Vibrio spp. Control at Brine Shrimp, Artemia , Hatching and Enrichment

JOURNAL OF THEWORLD AQUACULTURE SOCIETY

Vol. 45, No. 1February, 2014

doi: 10.1111/jwas.12096

Vibrio spp. Control at Brine Shrimp, Artemia , Hatchingand Enrichment

Juliana Aguiar Interaminense1, Nathalia Ferreira Calazans, BrunaCaritas do Valle, Joana Lyra Vogeley, Sílvio Peixoto and Roberta Soares

Laboratorio de Tecnologia em Aquicultura, Departamento de Pesca e Aquicultura,Universidade Federal Rural de Pernambuco, Recife, PE 52171-900, Brazil

Jose Vitor Lima Filho

Laboratorio de Microbiologia e Imunologia, Departamento de Biologia, Universidade FederalRural de Pernambuco, Recife, PE 52171-900, Brazil

AbstractExperiments were conducted to evaluate different prophylactic methods to control the bacterial

load in brine shrimp, Artemia , hatching. The first experiment evaluated three treatments to controlVibrio spp. during the Artemia hatching: microalgae (Chaetoceros calcitrans), probiotic (Bacillus spp.),and antibiotic (Florfenicol). In the second experiment, Artemia metanauplius were enriched in distincttreatments with C. calcitrans , probiotic, and emulsion rich in docosahexaenoic and eicosapentaenoicfatty 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, thewidespread use of brine shrimp, Artemia , aslive food is due to their positive characteristicssuch as high protein content and ability to pro-duce storable cysts (Leger et al. 1987). TheArtemia nutritional value can be furtherincreased by the enrichment process (bioen-capsulation). In addition, Artemia can modifythe fatty acid composition of the enrichmentproduct as well as its lipid classes. The lipidconversion during the Artemia enrichmentprocess is responsible for an extensive incorpo-ration of eicosapentaenoic fatty acid, importantto larval development (Navarro et al. 1999;Garcia et al. 2008). The enrichment techniqueexploits the fact that Artemia is a non-selectivefilter feeder organism in its second stage ofdevelopment (instar II or metanauplius), whichoccurs 8 h after hatching (Campbell et al. 1993;

1 Corresponding author.

Dixon et al. 1995; Sorgeloos et al. 2001). Thisfeature also allows the use of Artemia in diseasecontrol through the bioencapsulation of antimi-crobial agents. However, Artemia naupliushas been also reported as vector of pathogenicbacteria in shrimp hatcheries (Lopez-Torres andLizarraga-Partida 2001).

The high organic load during intensive livefood production induces a proportional growthof opportunistic bacteria. The disinfection canbe beneficial, but cannot prevent the live food tobe recolonized in a short period of time (Munroet al. 1999; Skjermo and Vadstein 1999). Thebacterial load of Artemia includes Vibrio spp.,which have been related to high mortality inpenaeid shrimp rearings worldwide (Lightnerand Lewis 1975; Baticados et al. 1990; Lavilla-Pitogo et al. 1990; Gomez-Gil et al. 2004).

Nicolas et al. (1989) observed among theVibrionaceae, which were the main constituentof the bacterial flora in the gut of turbot lar-vae, Scophthalmus maximus , that some were

© Copyright by the World Aquaculture Society 2014

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66 INTERAMINENSE ET AL.

probably introduced by rotifers. Munro et al.(1994) isolated potential pathogens from appar-ently healthy turbot larvae with high survivalrates. It is suggested that the bacterial floraplays an important role in determining larvalsurvival, but the criteria to establish a beneficialflora have still not been elucidated.

Frequent and inappropriate use of antibioticscan induce the selection and proliferation ofresistant bacterial strains. Therefore, alternativeprophylactic measures to reduce Vibrio spp.spreading should be adopted as they are morecost-effective and less dependent on the use ofchemicals (Planas and Cunha 1999; Witte et al.1999).

In this context, this study evaluated differentprophylactic methods to control the bacterialload in Artemia hatching and enrichment. Thebacterial load of white shrimp, Litopenaeusvannamei , postlarvae (PL) fed with Artemiawas also investigated.

Materials and Methods

Experiment I

The experiment evaluated three supplementsto control Vibrio spp. during the hatchingof Artemia cysts GSL INVE (Inve Technolo-gies, Dendermonde, Belgium), strain Francis-cana . A sequence of two trials tested capsu-lated and decapsulated cysts. A 12% sodiumhypochlorite solution was used to decapsu-late the cysts as recommended by Van Stap-pen (1996). Four treatments were established,according to the type of supplement: antibiotic,probiotic, microalgae, and control. All supple-ments were applied directly into the hatchingwater at the same time of the cysts addition.

The antibiotic treatment received 300 mg/Ldose of Florfenicol Aquaflor 50% Premix(Schering Plough, Kenilworth, NJ, USA),according to Roiha et al. (2010). A commercialprobiotic, Sanolife MIC INVE (Inve Technolo-gies) consisting of Bacillus subtilis , Bacilluspumilus , and Bacillus licheniformis (2 × 105

CFU/mL) was used in the probiotic treatment.The microalgae treatment received the marinediatom, Chaetoceros calcitrans (8 × 105

cells/mL), collected during exponential growth

phase from a non-axenic culture. Seawater (30g/L) for microalgae culture was previouslytreated with chlorine (15 ppm) during 24 h.Later, the seawater was dechlorinated withascorbic acid and enriched with modified Con-way medium. In the control treatment, onlyseawater disinfected with chlorine (15 ppm)during 24 h and dechlorinated with ascorbicacid was used.

Artemia cysts (1 g/L) were stocked in 12cylindrical-conical tanks filled with 20 L seawa-ter previously disinfected according to the sameprocedure described for the control treatment.Temperature, salinity, dissolved oxygen, andpH were maintained at 29.9 ± 0.3 C, 27.7 ± 0.1g/L, 5.7 ± 0.1 mg/L, and 8.1 ± 0.03, respec-tively, as recommended by Van Stappen (1996).The Artemia hatching tanks were illuminated(2000 lx) and aerated continuously. Three repli-cates were used for each treatment. After 24h of incubation, the hatched nauplii were har-vested and counted. The nauplii hatching effi-ciency (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 treatmentswere collected to quantify presumptive Vibriocolony forming units (CFUs) using the agarthiosulfate bile sucrose (TCBS, agar Himedia)(Himedia Laboratories, Mumbai, India). Watersamples were serially diluted (1/10) in sterilesaline solution (2.5% NaCl). Aliquots of 0.1mL from three dilutions were spread plated onTCBS agar and incubated for 24 h at 30 C.After incubation, the total CFUs for plates thatpresented between 30 and 300 colonies wereenumerated (Downes and Ito 2001).

The samples of Artemia (2 g) were asep-tically macerated, diluted, and plated usingthe same methodology described for thewater analysis. Additionally, 2 g samples ofdifferent Artemia treatments were frozen at atemperature of −25 C for 48 h and then thebacterial load was determined. This analysisdetermined the influence of temperature onthe bacteria viability, as commercial hatcheriesoccasionally use frozen Artemia .

From the nauplii samples, different bacte-rial morphotypes grown on TCBS agar were

VIBRIO SPP. CONTROL IN ARTEMIA 67

isolated and subjected to presumptive identifi-cation tests for the Vibrio genus (Gram stain,detection of glucose anaerobic fermentation,and oxidase). According to Alsina and Blanch(1994), Vibrio spp. from environmental isolatesare Gram-negative, give a positive oxidase test,grow on TCBS medium, and are facultativeanaerobes. Further, the morphotypes were iden-tified through their biochemical profiles pre-sented in the commercial bacterial identificationkit API 20 E Biomerieux (Biomerieux Diagnos-tics, Rhone-Alpes, France).

Experiment II

Vibrio concentration was evaluated inArtemia enriched with three different supple-ments used in distinct treatments: C . calcitrans(microalgae treatment), commercial probiotic(probiotic treatment), and a commercial emul-sion rich in fatty acids, DC DHA Selco INVE(Selco treatment, Inve Aquaculture, Salt Lake,UT, USA). The control was represented bynewly hatched nauplii with no added supple-ments. Three replicates were used for eachtreatment.

Capsulated Artemia cysts were hatched underthe same conditions of the control in the firstexperiment. The newly hatched nauplii werewashed with chlorine-disinfected seawater andstocked (100–300 nauplii/mL) in 5-L contain-ers filled with seawater and the different supple-ments. Artemia were submitted to enrichmentprocess during 28 h under constant aeration.Average water temperature, salinity, pH, anddissolved oxygen were maintained between 27and 31 C, 28 and 30 g/L, 8.5 and 9.0, and 4.69and 6.65 mg/L, respectively.

The microalgae treatment received 8 × 105

cells/mL of C . calcitrans and the probiotictreatment received 2 × 105 CFU/mL of com-mercial probiotic (B. subtilis , B . pumilus , andB . licheniformis). The Selco treatment received0.3 g/L every 12 h (Merchie et al. 1995) of com-mercial emulsion rich in docosahexaenoic andeicosapentaenoic fatty acids that can also pro-vide Vibrio control. This emulsion is known asa disinfectant-appended enrichment diet (Liaoet al. 2001). The hatching and enrichment pro-cesses were repeated for 12 d.

PL Rearing

Litopenaeus vannamei PL in PL7 stage(7 d in the PL stage) with wet mean weight of0.843 mg were randomly stocked in 12 plasticcontainers (10 L) at a density of 50 PL/L.Water was previously disinfected with chlorine(15 ppm) for 24 h and then neutralized withascorbic acid.

Enriched Artemia from the four treatments(microalgae, probiotic, Selco, and control) wereoffered to shrimp PL feeding during 12 d (threereplicates each). Shrimp were fed twice daily(0700 and 1900 h) at an initial rate of 5nauplii/mL reaching 12 nauplii/mL at the endof rearing, according to the larvae consumption.

Shrimp PL were maintained under constantaeration at a temperature of 27–28 C. Daily,50% of water in the experimental units wasexchanged. Temperature, dissolved oxygen, pH,and salinity were monitored daily by a multipa-rameter sensor YSI 556 (Yellow Springs Instru-ment Company, Yellow Springs, OH, USA).

Bacterial Analysis

Presumptive Vibrio spp. were quantified insamples of enriched Artemia , water of Artemiaenrichment, shrimp PL, and water of PLrearing.

The procedure for Vibrio analysis in waterand Artemia and PL samples followed themethodology presented in the Experiment I.The PL samples were weighted, macerated, anddiluted (1/10) and 0.1 mL of three dilutionswere plated in TCBS agar, incubated, andenumerated as described previously.

Additionally, the colonization of bacteriafrom commercial probiotic was verified bythe quantification of Bacillus CFU in enrichedArtemia and shrimp PL from the probiotic treat-ment. The samples were weighted, macerated,and diluted (1/10) and 0.1 mL of three dilutionswere plated in MYP agar (Mannitol Egg YolkAgar Polymyxin; Himedia), incubated, and enu-merated.

Data Analysis

The HE data were submitted to analysisof variance (ANOVA) and Tukey’s test. The


Student’s t-test was used to compare the bac-terial count of Artemia natural biomass andfrozen biomass. Bacterial counts data were sub-mitted to ANOVA and Fisher tests. Differenceswere considered at the 5% significance level.All analyzes were performed using Statistica7.0. (Statsoft, Inc., Tulsa, OK, USA).

Results

Experiment I

The HE was similar among treatments andmean values (±SE) ranged from 1.9 ± 0.1 ×105 to 2.7 × 105 nauplii/g.

The decapsulation process was not effectivein reducing the presumptive Vibrio spp. loadin nauplii and water. Except for the antibi-otic treatment, the Vibrio load was similar(P > 0.05) between treatments with capsulateand decapsulated cysts (Table 1).

The number of bacterial colonies in waterwas significantly lower in the antibiotic fol-lowed by the microalgae treatment of capsu-lated cysts (Table 1). Only the probiotic treat-ment did not differ significantly from the con-trol.

In the trial with decapsulated cysts, lowerVibrio count was recorded in water fromthe antibiotic treatment but it did not differsignificantly from the microalgae (Table 1).Mean Vibrio counts in the control, microalgae,and probiotic treatments were not significantlydifferent.

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 sig-nificantly lower contamination in the antibioticand probiotic treatments (Table 1).

The freezing nauplii process during 48 hresulted in a reduction of 70.3 to 99.8% of theVibrio spp. load (Table 2).

Of the 43 bacterial colony morphotypesisolated from nauplii of capsulated cysts,including all treatments, 54% was identified asVibrio alginolyticus and 36% as Gram-negativerods oxidase-negative. Vibrio parahaemolyti-cus , Aeromonas hydrophila , Aeromonassalmonicida , Ochrobactrum anthropi , and aGram-negative rods oxidase-positive isolateeach represented 2% of the total isolates.Vibrio parahaemolyticus and O . anthropi wereresistant to Florfenicol.

From the nauplii of decapsulated cysts, atotal of 31 isolates were obtained. Of these,42% were identified as V . alginolyticus , 42% asGram-negative rods oxidase-negative, and 16%as Gram-positive isolates.

Experiment II

The Vibrio spp. concentrations in the Artemiaenrichment water were significantly lower inthe control (newly hatched nauplii) and didnot differ among other treatments (Table 3).In Artemia , the lowest value of bacterialcolonies was also observed in the control,but it did not differ from the microalgaeand probiotic treatments. The Selco treatmentpresented the highest level of contaminationdiffering significantly from the control.

Table 1. Average values (±SE) of presumptive Vibrio count in Artemia hatching water and Artemia nauplii(capsulated and decapsulated cysts) from different treatments.1

Vibrio count

Capsulated cysts Decapsulated cysts

Water (105 CFU/mL) Artemia (107 CFU/g) Water (105 CFU/mL) Artemia (107 CFU/g)

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

CFU = colony forming unit.1Different superscript letters in the same column indicate significant differences between treatments (P < 0.05).

Asterisks in the same line indicate significant differences between treatments (P < 0.05).


Table 2. Average values (±SE) of presumptive Vibrio count in Artemia nauplii (capsulated and decapsulated cysts)from different treatments, before and after freezing.1

Vibrio count

Capsulated cysts Decapsulated cysts

Before (107 CFU/g) After (107 CFU/g) Before (107 CFU/g) After (107 CFU/g)

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

CFU = colony forming unit.1Different superscript letters in a row indicate significant differences (P < 0.05) between before and after freezing.

Table 3. Average values (±SE) of presumptive Vibrio count in Artemia rearing water, Artemia, Litopenaeus vannameipostlarvae (PL), and PL rearing water from different treatments.1

Vibrio count

Artemia water (106 CFU/mL) Artemia (107 CFU/g) PL water (106 CFU/mL) PL (107 CFU/g)

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

CFU = colony forming unit.1Different superscript letters in the same column indicate significant differences between treatments (P < 0.05).

The Vibrio spp. concentration in the PLrearing water from the probiotic treatment wassignificantly lower than the control (Table 3).No significant differences were observed inthe number of bacterial colonies in PL amongtreatments (Table 3).

Bacillus quantification (±SE) in metanau-plius and PL from the probiotic treatment was8.7 ± 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 treat-ments.

Discussion

Bacterial enumeration in TCBS mediumshows that Artemia hold a high number ofbacteria. According to Verdonck et al. (1994),Artemia are usually highly contaminated withbacteria (>107 CFU/g) and mostly identified asVibrio spp. Moreover, it is necessary to control

these bacterial loads before the use of Artemiain culture systems.

The use of Florfenicol resulted in the lowestVibrio spp. load among all the supplementstested in Artemia hatchery. The Florfenicolhas been authorized in several countries foraquaculture activities (FAO 2005). In Brazil, itis the only antibiotic registered for this purposein the Ministry of Agriculture, Livestock andSupply (Schering Plough Animal Health 2009).In fish farming, Florfenicol has potent activityagainst a broad range of pathogens (Samuelsenet al. 2003), including microorganisms resistantto other antibiotics (Nordmo et al. 1994; Rang-dale et al. 1997; Bruun et al. 2000; Thyssenand Ollevier 2001; Vue et al. 2002; Samuelsenand Bergh 2004). Our findings suggested thatthe Florfenicol dose (300 mg/L) was efficientin reducing Vibrio counts but, some potentiallypathogenic strains of Vibrio (V . alginolyticusand V . parahaemolyticus) remained in Artemianauplii. The proliferation of these resistantbacteria could make possible infections moredifficult to treat in hatchery systems.


The microalgae treatment was the secondmost efficient in reducing Vibrio load in waterand nauplii (capsulated). This effect may berelated to the bacteriostatic or bactericidalmicroalgae activity (Kellam and Walker 1989;Olsen et al. 2000). The microalgae antibacte-rial activity has been detected in microalgaeextracts (Duff and Bruce 1966; Austin and Day1990; Austin et al. 1992; Tendencia and delaPena 2003) and it may be related to the asso-ciated microflora, antimicrobial proteins, fattyacids, and oxygen free radicals produced bymicroalgae cells (Marshall et al. 2005; Makridiset al. 2006; Kokou et al. 2007).

Despite newly hatched Artemia nauplii areunable to bioencapsulate, probiotic bacteria canbe active in the gills and body surface bycompeting with other bacteria for adhesion sites(Gatesoupe 1991; Verschuere et al. 2000). Thisstudy has shown that probiotics reduced theVibrio load in Artemia from capsulated anddecapsulated cysts.

Freezing Artemia for 48 h reduced the Vibriocounts, but most values remained over 107

CFU. Reviewing the responses of mesophilicbacteria to cold stress, Panoff et al. (1998) listedthe membrane damage and DNA denaturationas possible causes of bacterial death afterfreezing and thawing. Sensitive bacterial cellsusually undergo a slow death rate upon freezingand their response can be described as anexponential function (Haines 1938; Panoff et al.1998). Alur and Grecz (1975) reported higherrates of DNA fragmentation after fast freezingrather than slow freezing. However, after 24h storage, the slow frozen cells yielded thesame results as fast frozen cells. The authorssuggested that death was related to DNA andmembrane degradation as DNA is attached toplasma membrane.

It has been suggested that Vibrio spp. arecapable of entering into a viable but non-culturable (VBNC) state when exposed to lowtemperatures (Jiang and Chai 1996; Johnsonand Brown 2002). Eventually, when temper-ature rises, bacterial cells are able to emergefrom the VBNC state and become culturableon bacteriological media. Thus, offering short-term frozen stored Artemia to shrimp larvae

may not reduce the potential of Vibrio spp. con-tamination, as cold-induced death of bacteriaonly occur after several days to weeks (Oliver1981).

The sodium hypochlorite used in the decap-sulation process is able to totally decontami-nate Artemia cysts, but they can be quicklyrecolonized during the rupture stage beforehatching (Sorgeloos et al. 2001). At this stage,the organic substrate glycerol is released fromthe cysts and offers an ideal culture mediumfor Vibrio spp. In this study, the decapsula-tion process did not effectively reduced Vibrioconcentration, but decreased potential bacte-rial pathogens species in nauplii and may beregarded as an auxiliary prophylactic treatment.

Hoj et al. (2009) characterized the bac-terial community present in Artemia , withmore than half of Vibrio isolates being iden-tified as V . alginolyticus . Lopez-Torres andLizarraga-Partida (2001) observed that evenwhen Artemia cysts were hatched under sterileconditions, V . alginolyticus was the dominantspecies. These authors suggested that V . algi-nolyticus and Vibrio spp. isolated from Artemiahatching tanks were associated with those iso-lated from tanks with zoea, mysis, and PL,indicating that these Vibrio spp. remain in dif-ferent development phases of shrimp hatchery.Buglione et al. (2010) observed that V . algi-nolyticus strain caused high mortality of L. van-namei larvae. The V . alginolyticus virulenceis correlated to enzyme collagenase activity,which can cause softening of shrimp muscletissue (Brauer et al. 2003; Yishan et al. 2011).

The presence of V . parahaemolyticus insamples of shrimp submitted to the antibiotictreatment was reported by Verschuere et al.(2000). Likewise, this bacteria was identifiedin Artemia treated with antibiotic (Florfenicol)in our study. According to Gomez-Gil et al.(2004), V . parahaemolyticus affects mainlyshrimps at juvenile and adult stage. Ochrobac-trum anthropi was also resistant to the Flor-fenicol treatment. Although this species has notbeen related to shrimp diseases, it has beenisolated from fresh and cryopreserved sper-matophores of the giant tiger prawn, Penaeusmonodon (Nimrat et al. 2008), and water


samples from mangrove receiving shrimp farmeffluents (Sousa et al. 2006). In another study,O. anthropi was isolated and identified by phy-logenetic analysis using 16S rDNA sequencesfrom inhabited marine biofilms. Such bacteriacan colonize Artemia and L. vannamei oncethat it has been observed in the marine environ-ment and furthermore, biofilms cover most sub-tidal and intertidal solid surfaces such as rocks,ships, loops, marine animals, and algae (Leeet al. 2003). Ochrobactrum anthropi was alsoisolated from blue crab body meat, Callinectessapidus (Ozogul et al. 2010), and intestinalsamples of zebra fish, Danio rerio (Cantas et al.2012).

Aeromonas spp. compose the normal micro-flora of wild and reared crustaceans and can beconsidered an opportunistic pathogen (Lightner1993). This genus has been associated to thesoft shell syndrome in P. monodon (Baticadoset al. 1986; Uddin et al. 2008).

The Artemia enrichment process increasedbacterial load in water and Artemia speciallyin the Selco treatment. This could be explainedby the organic input from supplements andArtemia excretion, which allows a suddenincrease of opportunistic bacteria (Igarashi et al.1989; Skjermo and Vadstein 1993; Verschuereet al. 1997; Olsen et al. 2000). Hoj et al. (2009)and Hache and Plante (2011) also observedhigh bacterial load in Artemia enriched withmicroalgae and lipid emulsions in combination.

The bacterial load in shrimp PL and rearingwater were not associated with the concentra-tions of Vibrio observed in newly hatched nau-plii (control) and enriched Artemia . The dailyrenewal (50%) of PL rearing water probablyreduced the abundance of Vibrio spp. Krish-nika and Ramasamy (2012) also recorded asignificant reduction of Vibrio after the waterexchange in Artemia rearing tanks. Silva et al.(2013) observed a presumptive Vibrio load of7.9 × 107 CFU/g in L. vannamei PL (PL10) fednewly hatched Artemia nauplii. In this study,this load was slightly lower for PL19 (0.17 ×107 CFU/g).

Overall, results of this study indicated thataddition of C . calcitrans in Artemia hatchingwater is an effective alternative to antibiotics.

Additionally, the use of probiotic must also beconsidered to control the Vibrio spp. load inArtemia nauplii. The enrichment supplementsincreased the bacterial load in Artemia butthey did not affect Vibrio concentration inshrimp PL.

Acknowledgments

This study was supported by the Improve-ment of Higher Education Personnel Coor-dination (CAPES) and the Brazilian Councilfor Scientific and Technological Development(CNPq).

Literature CitedAlsina, M. and A. R. Blanch. 1994. A set of keys

for biochemical identification of environmentalVibrio species. Journal of Applied Microbiology76:79–85.

Alur, M. D. and N. Grecz. 1975. Mechanism of injuryof Escherichia coli by freezing and thawing. Bio-chemical and Biophysical Research Communications62:308–312.

Austin, B. and J. G. Day. 1990. Inhibition of prawnpathogenic Vibrio spp. by a commercial spray-dried preparation of Tetraselmis suecica . Aquaculture90:389–392.

Austin, B., E. Baudet, and M. Stobie. 1992. Inhibitionof bacterial fish pathogens by Tetraselmis suecica .Journal of Fish Diseases 15:55–61.

Baticados, C. L., R. M. Coleso, and R. S. Duremdez.1986. Studies on the chronic soft shell syndrome inthe tiger prawn, Penaeus monodon Fabricius frombrackish water ponds. Aquaculture 56:271–285.

Baticados, M. C. L., C. R. Lavilla-Pitogo, E. R. Cruz-Lacierda, L. D. dela Pena, and N. A. Sunaz.1990. Studies on the chemical control of luminousbacteria Vibrio harveyi and V . splendidus isolatedfrom diseased Penaeus monodon larvae and rearingwater. Diseases of Aquatic Organisms 9:133–139.

Brauer, J. M. E., J. A. S. Leyva, L. B. Alvarado,and O. R. Sandez. 2003. Effect of dietary proteinon muscle collagen, collagenase and shear force offarmed white shrimp, (Litopenaeus vannamei ). Euro-pean Food Research and Technology 217:277–280.

Bruun, M. S., A. S. Schmidt, L. Madsen, and I.Dalsgaard. 2000. Antimicrobial resistance patternsin Danish isolates of Flavobacterium psychrophilum .Aquaculture 187:201–212.

Buglione, C. C., F. N. Vieira, J. L. P. Mourino, F.S. Pedrotti, A. Jatoba, and M. L. Martins. 2010.Experimental infection with different bacterial strainsin larvae and juvenile Litopenaeus vannamei reared


in Santa Catarina State, Brazil. Acta ScientiarumBiological Sciences Maringa 32:291–296.

Campbell, R., A. Adams, M. Tatner, M. Chair, andP. Sorgeloos. 1993. Uptake of Vibrio anguillarumvaccine by Artemia salina as a potential oral deliverysystem to fish fry. Fish and Shellfish Immunology3:451–459.

Cantas, L., J. R. T. Sørby, P. Alestrom, and H. Sørum.2012. Culturable gut microbiota diversity in zebrafish.Zebrafish 9:26–37.

Dixon, B. A., S. O. Vanpoucke, M. Chair, M. Dehasque,H. J. Nelis, P. Sorgeloos, and A. P. De Leen-heer. 1995. Biocapsulation of the antibacterial drugsarafloxacin in nauplii of brine shrimp, Artemia fran-ciscana . Journal of Aquatic Animal Health 7:42–45.

Downes, F. P. and K. Ito, editors. 2001. Compendium ofmethods for the microbiological examination of foods.American Public Health Association, Washington,D.C., USA.

Duff, D. C. B. and D. L. Bruce. 1966. The antibacterialactivity of marine planktonic algae. Canadian Journalof Microbiology 12:877–884.

FAO (Food and Agriculture Organization of the UnitedNations). 2005. Responsible use of antibiotics inaquaculture. FAO Fisheries Technical Paper, Rome,Italy.

Garcia, A. S., C. C. Parrish, and J. A. Brown.2008. Growth and lipid composition of Atlantic cod,(Gadus morhua) larvae in response to differentlyenriched Artemia franciscana . Fish Physiology andBiochemistry 31:77–94.

Gatesoupe, F. J. 1991. Managing the dietary value ofArtemia for larval turbot, Scophthalmus maximus;the effect of enrichment and distribution techniques.Aquacultural Engineering 10:111–119.

Gomez-Gil, B., F. L. Thompson, C. C. Thompson, A.Garcia-Gasca, A. Roque, and J. Swings. 2004. Vib-rio hispanicus sp. nov., isolated from Artemia sp. andsea water in Spain. International Journal of Systematicand Evolutionary Microbiology 54:261–265.

Hache, R. and S. Plante. 2011. The relationship betweenenrichment, fatty acid pro files and bacterial loadin cultured rotifers, (Brachionus plicatilis L-strain)and Artemia (Artemia salina strain Franciscana).Aquaculture 311:201–208.

Haines, R. B. 1938. The effect of freezing bacteria.Proceedings of the Royal Society B 124:451–463.

Hoj, L., D. G. Bourne, and M. R. Hall. 2009. Local-ization, abundance and community structure of bac-teria associated with Artemia: effects of naupliienrichment and antimicrobial treatment. Aquaculture293:278–285.

Igarashi, M. A., H. Sugita, and Y. Deguchi. 1989.Microflora associated with eggs and nauplii of Artemiasalina . Nippon Suisan Gakkaishi 55:2045.

Jiang, X. and T. Chai. 1996. Survival of Vibrio para-haemolyticus at low temperature under starvation con-ditions and subsequent resuscitation of viable, noncul-turable cells. Applied and Environmental Microbiol-ogy 62:1300–1305.

Johnson, M. D. and M. H. Brown. 2002. An investi-gation into the changed physiological state of Vibriobacteria as a survival mechanism in response to coldtemperature and studies on their sensitivity to heat-ing and freezing. Journal of Applied Microbiology92:1–12.

Kellam, S. J. and J. M. Walker. 1989. Antibacterialactivity from marine microalgae in laboratory culture.British Phycological Journal 24:191–194.

Kokou, F., T. Ferreira, C.S. Tsigenopoulos, P. Makridis,G. Kotoulas, A. Magoulas and P. Divanach. 2007.Identification of bacteria growing in association withbatch cultures of the microalgae Chlorella minutis-sima . Page 105 in 8th International Marine Biotech-nology Conference, Eilat, Israel.

Krishnika, A. and P. Ramasamy. 2012. Effect ofwater exchange to eliminate Vibrio sp. during thenaupliar development of Artemia franciscana . Journalof Fisheries and Aquatic Science 7:205–214.

Lavilla-pitogo, C. R., M. C. L. Baticados, E. R. Cruz-Lacierda, and L. D. de la Pena. 1990. Occurrence ofluminous bacterial disease of Penaeus monodon larvaein the Philippines. Aquaculture 91:1–13.

Lee, Y. K., K. Kwon, K. H. Cho, H. W. Kim,J. H. Park, and H. K. Lee. 2003. Culture andidentification of bacteria from marine biofilms. Journalof Microbiology 41:183–188.

Leger, P., D. A. Bengton, P. Sorgeloos, K. L. Simp-son, and A. D. Beck. 1987. The nutritional value ofArtemia: a review. Pages 357–372 in P. Sorgeloos,D. A. Bengtson, W. Decleir, and E. Jaspers, editors.Artemia Research and its Applications. Ecology, Cul-turing, Use in Aquaculture. Universa Press, Wetteren,Belgium.

Liao, I. C., H. M. Su, and E. Y. Chang. 2001. Tech-niques in finfish larviculture in Taiwan. Aquaculture200:1–31.

Lightner, D. V. 1993. Diseases of cultured penaeid shrimp.Pages 393–486 in J. P. Mc Vey, editor. Handbookof mariculture. CRC press Inc., Boca Raton, Florida,USA.

Lightner, D. V. and D. H. Lewis. 1975. A septicemiabacterial disease syndrome in penaeid shrimp. MarineFisheries Review 37:25–28.

Lopez-Torres, M. A. and M. L. Lizarraga-Partida.2001. Bacteria isolated on TCBS media associatedwith hatched Artemia cysts of commercial brands.Aquaculture 194:11–20.

Makridis, P., R. Alves Costa, and M. T. Dinis. 2006.Microbial conditions and antimicrobial activity incultures of two microalgae species. Tetraselmis chuiiand Chlorella minutissima , and effect on bacterialload of enriched Artemia metanauplii. Aquaculture255:76–81.


Marshall, J. A., M. De Salas, T. Oda, and G. Hallegraef.2005. Superoxide production by marine microalgae-I.Survey of 37 species from 6 classes. Marine Biology147:533–540.

Merchie, G., P. Lavens, J. Radull, H. Nelis, A. DeLeenheer, and P. Sorgeloos. 1995. Evaluation ofvitamin C – enriched Artemia nauplii for larvae ofthe giant freshwater prawn. Aquaculture International3:355–363.

Munro, P. D., A. Barbour, and T. H. Birkbeck. 1994.Comparison of the gut bacterial flora of start-feedinglarval turbot reared under different conditions. Journalof Applied Microbiology 77:560–566.

Munro, P. D., R. J. Henderson, A. Barbour, and T. H.Birkbeck. 1999. Partial decontamination of rotiferswith ultraviolet radiation: the effect of changes in thebacterial load and flora of rotifers on mortalities instart-feeding larval turbot. Aquaculture 170:229–244.

Navarro, J. C., J. R. Henderson, L. McEvoy, M. V.Bell, and F. Amat. 1999. Lipid conversions duringenrichment of Artemia . Aquaculture 174:155–166.

Nicolas, J. L., E. Robic, and D. Ansquer. 1989. Bac-terial flora associated with a trophic chain consist-ing of microalgae, rotifers and turbot larvae: influ-ence of bacteria on larval survival. Aquaculture83:237–248.

Nimrat, S., A. N. Bart, A. Keatsaksit, and B.Vuthiphandchai. 2008. Microbial flora of sper-matophores from black tiger shrimp, (Penaeusmonodon) declines over long-term cryostorage.Aquaculture 274:247–253.

Nordmo, R., K. J. Varma, I. H. Sutherland, and E.S. Brokken. 1994. Florfenicol in Atlantic salmon,Salmo salar L. Field evaluation of efficacy againstfurunculosis in Norway. Journal of Fish Diseases17:239–244.

Oliver, J. D. 1981. Lethal cold stress of Vibrio vulnificusin oysters. Applied and Environmental Microbiology41:710–717.

Olsen, A. I., Y. Olsen, Y. Attramadal, K. Christie,T. H. Birkbeck, J. Skjermo, and O. Vadstein.2000. Effects of short term feeding of microalgae onthe bacterial flora associated with juvenile Artemiafranciscana . Aquaculture 190:11–25.

Ozogul, F., E. Kuley, and Y. Ozogul. 2010. Usefulnessof API test strips for identification of bacterialflora in blue crab (Callinectes sapidus) caught fromakyatan lagoon (Adana-Turkey). Journal of FisheriesSciences.com 4:1–7.

Panoff, J. M., B. Thammavongs, M. Gueguen, andP. Boutibonnes. 1998. Cold stress responses inmesophilic bacteria. Cryobiology 36:75–83.

Planas, M. and I. Cunha. 1999. Larviculture ofmarine fish: problems and perspectives. Aquaculture177:171–190.

Rangdale, R. E., R. H. Richards, and D. J. Alderman.1997. Minimum inhibitory concentrations of selected

antimicrobial compounds against Flavobacterium psy-chrophilum the causal agent of rainbow trout fry syn-drome (RTFS). Aquaculture 158:193–201.

Roiha, I. S., E. Otterlei, and O. B. Samuelsen.2010. Bioencapsulation of florfenicol in brine shrimp,Artemia franciscana nauplii. Journal of Bioanalysisand Biomedicine 2:60–64.

Samuelsen, O. and O. Bergh. 2004. Efficacy of orallyadministered florfenicol and oxolinic acid for the treat-ment of vibriosis in cod, (Gadus morhua). Aquaculture235:27–35.

Samuelsen, O. B., O. Bergh, and A. Ervik. 2003.Pharmacokinetics of florfenicol in cod, Gadusmorhua and in vitro antibacterial activity againstVibrio anguillarum . Diseases of Aquatic Organisms56:127–133.

Schering Plough Animal Health. 2009. Aquaflor. Flor-fenicol. Technical Monograph for catfish health profes-sionals. Schering Plough Animal Health Corp., Sum-mit, New Jersey, USA.

Silva, E. F., M. A. Soares, N. F. Calazans, J. L.Vogeley, B. C. do Valle, R. Soares, and S. Peixoto.2013. Effect of probiotic (Bacillus spp.) additionduring larvae and postlarvae culture of the whiteshrimp, Litopenaeus vannamei . Aquaculture Research44:13–21.

Skjermo, J. and O. Vadstein. 1993. Characterization of thebacterial flora of mass cultivated Brachionus plicatilis .Hydrobiologia 255:185–191.

Skjermo, J. and O. Vadstein. 1999. Techniques formicrobial control in the intensive rearing of marinefish larvae. Aquaculture 177:333–343.

Sorgeloos, P., P. Dhert, and P. Candevra. 2001. Use ofbrine shrimp, Artemia spp. in marine fish larviculture.Aquaculture 200:147–159.

Sousa, O. V., A. Macrae, F. G. R. Menezes, N. C. M.Gomes, R. H. S. F. Vieira, and L. C. S. Mendonca-Hagler. 2006. The impact of shrimp farming effluenton bacterial communities in mangrove waters, Ceara,Brazil. Marine Pollution Bulletin 52:1725–1734.

Tendencia, E. A. and M. dela Pena. 2003. Investigationof some components of the greenwater system whichmakes it effective in the control of luminous bacteria.Aquaculture 218:115–119.

Thyssen, A. and F. Ollevier. 2001. In vitro antimicro-bial susceptibility of Photobacterium damselae subsp.piscicida to 15 different antimicrobial agents. Aqua-culture 200:259–269.

Uddin, S. A., M. Zafar, A. S. M. Noman, and A.Sharmin. 2008. Isolation and Identification of Vibriospp. and Aeromonas spp. in wild and hatchery rearedPenaeus monodon postlarvae from Cox’s Bazar,Bangladesh. Pakistan Journal of Marine Sciences17:161–167.

Van Stappen, G. 1996. Introduction, biology and ecologyof Artemia . In P. Lavens and P. Sorgeloos, editors.Manual on the production and use of live food foraquaculture. FAO Fisheries Technical paper, Rome,Italy.


Verdonck, L., L. Grisez, E. Sweetman, G. Minkoff,P. Sorgeloos, F. Ollevier, and J. Swings. 1994.Variability of the microbial environment of rotifer,Brachionus plicatilis and Artemia production systems.Journal of World Aquaculture Society 25:55–59.

Verschuere, L., J. Dhont, P. Sorgeloos, and W. Ver-straete. 1997. Monitoring biology patterns and r/K-strategists in the intensive culture of Artemia juveniles.Journal of Applied Microbiology 83:603–612.

Verschuere, L., G. Rombaut, P. Sorgeloos, and W. Ver-straete. 2000. Probiotic bacteria as biological controlagents in aquaculture. Microbiology and MolecularBiology Reviews 64:655–671.

Vue, C., L. J. Schmidt, G. R. Stehly, and W. H. Gin-gerich. 2002. Liquid chromatographic determinationof florfenicol in the plasma of multiple species of fish.Journal of Chromatography B 780:111–117.

Witte, W., I. Klare, and G. Werner. 1999. Selectivepressure by antibiotics as feed additives. Infection27:35–38.

Yishan, L., F. Jiaming, W. Zaohe, and J. Jichang.2011. Genotype analysis of collagenase gene byPCR-SSCP in Vibrio alginolyticus and its associationwith virulence to marine fish. Current Microbiology62:1697–1703.

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Vibrio spp. Control at Brine Shrimp, Artemia , Hatching and Enrichment