellow colonies were dominant for most strains subsequently identified by molecular methods. However, green colonies were related to Shewanella sp. and P. damselae, although the first one was only identified in shrimp reared under biofloc conditions. Transparent colonies were identified as related to Photobacterium sp. (Table 3, Fig. 3).
3.1. Identification of signs The signs observed in shrimp reared in biofloc and clear seawater varied in severity grade (G0-G4) and included lethargy, anorexia, incomplete appendages, expanded chromatophores, melanization, necrosis and luminescence in uropods (Fig. 1, Table 2). 3.2. Bacterial isolation and molecular identification of strains
3.3. Identification of pathogenicity and oxytetracycline resistance-related genes
The CFU determined in hepatopancreas tissues of shrimp reared in biofloc remained at normal levels (7.14 ± 6.74 × 104 CFU mg−1), whereas the CFU for shrimp reared in clear seawater suggested a probable vibriosis (6.22 ± 7.18 × 106 CFU mg−1) (Gómez-Gil et al., 2011). However, results did not significantly differ in CFU counted in the hepatopancreas tissues analyzed (P = 0.0919). The 16S ribosomal DNA gene sequences of the Vibrio isolated from hepatopancreas of shrimp reared in biofloc or clear seawater system showed > 99% of identity for five different strains, which included: V. harveyi, V. rotiferianus, Shewanella sp., Photobacterium sp. and P.
P. damselae, V. harveyi and Photobacterium sp. isolated from both rearing systems expressed the toxR pathogenicity gene (Table 4, Fig. 4). V. rotiferianus, P. damselae, V. harveyi and Photobacterium sp. expressed the tet(D) oxytetracycline resistance-related gene, whereas tet(B) gene was expressed in V. rotiferianus and V. harveyi. Furthermore, tet(G) gene not only amplified in Shewanella sp., but also was expressed in V. rotiferianus and P. damselae. The genes tet(A), tet(C) and tet(E) did not
Fig. 1. (A) Shrimp reared in clear seawater showing melanization and expanded chromatophores. (B) Shrimp reared in clear seawater showing necrosis in uropods. (C) Shrimp reared in biofloc showing redness in appendages. 3
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Table 3 Growth characteristics of the five dominant strains isolated from hepatopancreas of shrimp L. vannamei reared in biofloc and clear seawater system. Growth
Strain Vr
Sh
Pd
Vh
Ph
100 Biofloc
Color
100 Biofloc Clear seawater Yellow
100 Biofloc Clear seawater Green
100 Biofloc Clear seawater Yellow
99 Biofloc Clear seawater Transparent
Size (mm)
0.6–2.0
0.8–1.6
1.8–2.7
0.4–2.6
Translucency
Opaque
Translucent
Translucent
Translucent
Circumference
Irregulate
Smooth
Smooth
Smooth
Percentage of similitud (%) Rearing system
Green Yellow 0.7–2.6 1.9–2.7 Translucent Opaque Smooth
Vr: V. rotiferianus, Sh: Shewanella sp., Pd: P. damselae, Vh: V. harveyi, Ph: Photobacterium sp.
Fig. 2. Phylogenetic tree with different bacterial strains isolated from hepatopancreas of shrimp L. vannamei reared in biofloc and clear seawater system. Escherichia coli was used as outgroup.
gariepinus (Ariole and Ekeke, 2015), and V. parahaemolyticus and V. alginolyticus in P. monodon (Zadeh et al., 2010). This bacterium has been used as a nutritional supplement in L. vannamei, improving its growth, immune response and disease resistance against V. harveyi (Hao et al., 2014) by its influence on the natural microbiota composition of gut in wild and cultured shrimp (Nayak, 2010; Oxley et al., 2002). Many different types of antibiotics have been used as preventive and therapeutic agents, and include oxytetracycline, oxolinic acid, and chloramphenicol (Saproka et al., 2008). However, antibiotic-resistant bacterial pathogens might cause catastrophic losses due to the lack of drug efficacy for disease control and prevention (Dang et al., 2009). In this study, we administered oxytetracycline orally (in feed) during the vibriosis observed in shrimp reared in clear seawater. This broadspectrum, low toxicity antibiotic is widely used as a treatment for bacterial diseases; however, vibrios have developed resistance for this antibiotic due to the ability to readily develop antibiotic resistance in response to selective pressure, and the ability to spread resistance by horizontal genetic material exchange (Dang et al., 2009). The shrimp reared in biofloc demonstrated an improvement in its health status without the administration of the oxytetracycline. This result is possibly related to mechanisms of immune response that L. vannamei reared in biofloc express during vibriosis outbreaks (Aguilera-Rivera et al., 2018,
amplify in any of the strains identified in hepatopancreas of shrimp reared in biofloc or clear seawater system (Table 4, Fig. 5). 4. Discussion We report a vibriosis outbreak in shrimp reared in biofloc and its recovery without the administration of antibiotics. The seasonal vibriosis detected and described in this study has persisted for the last 6 years and during the warmest months in the study area (May and June). The signs observed coincide with others reported for vibriosis (reddening of the body, brown gills, reduce feeding, lethargy and luminescence) (Chen, 1992), and is commonly related to poor water quality, crowding, high water temperature, low dissolved oxygen and low water exchange (Brock and Lightner, 1990). V. harveyi, V. rotiferianus, Photobacterium sp. and P. damselae were identified by molecular analysis of the strains isolated from biofloc and clear seawater. Infection with these bacteria are related to luminescence in L. vannamei (Chen, 1992) and mass mortality and wounds in P. monodon (Vaseeharan et al., 2007). Shewanella sp. was exclusively identified in shrimp reared in biofloc. This bacterial genus has been isolated from healthy P. monodon intestine and was successfully tested for its antagonism effect against Aeromonas hydrophila in Clarias 4
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Fig. 3. Colony morphology for V. rotiferianus (A), Shewanella sp. (B1, B2), P. damselae (C), V. harveyi (D), and Photobacterium sp. (E) isolated from hepatopancreas of shrimp Litopenaeus vannamei and growth in TCBS agar plates.
2019). For clear seawater and biofloc systems, three of five (60%) tet-positive isolates harbored two or more tet genes, which are related to mechanisms of oxytetracycline resistance. The genes tet(B), tet(D) and tet(G) were identified in V. rotiferianus, V. harveyi, Shewanella sp., Photobaterium sp. and P. damselae isolated from shrimp with different degrees of severity of vibriosis and reared in clear seawater or biofloc. The tet(B) and tet(G) genes have been characterized as non-mobile and not associated with a transferable plasmid, whereas tet(D) has been associated with either conjugative or mobilizable elements, which could explain why tet(B) and tet(G) showed limited distribution (Lyu et al., 2004). On the other hand, tet(D) was identified in the most of strains analyzed (except Shewanella sp.), followed by tet(B), that was only identified in V. rotiferianus and V. harveyi, although tet(B) is considered the most commonly carried efflux gene identified in Gram-negative bacteria (Roberts, 2005). With the aim to identify phylogenetically related Vibrio species and to understand the pathogenicity mechanisms related to the ability to cause disease during the vibriosis outbreak described in this study, toxR gene was identified from strains isolated in shrimp for both rearing systems (clear seawater and biofloc). P. damselae, Photobacterium sp. and V. harveyi expressed this gene, which also has been identified in other strains, including V. tubiashii (Beaubrun et al., 2008), V. campbellii (Orata and Hedreyda, 2011), V. cholerae, V. anguillarum (Kaper et al., 1994) and V. parahaemolyticus (Asgarpoor et al., 2018). toxR is considered to be an ancestral gene of Vibrio species and is therefore present and relatively conserved in all members of Vibrionacea (DiRita and Mekalanos, 1996; Kim et al., 1999; Conejero and Hedreyta, 2003). Furthermore, this gene can be related to shrimp and fish diseases by its
Table 4 Presence of pathogenicity and oxytetracycline resistance genes identified in five strains isolated from hepatopancreas of shrimp L. vannamei reared in biofloc and clear seawater system. Strain
V. rotiferianus Shewanella sp. P. damselae V. harveyi Photobacterium sp.
Presence of toxR
tet(A)
tet(B)
tet(C)
tet(D)
tet(E)
tet(G)
– – x x x
– – – – –
x – – x –
– – – – –
x – x x x
– – – – –
x x x – –
Fig. 4. Agarose gel electrophoresis of the PCR products from nucleic acids of bacteria with specific pathogenicity primer. M, 2072 bp molecular size marker; Lane 1, P. damselae (toxR-382 bp); Lane 2, Photobacterium sp. (toxR-382 bp); Lane 3, V. harveyi (toxR-382 bp); Lane 4, negative control.
Fig. 5. Agarose gel electrophoresis of the PCR products from nucleic acids of bacteria with specific oxytetracycline resistance primers. M, 2072 bp molecular size marker; Lane 1, V. rotiferianus (tet(B)-171 bp); Lane 2, V. harveyi (tet(B)-171 bp); Lane 3, V. rotiferianus (tet(D)-484 bp); Lane 4, V. harveyi (tet(D)-484 bp); Lane 5, P. damselae (tet(D)484 bp); Lane 6, Photobacterium sp. (tet(D)484 bp); Lane 7, V. rotiferianus (tet(G)803 bp); Lane 8, Shewanella sp. (tet(G)803 bp); Lane 9, P. damselae (tet(G)-803 bp); Lane 10, negative control. 5
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5. Conclusions Although the occurrence of vibriosis in shrimp reared in biofloc is possible, the microbial dynamics in the system help to control the development of bacterial diseases produced by Vibrio. In comparison to the clear seawater rearing system, the presence of Shewanella sp. only in biofloc could be related to less severe lesions in shrimp and a low CFU in hepatopancreas tissue. Although oxytetracycline was used for the treatment of vibriosis in the clear seawater system, expression of tet(B), tet(C) and tet(D) genes confirmed the inefficiency of the antibiotic we observed by the presence of disease signs and mortality in shrimp. The diagnosis and identification of strains that can express patterns of pathogenicity and resistance at genetic level is crucial to monitor and prevent the occurrence of bacterial diseases that affect the aquaculture sector. Acknowledgments This work was supported by Consejo Nacional de Ciencia y Tecnología (Ciencia Básica No. 167670). We appreciate the collaboration of M.Sc. Miguel Arevalo, Dr. Manuel Valenzuela, Lic. Gabriela Palomino, Lic. Adriana Paredes, Patricia Balam, Dr. Carlos Maldonado and student members of “Programa Camarón” (UMDI-Sisal). Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jip.2019.107246. References Aguilera-Rivera, D., Prieto-Davó, A., Escalante, K., Chávez, C., Cuzon, G., Gaxiola, G., 2014. Probiotic effect of floc on Vibrios in the Pacific white shrimp Litopenaeus vannamei. Aquaculture 424–425, 215–219. https://doi.org/10.1016/j.aquaculture. 2014.01.008. Aguilera-Rivera, D., Escalante-Herrera, K., Gaxiola, G., Prieto-Davó, A., RodríguezFuentes, G., Guerra-Castro, E., Hernández-López, J., Chávez-Sánchez, M.C., Rodríguez-Canul, R., 2018. Immune response of the Pacific white shrimp, Litopenaeus vannamei, previously reared in biofloc and after an infection assay with Vibrio harveyi. J. World Aquacult Soc. 50, 119–136. https://doi.org/10.1111/jwas.12543. Aguilera-Rivera, D., Rodríguez-Fuentes, G., Escalante-Herrera, K.S., Guerra-Castro, E., Prieto-Davó, A., Gaxiola, G., 2019. Differential expression of immune-related genes in Pacific white shrimp, Litopenaeus vannamei, previously reared in biofloc and challenged with Vibrio harveyi. Aquac. Res. https://doi.org/10.1111/are.14063. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local aligment search tool. J. Mol. Biol. 215, 403–410. Ariole, C.N., Ekeke, J.I., 2015. Effect of an indigenous probiotic (Shewanella algae) isolated from healthy shrimp (Penaeus monodon) intestine on Clarias gariepinus. Int. J. Aquac. 5 (36), 1–9. https://doi.org/10.5376/ijms.2016.06.0059. Asgarpoor, D., Haghi, F., Zeighami, H., 2018. Detection and molecular characterization of Vibrio parahaemolyticus in shrimp samples. Th Open Biotechnol. J. 12, 46–50. https://doi.org/10.2174/1874070701812010046. Avnimelech, Y., 2015. Biofloc technology. A practical guidebook, Baton Rouge, Louisiana, United States. Baffone, W., Citterio, B., Vittoria, E., Casaroli, A., Campana, R., Falzano, L., Donelli, G., 2003. Retention of virulence in viable but non-culturable halophilic Vibrio spp. Int. J. Food Microbiol. 89 (1), 31–39. Beaubrun, J.J.G., Kothary, M.H., Curtis, S.K., Flores, N.C., Eribo, B.E., Tall, B.D., 2008. Isolation and characterization of Vibrio tubiashii outer membrane proteins and determination of a toxR homolog. Appl. Environ. Microbiol. 74 (3), 907–911. https:// doi.org/10.1128/AEM.02052-07. Brock, J.A., Lightner, D.V., 1990. Diseases of crustacea. Diseases caused by microorganisms. Dis. Marine Anim. 3, 245–349. Chen, D., 1992. An overview of the disease situation, diagnostic techniques, treatments and preventatives used on shrimp farms in China. In: Fuls, Main, K.L. (Eds.), Diseases of cultured penaeid shrimp in Asia and the United States. The Oceanic Institute Hawaii, pp. 47–55. Conejero, M.J.U., Hedreyda, C.T., 2003. Isolation of partial toxR gene of Vibrio harveyi and design of toxR-targeted PCR primers for species detection. J. Appl. Microbiol. 95, 602–611.
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Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28 (10), 2731–2739. https://doi.org/10.1093/molbev/msr121. Thompson, F.L., Iida, T., Swings, J., 2004. Biodiversity of vibrios. Microbiol. Mol. Biol. Rev. 68 (3), 403–431. Tran, L., Nunan, L., Redman, R., Mohney, L., Pantoja, C., Fitzsimmons, K., Lightner, D., 2013. Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp. Dis. Aquatic Organisms 105 (1), 45–55. https://doi.org/10.3354/dao02621. Urakawa, H., Yoshida, T., 2000. Characterization of depth-related population variation in microbial communities of a coastal marine sediment using 16S Rdna-based approaches and quinone profiling. Environ. Microbiol. 2, 542–554. Vandenberghe, J., Verdonck, L., Robles-Arozarena, R., Rivera, G., Bolland, A., Balladares, M., Gómez-Gil, B., Sorgeloos, P., Swings, J., 1999. Vibrios associated with Litopenaeus vannamei larvae, postlarvae, broodstock, and hatchery probionts. Appl. Environ. Microbiol. 65 (6), 2592–2597. Vandenberghe, J., Thompson, F.L., Gomez-Gil, B., Swings, J., 2003. Phenotypic diversity amongst Vibrio isolates from marine aquaculture systems. Aquaculture 219, 9–20. https://doi.org/10.1016/S0044-8486(02)00312-5. Vaseeharan, B., Sundararaj, S., Murugan, T., Chen, J.C., 2007. Photobacterium damselae ssp. Damselae associated with diseased black tiger shrimp Penaeus monodon Fabricius in India. Lett. Appl. Microbiol. 45 (1), 82–86. https://doi.org/10.1111/j.1472-765X. 2007.02139.x. Zadeh, S.S., Saad, C.R., Christianus, A., Kamarudin, M.S., Sijam, K., Shamsudin, M.N., Neela, V.K., 2010. Assessment of growth condition for a candidate probiotic, Shewanella algae, isolated from digestive system of a healthy juvenile Penaeus monodon. Aquacult. Int. 18 (6), 1017–1026. https://doi.org/10.1007/s10499-0109319-6.
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A vibriosis outbreak in the Pacific white shrimp, Litopenaeus vannamei reared in biofloc and clear seawater
T
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Diana Aguilera-Riveraa, , Alejandra Prieto-Davóc, Gabriela Rodríguez-Fuentesc, Karla Susana Escalante-Herrerab, Gabriela Gaxiolab a
Escuela Nacional de Estudios Superiores, Unidad Mérida, Universidad Nacional Autónoma de México, Mérida, Yucatán 97205, Mexico Unidad Multidisciplinaria de Docencia e Investigación de Sisal, Facultad de Ciencias, Universidad Nacional Autónoma de México, Sisal, Yucatán 97356, Mexico c Unidad de Química-Sisal, Facultad de Química, Universidad Nacional Autónoma de México, Sisal, Yucatán 97356, Mexico b
A R T I C LE I N FO
A B S T R A C T
Keywords: Vibriosis Biofloc Pathogen Oxytetracycline Resistance
In May and June 2015, moderate and severe lesions were observed in Litopenaeus vannamei reared in clear seawater while, at the same time, lesions in shrimp reared in biofloc were considerably fewer. The signs of disease included anorexia, lethargy, melanization, expanded chromatophores, luminescence and necrotic areas in the uropods, suggesting a possible vibriosis. However, lesions observed in shrimp reared in biofloc disappeared after a certain time and without mortality in tanks, whereas mortality and severe signs continued to be observed in shrimp reared in clear seawater. To treat the possible vibriosis, oxytetracycline was administered only in clear seawater tanks, but the results were not successful. Bacterial cultures from hepatopancreas tissues of shrimp from both rearing systems confirmed a vibriosis outbreak only in the clear seawater system. Subsequently, Vibrio harveyi, Vibrio rotiferianus, Photobacterium sp. and Photobacterium damselae were identified from bacterial culture previously isolated for both rearing systems by molecular methods. Shewanella sp. was isolated and identified only in biofloc. To understand the possible pathogenicity and resistance mechanisms of the Vibiro strains for both rearing systems, pathogenicity (toxR) and oxytetracycline resistance-related genes (tet (B), tet(D), tet(G)) were determined. Although these genes were expressed for both rearing systems, biofloc proved to have the ability to control the development of the disease, in comparison to clear water, where the vibriosis was evident regardless of the administration of oxytetracycline as a treatment.
1. Introduction One of the major problems for aquaculture farms are diseases caused by infectious agents and non-infectious factors. Vibrio bacteria are particularly important due the relationship between the pathogens, the environment and the aquatic invertebrate hosts. Vibrio spp. are Gram-negative, mobile (with 1-2 flagella), mesophilic and chemoorganotrophic bacteria, with a heterotrophic facultative fermentative metabolism. They are oxidase positive and can grow in selective media (TCBS) (Thompson et al., 2004). Vibrios are part of the microbiome of estuarine environments (Heidelberg et al., 2000; Urakawa and Yoshida, 2000; Denner et al., 2002; Potasman et al., 2002; Baffone et al., 2003). However, are considered as opportunistic microorganisms for aquatic organisms (corals, fishes, molluscs, crustaceans, zooplankton) (Rosenberg and Ben‐Haim, 2002; Huys et al., 2001; Sawabe et al., 2003; Vandenberghe et al., 2003; Heidelberg et al., 2002) by the environmental disturbances produced by the salinity,
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temperature or dissolved oxygen (Baffone et al., 2003; Páez-Osuna et al., 2003). Several species of vibrios are well documented as pathogens in shrimp, and include Vibrio harveyi (Mirbakhsh et al., 2014; SotoRodríguez et al., 2012), Vibrio alginolyticus (Vandenberghe et al., 1999), Vibrio parahaemolyticus (Tran et al., 2013), Vibrio campbellii (SotoRodríguez et al., 2006), Vibrio nigripulchritudo (Goarant et al., 2006) and Vibrio penaecida (Goarant and Merien, 2006). Traditional shrimp rearing systems often use antibiotics, chemical substances produced by microorganisms that have the capacity to inhibit or remove pathogens (Santiago et al., 2009) to treat diseases, especially those caused by Vibrio strains. Unfortunately, the use of these antibiotics is inefficient because of inaccurate dosages and time of exposure, affecting the mechanism of action and producing resistance in bacteria. As a sustainable alternative disease control in aquaculture, biofloc rearing systems are used to maintain the water quality. The medium is
Corresponding author. E-mail addresses: [email protected], [email protected] (D. Aguilera-Rivera).
https://doi.org/10.1016/j.jip.2019.107246 Received 28 April 2019; Received in revised form 11 September 2019; Accepted 11 September 2019 Available online 12 September 2019 0022-2011/ © 2019 Published by Elsevier Inc.
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G2 = moderate lesions, severe; G3 = moderate to severe lesions, more severe; and G4 = severe lesions, lethal (Lightner, 1996).
rich in organic matter composed of particulate biomass and colonized by bacteria with a probiotic effect (Aguilera-Rivera et al., 2014). The ability of biofloc to prevent disease outbreaks has been attributed to its protective effect on intestinal epithelium by the presence of short chain fatty acids (Nhan et al., 2010), disruption of the quorum sensing of pathogenic V. harveyi (Avnimelech, 2015) and a contribution to shrimp immune system improvement before and during vibriosis outbreaks (Aguilera-Rivera et al., 2018). For this reason, biofloc is considered to be an alternative to disease reduction in aquaculture farms. Here we report a vibriosis outbreak in a biofloc rearing system and the recovery of shrimp without the administration of antibiotics. In addition, a similar outbreak was observed in shrimp reared in clear seawater and, in this case, the use of oxytetracycline as a treatment was inefficient. To understand the mechanisms related to the susceptibility or resistance to Vibrio of Litopenaeus vannamei juveniles reared in biofloc or clear seawater during a vibriosis outbreak, we isolated and identified Vibrio species through the CFU determination in hepatopancreas tissue, molecular identification and expression of pathogenicity and oxytetracycline resistance-related genes of Vibrio strains.
2.4. Bacterial isolation and molecular identification of strains Hepatopancreas tissues of the moribund shrimp previously analyzed were extracted (100 mg) and homogenized under aseptic conditions in sterile saline solution (0.85% NaCl). Sample solutions were serially diluted from 10−1 to 10−5. Three spread plates (100 μL) were made from each sample on thiosulfate-citrate-bile salts-sucrose (TCBS) and incubated at 30 °C for 24 h (Thompson et al., 2004). Subsequently, colonies were counted to estimate colony forming units (CFU) per mg of hepatopancreas tissue. The colony morphology of the bacteria was visually inspected after its growth on agar plates: colony size (measured in mm), color (green or yellow), translucency (opaque and translucent), and colony circumference (smooth or irregulate). Representative morphologies were selected for further analyses. Representative bacteria previously isolated were processed for its DNA extraction (Rojas-Herrera et al., 2008). PCR amplification of the 16S ribosomal RNA, purification and sequencing were conducted as previously described (Aguilera-Rivera et al., 2014). Sequences analyses were conducted using MEGA v5 interphase (Tamura et al., 2011), along with the 16S ribosomal gene sequences from Vibrio strains available in the GenBank data base, using nucleotide BLAST comparison software (Altschul et al., 1990) from the National Center for Biotechnology Information (NCBI). A phylogenetic tree was constructed on alignments using 590 bp. The phylogenetic tree was generated using the NeighborJoining statistical method and a Jukes–Cantor substitution model. The phylogenetic tree generated was validated by a bootstrap method as a support test of their phylogeny.
2. Materials and methods 2.1. Culture conditions Juvenile L. vannamei were reared at Unidad Multidisciplinaria de Docencia e Investigación, Facultad de Ciencias (Sisal, Yucatan, Mexico) in geomembrane circular lined tanks (20,000 L, deep 1 m). Shrimp were maintained 4 months under biofloc (13.98 ± 2.94 g) and clear seawater conditions (11.34 ± 1.54 g). The biofloc rearing system consisted of zero water exchange, heterotrophic medium (filamentous cyanobacteria, protozoa, nematodes, rotifer and copepods), constant aeration, ad libitum commercial feed with 35% of crude protein (Malta Cleyton, Culiacan, Sinaloa, Mexico) and organic fertilization with molasses (Emerenciano et al., 2013). The clear seawater rearing system consisted of daily water exchange of ~25% per day, and the same commercial feeding scheme as for the biofloc system. Water quality parameters were recorded before and during the appearance of lesions and mortality in tanks for both biofloc and clear seawater systems (temperature: 29.3 ± 1.34 and 29.8 ± 1.85 °C; dissolved oxygen: 6.32 ± 2.48 and 6.19 ± 1.89 mg L−1; salinity: 39%o; pH: 8.2 and 8.3, respectively). Total ammonia nitrogen (TAN) and nitrite (NO2) in biofloc also were recorded in the tank (TAN: 1.34 ± 0.43 mg L−1; NO2-N: 0.8 ± 1.09 mg L−1).
2.5. Identification of pathogenicity and oxytetracycline resistance-related genes In order to confirm the presence of the pathogenicity (Pang et al., 2006) and resistance oxytetracycline genes (Lyu et al., 2004) expressed in the strains previously identified by molecular methods, PCR analyzes was performed using primers shown in Table 1. The PCR mixture consisted of 25 µL reaction containing 2 µL of DNA sample, 5 µL of PCR buffer, 2 µL of 25 mmolL−1 MgCl2, 0.5 µL of 2.5 mmol L−1 dNTPs, 1 μL of each gene-specific forward and reverse primers, 0.2 µL of Taq polymerase (ThermoFisher Scientific, Waltham, MA, USA) and 13.3 µL of pyrogen free water. The PCR was carried out in a fluorometric thermocycler IQ5 (BIO-RAD®, Philadelphia, PA, USA). The amplification conditions for toxR were an initial denaturation at 94 °C for 5 min, followed by 30 cycles of amplification with a
2.2. History and gross signs In May and June 2015, mortalities were detected in shrimp reared in biofloc and clear seawater (25 and 80%, respectively). Moderate and severe signs observed in shrimp reared in clear seawater included anorexia, lethargy, melanization, expanded chromatophores, luminescence and necrotic areas in the uropods. Treatment with oxytetracycline (150 mg kg−1) (Gómez-Jiménez et al., 2008) was applied in the feed by repelletization with only occasionally positive results, possibly due to resistance that the bacteria developed against the antibiotic. Interestingly, shrimp reared in biofloc only were observed to have less multifocal melanization in the exoskeleton and redness in appendages. These signs then disappeared without the addition of antibiotics to treat the possible infection.
Table 1 Nucleotide sequences used to amplified toxR, tet(A), tet(B), tet(C), tet(D), tet(E) and tet(G) genes of strains isolated from diseased L. vannamei by conventional PCR. Gene
Sequence Forward/Reverse
Size (bp)
Reference
toxR
5′- GAAGCAGCACTCACCGAT – 3′ 5′- GGTGAAGACTCATCAGCA – 3′ 5′- GCGCTNTATGCGTTGATGCA – 3′ 5′- ACAGCCCGTCAGGAAATT – 3′ 5′- GCGCTNTATGCGTTGATGCA −3′ 5′-TGAAAGCAAACGGCCTAA-3′ 5′- GCGCTNTATGCGTTGATGCA – 3′ 5′ - CGTGCAAGATTCCGAATA – 3′ 5′ – GCGCTNTATGCGTTGATGCA – 3′ 5′- CCAGAGGTTTAAGCAGTGT – 3 5′- GCGCTNTATGCGTTGATGCA – 3′ 5′- ATGTGTCCTGGATTCCT – 3′ 5′- GCGCTNTATGCGTTGATGCA – 3′ 5′- ATGCCAACACCCCCGGCG −3′
382
Pang et al. (2006)
387
Lyu et al. (2004)
tet(A) tet(B) tet(C)
2.3. Signs in shrimp
tet(D)
Ten moribund shrimp with signs of disease were collected from each rearing system and were placed in an ice chest at 23 °C (~5 °C less than the temperature measured in the rearing tanks). A laboratory assay was conducted to determine the severity grade of lesions identified by the following scale: G0 = without lesions; G1 = few lesions, less severe;
tet(E) tet(G)
2
171 631 484 246 803
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denaturation at 94 °C for 1 min, alignment at 58 °C for 1 min and an extension at 72 °C for 1 min (Luan et al., 2006). For tet(A), tet(B), tet(C), tet(D), tet(E) and tet(G), amplification conditions were an initial denaturation at 94 °C for 3 min, followed by 30 cycles of amplification with a denaturation at 94 °C for 30 sec, alignment at 55 °C for 30 sec and an extension at 72 °C for 30 sec (Lyu et al., 2004).
Table 2 Signs identified in the shrimp L. vannamei reared in biofloc and clear seawater systems. Characterization of signs observed: G0 = without signs; G1 = few signs, less severe; G2 = moderate signs, severe; G3 = moderate to severe signs, more severe; and G4 = severe signs, lethal (Lightner, 1996).
2.6. Statistical analyses
Signs
Biofloc
Clear seawater
Lethargy
8 (G0) 2 (G1) 10 (G0)
7 (G2) 3 (G1) 4 (G2) 6 (G3) 3 (G0) 7 (G3) 2 (G0) 8 (G3) 1 (G2) 3 (G3) 6 (G4) 10 (G3)
Incomplete appendages
For CFU analyzes, data were checked for normality (KolmogorovSmirnov test) and homogeneity of variances (Cochran-C test). One-way ANOVA was used to determine significative differences between CFU counted in shrimp reared in clear seawater and biofloc. If the effect was significant, the ANOVA was followed by Tukeýs test. The significance level was set at 0.05. We used the software package STATISTICA v10 for Windows (StatSoft Inc., 2011) for statistical analyses.
Chromatophores expanded Melanization Necrosis in uropods
Luminescence in uropods
3. Results
7 3 6 4 9 1
(G0) (G1) (G0) (G1) (G0) (G1)
10 (G0)
damselae (Table 3, Fig. 2). The five most dominant morphological characteristics in bacteria grown on agar plates included yellow, green and transparent colonies with different sizes, translucency, and circumferences. The yellow colonies were dominant for most strains subsequently identified by molecular methods. However, green colonies were related to Shewanella sp. and P. damselae, although the first one was only identified in shrimp reared under biofloc conditions. Transparent colonies were identified as related to Photobacterium sp. (Table 3, Fig. 3).
3.1. Identification of signs The signs observed in shrimp reared in biofloc and clear seawater varied in severity grade (G0-G4) and included lethargy, anorexia, incomplete appendages, expanded chromatophores, melanization, necrosis and luminescence in uropods (Fig. 1, Table 2). 3.2. Bacterial isolation and molecular identification of strains
3.3. Identification of pathogenicity and oxytetracycline resistance-related genes
The CFU determined in hepatopancreas tissues of shrimp reared in biofloc remained at normal levels (7.14 ± 6.74 × 104 CFU mg−1), whereas the CFU for shrimp reared in clear seawater suggested a probable vibriosis (6.22 ± 7.18 × 106 CFU mg−1) (Gómez-Gil et al., 2011). However, results did not significantly differ in CFU counted in the hepatopancreas tissues analyzed (P = 0.0919). The 16S ribosomal DNA gene sequences of the Vibrio isolated from hepatopancreas of shrimp reared in biofloc or clear seawater system showed > 99% of identity for five different strains, which included: V. harveyi, V. rotiferianus, Shewanella sp., Photobacterium sp. and P.
P. damselae, V. harveyi and Photobacterium sp. isolated from both rearing systems expressed the toxR pathogenicity gene (Table 4, Fig. 4). V. rotiferianus, P. damselae, V. harveyi and Photobacterium sp. expressed the tet(D) oxytetracycline resistance-related gene, whereas tet(B) gene was expressed in V. rotiferianus and V. harveyi. Furthermore, tet(G) gene not only amplified in Shewanella sp., but also was expressed in V. rotiferianus and P. damselae. The genes tet(A), tet(C) and tet(E) did not
Fig. 1. (A) Shrimp reared in clear seawater showing melanization and expanded chromatophores. (B) Shrimp reared in clear seawater showing necrosis in uropods. (C) Shrimp reared in biofloc showing redness in appendages. 3
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Table 3 Growth characteristics of the five dominant strains isolated from hepatopancreas of shrimp L. vannamei reared in biofloc and clear seawater system. Growth
Strain Vr
Sh
Pd
Vh
Ph
100 Biofloc
Color
100 Biofloc Clear seawater Yellow
100 Biofloc Clear seawater Green
100 Biofloc Clear seawater Yellow
99 Biofloc Clear seawater Transparent
Size (mm)
0.6–2.0
0.8–1.6
1.8–2.7
0.4–2.6
Translucency
Opaque
Translucent
Translucent
Translucent
Circumference
Irregulate
Smooth
Smooth
Smooth
Percentage of similitud (%) Rearing system
Green Yellow 0.7–2.6 1.9–2.7 Translucent Opaque Smooth
Vr: V. rotiferianus, Sh: Shewanella sp., Pd: P. damselae, Vh: V. harveyi, Ph: Photobacterium sp.
Fig. 2. Phylogenetic tree with different bacterial strains isolated from hepatopancreas of shrimp L. vannamei reared in biofloc and clear seawater system. Escherichia coli was used as outgroup.
gariepinus (Ariole and Ekeke, 2015), and V. parahaemolyticus and V. alginolyticus in P. monodon (Zadeh et al., 2010). This bacterium has been used as a nutritional supplement in L. vannamei, improving its growth, immune response and disease resistance against V. harveyi (Hao et al., 2014) by its influence on the natural microbiota composition of gut in wild and cultured shrimp (Nayak, 2010; Oxley et al., 2002). Many different types of antibiotics have been used as preventive and therapeutic agents, and include oxytetracycline, oxolinic acid, and chloramphenicol (Saproka et al., 2008). However, antibiotic-resistant bacterial pathogens might cause catastrophic losses due to the lack of drug efficacy for disease control and prevention (Dang et al., 2009). In this study, we administered oxytetracycline orally (in feed) during the vibriosis observed in shrimp reared in clear seawater. This broadspectrum, low toxicity antibiotic is widely used as a treatment for bacterial diseases; however, vibrios have developed resistance for this antibiotic due to the ability to readily develop antibiotic resistance in response to selective pressure, and the ability to spread resistance by horizontal genetic material exchange (Dang et al., 2009). The shrimp reared in biofloc demonstrated an improvement in its health status without the administration of the oxytetracycline. This result is possibly related to mechanisms of immune response that L. vannamei reared in biofloc express during vibriosis outbreaks (Aguilera-Rivera et al., 2018,
amplify in any of the strains identified in hepatopancreas of shrimp reared in biofloc or clear seawater system (Table 4, Fig. 5). 4. Discussion We report a vibriosis outbreak in shrimp reared in biofloc and its recovery without the administration of antibiotics. The seasonal vibriosis detected and described in this study has persisted for the last 6 years and during the warmest months in the study area (May and June). The signs observed coincide with others reported for vibriosis (reddening of the body, brown gills, reduce feeding, lethargy and luminescence) (Chen, 1992), and is commonly related to poor water quality, crowding, high water temperature, low dissolved oxygen and low water exchange (Brock and Lightner, 1990). V. harveyi, V. rotiferianus, Photobacterium sp. and P. damselae were identified by molecular analysis of the strains isolated from biofloc and clear seawater. Infection with these bacteria are related to luminescence in L. vannamei (Chen, 1992) and mass mortality and wounds in P. monodon (Vaseeharan et al., 2007). Shewanella sp. was exclusively identified in shrimp reared in biofloc. This bacterial genus has been isolated from healthy P. monodon intestine and was successfully tested for its antagonism effect against Aeromonas hydrophila in Clarias 4
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Fig. 3. Colony morphology for V. rotiferianus (A), Shewanella sp. (B1, B2), P. damselae (C), V. harveyi (D), and Photobacterium sp. (E) isolated from hepatopancreas of shrimp Litopenaeus vannamei and growth in TCBS agar plates.
2019). For clear seawater and biofloc systems, three of five (60%) tet-positive isolates harbored two or more tet genes, which are related to mechanisms of oxytetracycline resistance. The genes tet(B), tet(D) and tet(G) were identified in V. rotiferianus, V. harveyi, Shewanella sp., Photobaterium sp. and P. damselae isolated from shrimp with different degrees of severity of vibriosis and reared in clear seawater or biofloc. The tet(B) and tet(G) genes have been characterized as non-mobile and not associated with a transferable plasmid, whereas tet(D) has been associated with either conjugative or mobilizable elements, which could explain why tet(B) and tet(G) showed limited distribution (Lyu et al., 2004). On the other hand, tet(D) was identified in the most of strains analyzed (except Shewanella sp.), followed by tet(B), that was only identified in V. rotiferianus and V. harveyi, although tet(B) is considered the most commonly carried efflux gene identified in Gram-negative bacteria (Roberts, 2005). With the aim to identify phylogenetically related Vibrio species and to understand the pathogenicity mechanisms related to the ability to cause disease during the vibriosis outbreak described in this study, toxR gene was identified from strains isolated in shrimp for both rearing systems (clear seawater and biofloc). P. damselae, Photobacterium sp. and V. harveyi expressed this gene, which also has been identified in other strains, including V. tubiashii (Beaubrun et al., 2008), V. campbellii (Orata and Hedreyda, 2011), V. cholerae, V. anguillarum (Kaper et al., 1994) and V. parahaemolyticus (Asgarpoor et al., 2018). toxR is considered to be an ancestral gene of Vibrio species and is therefore present and relatively conserved in all members of Vibrionacea (DiRita and Mekalanos, 1996; Kim et al., 1999; Conejero and Hedreyta, 2003). Furthermore, this gene can be related to shrimp and fish diseases by its
Table 4 Presence of pathogenicity and oxytetracycline resistance genes identified in five strains isolated from hepatopancreas of shrimp L. vannamei reared in biofloc and clear seawater system. Strain
V. rotiferianus Shewanella sp. P. damselae V. harveyi Photobacterium sp.
Presence of toxR
tet(A)
tet(B)
tet(C)
tet(D)
tet(E)
tet(G)
– – x x x
– – – – –
x – – x –
– – – – –
x – x x x
– – – – –
x x x – –
Fig. 4. Agarose gel electrophoresis of the PCR products from nucleic acids of bacteria with specific pathogenicity primer. M, 2072 bp molecular size marker; Lane 1, P. damselae (toxR-382 bp); Lane 2, Photobacterium sp. (toxR-382 bp); Lane 3, V. harveyi (toxR-382 bp); Lane 4, negative control.
Fig. 5. Agarose gel electrophoresis of the PCR products from nucleic acids of bacteria with specific oxytetracycline resistance primers. M, 2072 bp molecular size marker; Lane 1, V. rotiferianus (tet(B)-171 bp); Lane 2, V. harveyi (tet(B)-171 bp); Lane 3, V. rotiferianus (tet(D)-484 bp); Lane 4, V. harveyi (tet(D)-484 bp); Lane 5, P. damselae (tet(D)484 bp); Lane 6, Photobacterium sp. (tet(D)484 bp); Lane 7, V. rotiferianus (tet(G)803 bp); Lane 8, Shewanella sp. (tet(G)803 bp); Lane 9, P. damselae (tet(G)-803 bp); Lane 10, negative control. 5
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toxic properties elicited by the expression of virulence genes (hemolysin, protease, neuraminidase, cytolysin, lipases, adhesins, lipopolysaccharide, fimbriae, tdh) (DiRita and Mekalanos, 1996; Nor-Najwa et al., 2015).
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5. Conclusions Although the occurrence of vibriosis in shrimp reared in biofloc is possible, the microbial dynamics in the system help to control the development of bacterial diseases produced by Vibrio. In comparison to the clear seawater rearing system, the presence of Shewanella sp. only in biofloc could be related to less severe lesions in shrimp and a low CFU in hepatopancreas tissue. Although oxytetracycline was used for the treatment of vibriosis in the clear seawater system, expression of tet(B), tet(C) and tet(D) genes confirmed the inefficiency of the antibiotic we observed by the presence of disease signs and mortality in shrimp. The diagnosis and identification of strains that can express patterns of pathogenicity and resistance at genetic level is crucial to monitor and prevent the occurrence of bacterial diseases that affect the aquaculture sector. Acknowledgments This work was supported by Consejo Nacional de Ciencia y Tecnología (Ciencia Básica No. 167670). We appreciate the collaboration of M.Sc. Miguel Arevalo, Dr. Manuel Valenzuela, Lic. Gabriela Palomino, Lic. Adriana Paredes, Patricia Balam, Dr. Carlos Maldonado and student members of “Programa Camarón” (UMDI-Sisal). Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jip.2019.107246. References Aguilera-Rivera, D., Prieto-Davó, A., Escalante, K., Chávez, C., Cuzon, G., Gaxiola, G., 2014. Probiotic effect of floc on Vibrios in the Pacific white shrimp Litopenaeus vannamei. Aquaculture 424–425, 215–219. https://doi.org/10.1016/j.aquaculture. 2014.01.008. Aguilera-Rivera, D., Escalante-Herrera, K., Gaxiola, G., Prieto-Davó, A., RodríguezFuentes, G., Guerra-Castro, E., Hernández-López, J., Chávez-Sánchez, M.C., Rodríguez-Canul, R., 2018. Immune response of the Pacific white shrimp, Litopenaeus vannamei, previously reared in biofloc and after an infection assay with Vibrio harveyi. J. World Aquacult Soc. 50, 119–136. https://doi.org/10.1111/jwas.12543. Aguilera-Rivera, D., Rodríguez-Fuentes, G., Escalante-Herrera, K.S., Guerra-Castro, E., Prieto-Davó, A., Gaxiola, G., 2019. Differential expression of immune-related genes in Pacific white shrimp, Litopenaeus vannamei, previously reared in biofloc and challenged with Vibrio harveyi. Aquac. Res. https://doi.org/10.1111/are.14063. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local aligment search tool. J. Mol. Biol. 215, 403–410. Ariole, C.N., Ekeke, J.I., 2015. Effect of an indigenous probiotic (Shewanella algae) isolated from healthy shrimp (Penaeus monodon) intestine on Clarias gariepinus. Int. J. Aquac. 5 (36), 1–9. https://doi.org/10.5376/ijms.2016.06.0059. Asgarpoor, D., Haghi, F., Zeighami, H., 2018. Detection and molecular characterization of Vibrio parahaemolyticus in shrimp samples. Th Open Biotechnol. J. 12, 46–50. https://doi.org/10.2174/1874070701812010046. Avnimelech, Y., 2015. Biofloc technology. A practical guidebook, Baton Rouge, Louisiana, United States. Baffone, W., Citterio, B., Vittoria, E., Casaroli, A., Campana, R., Falzano, L., Donelli, G., 2003. Retention of virulence in viable but non-culturable halophilic Vibrio spp. Int. J. Food Microbiol. 89 (1), 31–39. Beaubrun, J.J.G., Kothary, M.H., Curtis, S.K., Flores, N.C., Eribo, B.E., Tall, B.D., 2008. Isolation and characterization of Vibrio tubiashii outer membrane proteins and determination of a toxR homolog. Appl. Environ. Microbiol. 74 (3), 907–911. https:// doi.org/10.1128/AEM.02052-07. Brock, J.A., Lightner, D.V., 1990. Diseases of crustacea. Diseases caused by microorganisms. Dis. Marine Anim. 3, 245–349. Chen, D., 1992. An overview of the disease situation, diagnostic techniques, treatments and preventatives used on shrimp farms in China. In: Fuls, Main, K.L. (Eds.), Diseases of cultured penaeid shrimp in Asia and the United States. The Oceanic Institute Hawaii, pp. 47–55. Conejero, M.J.U., Hedreyda, C.T., 2003. Isolation of partial toxR gene of Vibrio harveyi and design of toxR-targeted PCR primers for species detection. J. Appl. Microbiol. 95, 602–611.
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