Vibrio spp. Control at Brine Shrimp, Artemia, Hatching and Enrichment
Experiments were conducted to evaluate different prophylactic methods to control the bacterial load in brine shrimp, Artemia, hatching. The first experiment evaluated three treatments to control Vibrio spp. during the Artemia hatching: microalgae (Chaetoceros calcitrans), probiotic (Bacillus spp.), and antibiotic (Florfenicol). In the second experiment, Artemia metanauplius were enriched in distinct treatments with C. calcitrans, probiotic, and emulsion rich in docosahexaenoic and eicosapentaenoic fatty acids. Enriched Artemia metanauplius and nauplii (control) were offered to white shrimp, Litopenaeus vannamei, postlarvae (PL7–PL19). Presumptive Vibrio were quantified in Artemia, PL, and rearing water. Microalgae and probiotic were effective to control Vibrio spp. in Artemia nauplii. The enrichment process increased the Artemia bacterial load but did not affect Vibrio load in L. vannamei.
In fish and shellfish hatcheries, the widespread use of brine shrimp, Artemia, as live food is due to their positive characteristics such as high protein content and ability to produce storable cysts (Léger et al. 1987). The Artemia nutritional value can be further increased by the enrichment process (bioencapsulation). In addition, Artemia can modify the fatty acid composition of the enrichment product as well as its lipid classes. The lipid conversion during the Artemia enrichment process is responsible for an extensive incorporation of eicosapentaenoic fatty acid, important to larval development (Navarro et al. 1999; Garcia et al. 2008). The enrichment technique exploits the fact that Artemia is a non-selective filter feeder organism in its second stage of development (instar II or metanauplius), which occurs 8 h after hatching (Campbell et al. 1993; Dixon et al. 1995; Sorgeloos et al. 2001). This feature also allows the use of Artemia in disease control through the bioencapsulation of antimicrobial agents. However, Artemia nauplius has been also reported as vector of pathogenic bacteria in shrimp hatcheries (López-Torres and Lizárraga-Partida 2001).
The high organic load during intensive live food production induces a proportional growth of opportunistic bacteria. The disinfection can be beneficial, but cannot prevent the live food to be recolonized in a short period of time (Munro et al. 1999; Skjermo and Vadstein 1999). The bacterial load of Artemia includes Vibrio spp., which have been related to high mortality in penaeid shrimp rearings worldwide (Lightner and Lewis 1975; Baticados et al. 1990; Lavilla-Pitogo et al. 1990; Gomez-Gil et al. 2004).
Nicolas et al. (1989) observed among the Vibrionaceae, which were the main constituent of the bacterial flora in the gut of turbot larvae, Scophthalmus maximus, that some were probably introduced by rotifers. Munro et al. (1994) isolated potential pathogens from apparently healthy turbot larvae with high survival rates. It is suggested that the bacterial flora plays an important role in determining larval survival, but the criteria to establish a beneficial flora have still not been elucidated.
Frequent and inappropriate use of antibiotics can induce the selection and proliferation of resistant bacterial strains. Therefore, alternative prophylactic measures to reduce Vibrio spp. spreading should be adopted as they are more cost-effective and less dependent on the use of chemicals (Planas and Cunha 1999; Witte et al. 1999).
In this context, this study evaluated different prophylactic methods to control the bacterial load in Artemia hatching and enrichment. The bacterial load of white shrimp, Litopenaeus vannamei, postlarvae (PL) fed with Artemia was also investigated.
Materials and Methods
The experiment evaluated three supplements to control Vibrio spp. during the hatching of Artemia cysts GSL INVE (Inve Technologies, Dendermonde, Belgium), strain Franciscana. A sequence of two trials tested capsulated and decapsulated cysts. A 12% sodium hypochlorite solution was used to decapsulate the cysts as recommended by Van Stappen (1996). Four treatments were established, according to the type of supplement: antibiotic, probiotic, microalgae, and control. All supplements were applied directly into the hatching water at the same time of the cysts addition.
The antibiotic treatment received 300 mg/L dose of Florfenicol Aquaflor 50% Premix (Schering Plough, Kenilworth, NJ, USA), according to Roiha et al. (2010). A commercial probiotic, Sanolife MIC INVE (Inve Technologies) consisting of Bacillus subtilis, Bacillus pumilus, and Bacillus licheniformis (2 × 105 CFU/mL) was used in the probiotic treatment. The microalgae treatment received the marine diatom, Chaetoceros calcitrans (8 × 105 cells/mL), collected during exponential growth phase from a non-axenic culture. Seawater (30 g/L) for microalgae culture was previously treated with chlorine (15 ppm) during 24 h. Later, the seawater was dechlorinated with ascorbic acid and enriched with modified Conway medium. In the control treatment, only seawater disinfected with chlorine (15 ppm) during 24 h and dechlorinated with ascorbic acid was used.
Artemia cysts (1 g/L) were stocked in 12 cylindrical-conical tanks filled with 20 L seawater previously disinfected according to the same procedure described for the control treatment. Temperature, salinity, dissolved oxygen, and pH were maintained at 29.9 ± 0.3 C, 27.7 ± 0.1 g/L, 5.7 ± 0.1 mg/L, and 8.1 ± 0.03, respectively, as recommended by Van Stappen (1996). The Artemia hatching tanks were illuminated (2000 lx) and aerated continuously. Three replicates were used for each treatment. After 24 h of incubation, the hatched nauplii were harvested and counted. The nauplii hatching efficiency (HE) was calculated using the formula: HE = (mean number of nauplii hatched / mL × water volume) / grams of cysts added.
Water and Artemia samples of all treatments were collected to quantify presumptive Vibrio colony forming units (CFUs) using the agar thiosulfate bile sucrose (TCBS, agar Himedia) (Himedia Laboratories, Mumbai, India). Water samples were serially diluted (1/10) in sterile saline solution (2.5% NaCl). Aliquots of 0.1 mL from three dilutions were spread plated on TCBS agar and incubated for 24 h at 30 C. After incubation, the total CFUs for plates that presented between 30 and 300 colonies were enumerated (Downes and Ito 2001).
The samples of Artemia (2 g) were aseptically macerated, diluted, and plated using the same methodology described for the water analysis. Additionally, 2 g samples of different Artemia treatments were frozen at a temperature of −25 C for 48 h and then the bacterial load was determined. This analysis determined the influence of temperature on the bacteria viability, as commercial hatcheries occasionally use frozen Artemia.
From the nauplii samples, different bacterial morphotypes grown on TCBS agar were isolated and subjected to presumptive identification tests for the Vibrio genus (Gram stain, detection of glucose anaerobic fermentation, and oxidase). According to Alsina and Blanch (1994), Vibrio spp. from environmental isolates are Gram-negative, give a positive oxidase test, grow on TCBS medium, and are facultative anaerobes. Further, the morphotypes were identified through their biochemical profiles presented in the commercial bacterial identification kit API 20 E Biomerieux (Biomerieux Diagnostics, Rhone-Alpes, France).
Vibrio concentration was evaluated in Artemia enriched with three different supplements used in distinct treatments: C. calcitrans (microalgae treatment), commercial probiotic (probiotic treatment), and a commercial emulsion rich in fatty acids, DC DHA Selco INVE (Selco treatment, Inve Aquaculture, Salt Lake, UT, USA). The control was represented by newly hatched nauplii with no added supplements. Three replicates were used for each treatment.
Capsulated Artemia cysts were hatched under the same conditions of the control in the first experiment. The newly hatched nauplii were washed with chlorine-disinfected seawater and stocked (100–300 nauplii/mL) in 5-L containers filled with seawater and the different supplements. Artemia were submitted to enrichment process during 28 h under constant aeration. Average water temperature, salinity, pH, and dissolved oxygen were maintained between 27 and 31 C, 28 and 30 g/L, 8.5 and 9.0, and 4.69 and 6.65 mg/L, respectively.
The microalgae treatment received 8 × 105 cells/mL of C. calcitrans and the probiotic treatment received 2 × 105 CFU/mL of commercial probiotic (B. subtilis, B. pumilus, and B. licheniformis). The Selco treatment received 0.3 g/L every 12 h (Merchie et al. 1995) of commercial emulsion rich in docosahexaenoic and eicosapentaenoic fatty acids that can also provide Vibrio control. This emulsion is known as a disinfectant-appended enrichment diet (Liao et al. 2001). The hatching and enrichment processes were repeated for 12 d.
Litopenaeus vannamei PL in PL7 stage (7 d in the PL stage) with wet mean weight of 0.843 mg were randomly stocked in 12 plastic containers (10 L) at a density of 50 PL/L. Water was previously disinfected with chlorine (15 ppm) for 24 h and then neutralized with ascorbic acid.
Enriched Artemia from the four treatments (microalgae, probiotic, Selco, and control) were offered to shrimp PL feeding during 12 d (three replicates each). Shrimp were fed twice daily (0700 and 1900 h) at an initial rate of 5 nauplii/mL reaching 12 nauplii/mL at the end of rearing, according to the larvae consumption.
Shrimp PL were maintained under constant aeration at a temperature of 27–28 C. Daily, 50% of water in the experimental units was exchanged. Temperature, dissolved oxygen, pH, and salinity were monitored daily by a multiparameter sensor YSI 556 (Yellow Springs Instrument Company, Yellow Springs, OH, USA).
Presumptive Vibrio spp. were quantified in samples of enriched Artemia, water of Artemia enrichment, shrimp PL, and water of PL rearing.
The procedure for Vibrio analysis in water and Artemia and PL samples followed the methodology presented in the Experiment I. The PL samples were weighted, macerated, and diluted (1/10) and 0.1 mL of three dilutions were plated in TCBS agar, incubated, and enumerated as described previously.
Additionally, the colonization of bacteria from commercial probiotic was verified by the quantification of Bacillus CFU in enriched Artemia and shrimp PL from the probiotic treatment. The samples were weighted, macerated, and diluted (1/10) and 0.1 mL of three dilutions were plated in MYP agar (Mannitol Egg Yolk Agar Polymyxin; Himedia), incubated, and enumerated.
The HE data were submitted to analysis of variance (ANOVA) and Tukey's test. The Student's t-test was used to compare the bacterial count of Artemia natural biomass and frozen biomass. Bacterial counts data were submitted to ANOVA and Fisher tests. Differences were considered at the 5% significance level. All analyzes were performed using Statistica 7.0. (Statsoft, Inc., Tulsa, OK, USA).
The HE was similar among treatments and mean values (±SE) ranged from 1.9 ± 0.1 × 105 to 2.7 × 105 nauplii/g.
The decapsulation process was not effective in reducing the presumptive Vibrio spp. load in nauplii and water. Except for the antibiotic treatment, the Vibrio load was similar (P > 0.05) between treatments with capsulate and decapsulated cysts (Table 1).
Table 1. Average values (±SE) of presumptive Vibrio count in Artemia hatching water and Artemia nauplii (capsulated and decapsulated cysts) from different treatments.a
|Control||620.0 ± 450.0c||120.0 ± 90.0b||200.0 ± 150.0b||45.0 ± 24.0b|
|Antibiotic||0.008 ± 0.007a*||0.02 ± 0.007a||7.50 ± 2.90a*||3.10 ± 0.70a|
|Microalgae||37.0 ± 21.0b||0.40 ± 0.30a||44.0 ± 1.90ab||29.0 ± 10.0ab|
|Probiotic||590.0 ± 230.0c||15.0 ± 15.0a||320.0 ± 190.0b||15.0 ± 9.0a|
The number of bacterial colonies in water was significantly lower in the antibiotic followed by the microalgae treatment of capsulated cysts (Table 1). Only the probiotic treatment did not differ significantly from the control.
In the trial with decapsulated cysts, lower Vibrio count was recorded in water from the antibiotic treatment but it did not differ significantly from the microalgae (Table 1). Mean Vibrio counts in the control, microalgae, and probiotic treatments were not significantly different.
In the trial with capsulated cysts, Vibrio spp. load of supplemented Artemia was significantly lower than the control (Table 1). However, Artemia from decapsulated cysts showed significantly lower contamination in the antibiotic and probiotic treatments (Table 1).
The freezing nauplii process during 48 h resulted in a reduction of 70.3 to 99.8% of the Vibrio spp. load (Table 2).
Table 2. Average values (±SE) of presumptive Vibrio count in Artemia nauplii (capsulated and decapsulated cysts) from different treatments, before and after freezing.a
|Control||120.0 ± 90.0a||25.0 ± 23.0b||45.0 ± 24.0a||2.60 ± 1.00b|
|Antibiotic||0.02 ± 0.007a||0.00005 ± 0.00003b||3.10 ± 0.70a||0.92 ± 0.43b|
|Microalgae||0.40 ± 0.30a||1.30 ± 1.10a||29.0 ± 10.0a||6.00 ± 1.60b|
|Probiotic||15.0 ± 15.0a||1.40 ± 0.90b||15.0 ± 9.0a||2.50 ± 0.70b|
Of the 43 bacterial colony morphotypes isolated from nauplii of capsulated cysts, including all treatments, 54% was identified as Vibrio alginolyticus and 36% as Gram-negative rods oxidase-negative. Vibrio parahaemolyticus, Aeromonas hydrophila, Aeromonas salmonicida, Ochrobactrum anthropi, and a Gram-negative rods oxidase-positive isolate each represented 2% of the total isolates. Vibrio parahaemolyticus and O. anthropi were resistant to Florfenicol.
From the nauplii of decapsulated cysts, a total of 31 isolates were obtained. Of these, 42% were identified as V. alginolyticus, 42% as Gram-negative rods oxidase-negative, and 16% as Gram-positive isolates.
The Vibrio spp. concentrations in the Artemia enrichment water were significantly lower in the control (newly hatched nauplii) and did not differ among other treatments (Table 3). In Artemia, the lowest value of bacterial colonies was also observed in the control, but it did not differ from the microalgae and probiotic treatments. The Selco treatment presented the highest level of contamination differing significantly from the control.
Table 3. Average values (±SE) of presumptive Vibrio count in Artemia rearing water, Artemia, Litopenaeus vannamei postlarvae (PL), and PL rearing water from different treatments.a
|Control||3.4 ± 3.0a||6.5 ± 3.0a||0.25 ± 0.00b||0.17 ± 0.07a|
|Selco||100.0 ± 30.0b||160.0 ± 140.0b||0.11 ± 0.03ab||0.87 ± 0.86a|
|Microalgae||17.0 ± 5.0b||40.0 ± 7.0ab||0.15 ± 0.09ab||0.04 ± 0.02a|
|Probiotic||67.0 ± 23.0b||23.0 ± 2.0ab||0.05 ± 0.02a||0.06 ± 0.02a|
The Vibrio spp. concentration in the PL rearing water from the probiotic treatment was significantly lower than the control (Table 3). No significant differences were observed in the number of bacterial colonies in PL among treatments (Table 3).
Bacillus quantification (±SE) in metanauplius and PL from the probiotic treatment was 8.7 ± 4.2 × 105 and 1.4 ± 0.8 × 106 CFU/g, respectively.
In the PL rearing water, the mean values (±SE) of temperature (28.7 ± 0.04 C), OD (5.5 ± 0.1 mg/L), pH (8.50 ± 0.04), and salinity (29.5 ± 0.06 g/L) did not differ among treatments.
Bacterial enumeration in TCBS medium shows that Artemia hold a high number of bacteria. According to Verdonck et al. (1994), Artemia are usually highly contaminated with bacteria (>107 CFU/g) and mostly identified as Vibrio spp. Moreover, it is necessary to control these bacterial loads before the use of Artemia in culture systems.
The use of Florfenicol resulted in the lowest Vibrio spp. load among all the supplements tested in Artemia hatchery. The Florfenicol has been authorized in several countries for aquaculture activities (FAO 2005). In Brazil, it is the only antibiotic registered for this purpose in the Ministry of Agriculture, Livestock and Supply (Schering Plough Animal Health 2009). In fish farming, Florfenicol has potent activity against a broad range of pathogens (Samuelsen et al. 2003), including microorganisms resistant to other antibiotics (Nordmo et al. 1994; Rangdale et al. 1997; Bruun et al. 2000; Thyssen and Ollevier 2001; Vue et al. 2002; Samuelsen and Bergh 2004). Our findings suggested that the Florfenicol dose (300 mg/L) was efficient in reducing Vibrio counts but, some potentially pathogenic strains of Vibrio (V. alginolyticus and V. parahaemolyticus) remained in Artemia nauplii. The proliferation of these resistant bacteria could make possible infections more difficult to treat in hatchery systems.
The microalgae treatment was the second most efficient in reducing Vibrio load in water and nauplii (capsulated). This effect may be related to the bacteriostatic or bactericidal microalgae activity (Kellam and Walker 1989; Olsen et al. 2000). The microalgae antibacterial activity has been detected in microalgae extracts (Duff and Bruce 1966; Austin and Day 1990; Austin et al. 1992; Tendencia and dela Peña 2003) and it may be related to the associated microflora, antimicrobial proteins, fatty acids, and oxygen free radicals produced by microalgae cells (Marshall et al. 2005; Makridis et al. 2006; Kokou et al. 2007).
Despite newly hatched Artemia nauplii are unable to bioencapsulate, probiotic bacteria can be active in the gills and body surface by competing with other bacteria for adhesion sites (Gatesoupe 1991; Verschuere et al. 2000). This study has shown that probiotics reduced the Vibrio load in Artemia from capsulated and decapsulated cysts.
Freezing Artemia for 48 h reduced the Vibrio counts, but most values remained over 107 CFU. Reviewing the responses of mesophilic bacteria to cold stress, Panoff et al. (1998) listed the membrane damage and DNA denaturation as possible causes of bacterial death after freezing and thawing. Sensitive bacterial cells usually undergo a slow death rate upon freezing and their response can be described as an exponential function (Haines 1938; Panoff et al. 1998). Alur and Grecz (1975) reported higher rates of DNA fragmentation after fast freezing rather than slow freezing. However, after 24 h storage, the slow frozen cells yielded the same results as fast frozen cells. The authors suggested that death was related to DNA and membrane degradation as DNA is attached to plasma membrane.
It has been suggested that Vibrio spp. are capable of entering into a viable but non-culturable (VBNC) state when exposed to low temperatures (Jiang and Chai 1996; Johnson and Brown 2002). Eventually, when temperature rises, bacterial cells are able to emerge from the VBNC state and become culturable on bacteriological media. Thus, offering short-term frozen stored Artemia to shrimp larvae may not reduce the potential of Vibrio spp. contamination, as cold-induced death of bacteria only occur after several days to weeks (Oliver 1981).
The sodium hypochlorite used in the decapsulation process is able to totally decontaminate Artemia cysts, but they can be quickly recolonized during the rupture stage before hatching (Sorgeloos et al. 2001). At this stage, the organic substrate glycerol is released from the cysts and offers an ideal culture medium for Vibrio spp. In this study, the decapsulation process did not effectively reduced Vibrio concentration, but decreased potential bacterial pathogens species in nauplii and may be regarded as an auxiliary prophylactic treatment.
Hoj et al. (2009) characterized the bacterial community present in Artemia, with more than half of Vibrio isolates being identified as V. alginolyticus. López-Torres and Lizárraga-Partida (2001) observed that even when Artemia cysts were hatched under sterile conditions, V. alginolyticus was the dominant species. These authors suggested that V. alginolyticus and Vibrio spp. isolated from Artemia hatching tanks were associated with those isolated from tanks with zoea, mysis, and PL, indicating that these Vibrio spp. remain in different development phases of shrimp hatchery. Buglione et al. (2010) observed that V. alginolyticus strain caused high mortality of L. vannamei larvae. The V. alginolyticus virulence is correlated to enzyme collagenase activity, which can cause softening of shrimp muscle tissue (Brauer et al. 2003; Yishan et al. 2011).
The presence of V. parahaemolyticus in samples of shrimp submitted to the antibiotic treatment was reported by Verschuere et al. (2000). Likewise, this bacteria was identified in Artemia treated with antibiotic (Florfenicol) in our study. According to Gomez-Gil et al. (2004), V. parahaemolyticus affects mainly shrimps at juvenile and adult stage. Ochrobactrum anthropi was also resistant to the Florfenicol treatment. Although this species has not been related to shrimp diseases, it has been isolated from fresh and cryopreserved spermatophores of the giant tiger prawn, Penaeus monodon (Nimrat et al. 2008), and water samples from mangrove receiving shrimp farm effluents (Sousa et al. 2006). In another study, O. anthropi was isolated and identified by phylogenetic analysis using 16S rDNA sequences from inhabited marine biofilms. Such bacteria can colonize Artemia and L. vannamei once that it has been observed in the marine environment and furthermore, biofilms cover most subtidal and intertidal solid surfaces such as rocks, ships, loops, marine animals, and algae (Lee et al. 2003). Ochrobactrum anthropi was also isolated from blue crab body meat, Callinectes sapidus (Özogul et al. 2010), and intestinal samples of zebra fish, Danio rerio (Cantas et al. 2012).
Aeromonas spp. compose the normal microflora of wild and reared crustaceans and can be considered an opportunistic pathogen (Lightner 1993). This genus has been associated to the soft shell syndrome in P. monodon (Baticados et al. 1986; Uddin et al. 2008).
The Artemia enrichment process increased bacterial load in water and Artemia specially in the Selco treatment. This could be explained by the organic input from supplements and Artemia excretion, which allows a sudden increase of opportunistic bacteria (Igarashi et al. 1989; Skjermo and Vadstein 1993; Verschuere et al. 1997; Olsen et al. 2000). Hoj et al. (2009) and Haché and Plante (2011) also observed high bacterial load in Artemia enriched with microalgae and lipid emulsions in combination.
The bacterial load in shrimp PL and rearing water were not associated with the concentrations of Vibrio observed in newly hatched nauplii (control) and enriched Artemia. The daily renewal (50%) of PL rearing water probably reduced the abundance of Vibrio spp. Krishnika and Ramasamy (2012) also recorded a significant reduction of Vibrio after the water exchange in Artemia rearing tanks. Silva et al. (2013) observed a presumptive Vibrio load of 7.9 × 107 CFU/g in L. vannamei PL (PL10) fed newly hatched Artemia nauplii. In this study, this load was slightly lower for PL19 (0.17 × 107 CFU/g).
Overall, results of this study indicated that addition of C. calcitrans in Artemia hatching water is an effective alternative to antibiotics. Additionally, the use of probiotic must also be considered to control the Vibrio spp. load in Artemia nauplii. The enrichment supplements increased the bacterial load in Artemia but they did not affect Vibrio concentration in shrimp PL.
This study was supported by the Improvement of Higher Education Personnel Coordination (CAPES) and the Brazilian Council for Scientific and Technological Development (CNPq).