Clinical observations of black disease in fairy shrimps,

Streptocephalus sirindhornae and Branchinella

thailandensis, from Thailand and pathogen verification

C Saejung1, K Hatai2, S Wada2, O Kurata2 and L Sanoamuang1,3

1 Applied Taxonomic Research Center, Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen,

Thailand

2 Laboratory of Fish Diseases, Nippon Veterinary and Life Science University, Tokyo, Japan

3 Faculty of Science, Mahasarakham University, Mahasarakham, Thailand

Abstract

In this study, black disease infecting fairy shrimps,Streptocephalus sirindhornae Sanoamuang, Mur-ugan, Weekers & Dumont, and Branchinella thai-landensis Sanoamuang, Saengphan & Murugan, inThailand, was investigated. The typical signs of thedisease are the appearance of black spots on thecuticle, located mainly on the dorsal side andthoracopods. A number of rod-shaped bacteriaaggregated in the black spots and were visualized byscanning electron microscopy. The histopathologi-cal results showed that a haemocytic response to theinfection resulted in a dense melanized core ofbacteria. In addition, generalized septicaemia byrod-shaped bacteria was also observed in the in-fected tissue. Of the 31 isolates, Aeromonas spp.were predominantly isolated and six strains wereselected for the experimental infections. The mostpathogenic strain was identified molecularly asA. hydrophila. When fairy shrimp were infected atbacterial concentrations of 104 and 106 cfu mL)1,the overall infection levels were 73.33 � 6.67%and 93.33 � 6.67%, respectively. The experimen-tally infected fairy shrimp showed abnormalswimming and died within 24–48 h after theappearance of the dark pigment.

Keywords: Aeromonas spp., black spot, Branchinellathailandensis, fairy shrimp, haemocytic encapsula-tion, Streptocephalus sirindhornae.

Introduction

Fairy shrimp are freshwater anostracans, which aresourcesof carotenoidpigments includingastaxanthin,canthaxanthin, antheraxanthin, lutein, b-cryptoxan-thin, violaxanthin (Velu & Munuswamy 2003;Sriputhorn & Sanoamuang 2011) and fatty acids(Mura,Ferrana,Fabietti,Delise&Bocca1997;Mura,Zarattini, Delise, Fabietti & Bocca 1999; Mura,Zarattini, Delise, Fabietti & Bocca 2000). Recently,the fairy shrimps, Streptocephalus sirindhornae Sano-amuang, Murugan, Weekers & Dumont andBranchinella thailandensis Sanoamuang, Saengphan& Murugan, have been discovered from temporaryponds in Thailand (Sanoamuang, Murugan, Wee-kers & Dumont 2000; Sanoamuang, Saengphan &Murugan 2002). They have been reared as a source oflive food for ornamental fish within Thailand(Dararat, Starkweather & Sanoamuang 2011) andhave been exported to Hong Kong. Although thecultivation of fairy shrimp has been developed, theproductivity is still limited. The presence of blackspots on the infected cuticle, i.e. black disease, is amajor factor limiting their cultivation. The appear-ance of the disease found on fairy shrimp in Thailandis similar to that found in other countries includingSpain, Algeria, Germany, America, Belgium (Dierc-kens, Vandenberghe, Beladjal, Huys, Mertens &Swings 1998) and Japan (Nambu, Tanaka & Nambu2007). The disease is considered to be a cause ofmortality in fairy shrimp and also has an adverseeffect on commercial cultivation. However, there islittle reference to black disease affecting fairy shrimpin the literature and the cause remains unclear.

Journal of Fish Diseases 2011, 34, 911–920 doi:10.1111/j.1365-2761.2011.01314.x

Correspondence Dr L Sanoamuang, Applied Taxonomic

Research Center, Khon Kaen University, Khon Kaen, Thailand

(e-mail: la_orsri@kku.ac.th)

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Hence, in the present study, the clinical signs of fairyshrimps, S. sirindhornae and B. thailandensis, withblack disease were evaluated and the bacterialpathogen was investigated by experimental infection.

Materials and methods

Sample collection

Fairy shrimps, S. sirindhornae and B. thailandensis,naturally infected with black disease were collectedfrom earthen ponds and the experimental station ofthe Applied Taxonomic Research Center, Faculty ofScience, Khon Kaen University from June 2009 toMay 2010. To determine the prevalence of infec-tion during each month, fairy shrimp were sampledrandomly 1–2 times a month. The prevalence ratewas calculated by dividing the number of infectedfairy shrimp by the total number of shrimp in thecollected samples expressed as a percentage. Thenumber of shrimp collected in samples each monthwas different because of the availability of shrimp.The rate of infection was also categorized into threeseasons (rainy, winter and summer). Infected bodyparts from diseased fairy shrimps were used forelectron microscopic examination, histopathologyand bacterial isolation.

Scanning electron microscopic (SEM)examination

Fairy shrimp with black disease were preserved in4% formalin solution. The lesions were dissectedbefore dehydration in a graded alcohol series and100% amyl acetate. The specimens were criticalpoint dried, mounted on a stub and sputter-coatedwith gold. SEM micrographs were taken using aLEO 1450VP (LEO Electron Microscopy Ltd).

Histopathology of infected fairy shrimp

Diseased fairy shrimp were fixed in 10% phosphate-buffered formalin solution. The samples were rou-tinely processed, embedded in paraffin, sectioned at5 lm and sections stained with haematoxylin andeosin (H&E) (Wada, Nakamura & Hatai 1995).

Isolation of bacteria

Moribund fairy shrimps, S. sirindhornae andB. thailandensis, were used for bacterial isolation.After they were cleaned using sterile distilled water

and sterile normal saline solution, the infected partswere inoculated onto trypticase soy agar (TSA;HiMedia Laboratories Pvt Ltd) and incubated at25 �C for 48 h, then different colonies werepurified. All isolates were frozen at )70 �C in20% (v/v) glycerol for long-term storage.

Phenotypic characterization of the isolatedbacteria

All isolates were biochemically identified to thegenus level according to Holt, Krieg, Sneath, Staley& Williams (1994) and Graevenitz (1985).

Experimental infection of fairy shrimp

Branchinella thailandensis used in this experimentwere cultivated in the laboratory. Prior to theexperiment, they were fed once a day with an excessof axenic cultures of Chlorella sp. The water in theplastic basin was partially exchanged (40–50%)every day, and shrimp faeces were siphoned daily tomaintain the water quality. A visible light intensityof approximate 1140 lux was maintained for thisstudy. The pH and temperature of the water were7.2 and 26 �C, respectively. The fairy shrimp werecultured until they reached maturation (approxi-mately 14–20 days). One hundred and ninety-fivehealthy fairy shrimp were selected for the experi-mental infection. They were washed three timeswith sterile dechlorinated tap water and thencarefully transferred to 39 Erlenmeyer flasks con-taining sterile dechlorinated tap water (five indi-viduals/flask). They were acclimatized for 1–2 h(Puente, Vega-Villasante, Holguin & Bashan1992). To avoid contamination, air was pumpedinto the flasks via polyethylene filters with 5- and0.3-lm-pore sizes. The aeration was adjusted byvalves.

Bacterial strains, Aeromonas C03, AeromonasC24, Aeromonas C26, Aeromonas C29, AcinetobacterC17 and Corynebacterium C16 were used. Suspen-sions were obtained by growing the strains intrypticase soy broth and centrifuging at relativecentrifugal force ¼ 4,427 g at 4 �C for 20 min.Bacterial cells were re-suspended in sterile normalsaline solution (0.85% NaCl), and the concentra-tion was adjusted using a 0.5 McFarland standard.

Healthy fairy shrimp were experimentally in-fected with re-suspended cells of the selectedbacterial strains to give a final concentration of104 (low dose) and 106 (high dose) cfu mL)1. The

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bacterial concentration was enumerated by the platecount technique. Normal saline solution was usedas a control. All treatments were conducted intriplicate. Axenic cultures of Chlorella sp. were usedas food. The fairy shrimp were then exposed tobacterial strains for 5 days (Dierckens et al. 1998)and their behaviour was observed daily. Infectedparts of diseased shrimp were examined andbacteria re-isolated after the experimental period.All procedures were carried out aseptically, and theexperiment was repeated twice.

Statistical analyses

Pathogenicity of all isolates was analysed statisti-cally. Descriptive statistics, including means andstandard error (SE), were calculated by SPSS withsignificance values given at P < 0.05.

Molecular identification of the pathogen

Bacteria showing pathogenicity from the experi-mental infection were characterized by molecularmethods. The strains used were Aeromonas C03,Aeromonas C24, Aeromonas C26, Aeromonas C29and Acinetobacter C17. All strains were grown onheart infusion agar (Nissui). Genomic DNA wasobtained by the modified method of Weerakhun,Aoki, Kurata, Hatai, Nibe & Hirae (2007); 1 lL ofthe sample was used. Amplification was carried outin a PCR thermal cycler (Takara Biomedicals) usingthe forward primer 20F (AGAGTTTGATC-MTGGCTCAG) and the reverse primer 1500R(GGTTACCTTGTTACGACTT). The conditionswere as follows: initial denaturation for 5 min at98 �C; 30 cycles of 10 s at 98 �C, 5 s at 55 �C and1.3 min at 72 �C; and a final extension of 5 min at72 �C. A negative control (no template DNA) wasincluded in the PCR. The PCR products wereanalysed by 0.8% agarose gel electrophoresis, and a500-bp DNA molecular marker was included ineach electrophoresis (EZ Load�; BIO-RAD). Theelectrophoresis bands were visualized and photo-graphed under ultraviolet (UV) illumination (FASIII; Toyobo). Subsequently, 20 lL of each PCRproduct was purified using the QIAquick GelExtraction Kit (Qiagen). The PCR productsequencing was performed using the Big DyeTerminator v1.1 Cycle Sequencing Kit (AppliedBiosystems) and an automated DNA sequencer(ABI Prism 3100 Genetic Analyzer) according tostandard manufacturer�s protocols. The DNA

sequences of all bacteria were compared with thosefrom the DNA Data Bank of Japan (DDBJ).

Results

Clinical signs

Black disease was characterized by the appearance ofblack spots. The infected areas were localizedmainly on the dorsal side of the body andthoracopods (Fig. 1). After the development ofthe black spots, abnormal swimming was observedin the moribund fairy shrimp and they diedwithin 5–7 days. Scanning electron micrographsof S. sirindhornae and B. thailandensis withblack disease are shown in Fig. 2. Rod-shapedbacteria were found in the lesions of both species(Fig. 2a,b), but on the cuticle of healthy fairyshrimp (Fig. 2c).

Histopathological examination

Rod-shaped bacteria were found in the black spotswhen observed microscopically. Histopathologydemonstrated that haemocytes were found aroundthe lesion and bacteria were encapsulated (Fig. 3a).Generalized septicaemia caused by bacteria was alsoobserved in the thoracopods and body cavity(Fig. 3b).

Seasonal infection of fairy shrimp

The prevalence of infection of black disease foundin S. sirindhornae and B. thailandensis is presentedin Table 1. The data indicate that S. sirindhornaehad slightly greater levels of infection than B. thai-landensis. The combined investigation (from June2009 to May 2010) showed that the prevalence ofinfection of S. sirindhornae and B. thailandensis was41.1 and 30.5%, respectively. The highest infectionand mortality rates of both species were foundduring November 2009–January 2010, which is thecoldest period (water temperatures 20–26 �C) inThailand. The prevalence of infection in S. sirind-hornae and B. thailandensis ranged from 46.7 to72.2 and 50 to 67.9, respectively. In the rainyseason (June–October 2009, water temperatures26–30 �C), fairy shrimp were infected at mild tomoderate prevalences (10–36.7%). However, thehighest prevalence rate of 64.6% in S. sirindhornaewas observed in June 2009 because of the heavyrainfall at that time. On the other hand, diseased

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fairy shrimp were rarely collected in summer(February–May 2010, water temperatures 27–34 �C) and the prevalence of infection was verylow (0–5%). The prevalence of infection in differ-ent seasons is summarized in Fig. 4.

Phenotypic characterization of the isolatedbacteria

Bacteria were isolated from the blackened areas ofaffected shrimp and classified according to Bergey�sManual of Determinative Bacteriology. Of 31isolates, 20, 5 and 6 isolates were identified asAeromonas sp., Acinetobacter sp. and Corynebacte-rium sp., respectively. The population of isolatedbacteria predominantly consisted of Aeromonas spp.The members of this genus were motile and ureasenegative, but positive for catalase, oxidase, gelati-nase, nitrate reduction and in the oxidation andfermentation test (OF). The biological and bio-chemical characteristics of Aeromonas spp. isolatedfrom fairy shrimp with black disease are summa-rized in Table 2.

Experimental infection of fairy shrimp

The effect of infection was evident 24–72 h afterthe first inoculation. Atypical swimming could beobserved in all infected fairy shrimp. Mortality wasseen within 1–2 days after the appearance of theblack spots in the fairy shrimp infected with bothbacterial concentrations. The clinical signs of theexperimentally infected fairy shrimp were identical

to those with natural infections. Gram-negativerod-shaped bacteria were re-isolated from the blackparts and they were biochemically identified as theoriginal strains. However, no lesions were observedin the control group. The black spots resemblingdense melanin surrounding bacteria are shown inFig. 5.

Only fairy shrimp with black spots on the cuticlewere evaluated. The data was analysed statisticallyby ANOVA, using SPSS and were expressed asmean � SE. Experimental infection of healthy fairyshrimp with Aeromonas C03, Aeromonas C24,Aeromonas C26, Aeromonas C29 and AcinetobacterC17 resulted in a high pathogenicity. The highestpathogenicity was observed with Aeromonas C03,the cumulative percentages of infected fairy shrimpat bacterial concentrations of 104 and 106cfu mL)1

were 73.3 � 6.7% and 93.3 � 6.7%, respectively.There were no differences between the mean valuesof pathogenicity of Aeromonas C29 at both con-centrations. Comparing least significant differencestatistics of the pathogenicity of each strain, Aero-monas C03, Aeromonas C26 and Aeromonas C29did not show any significant differences in patho-genicity (P > 0.05), whereas the pathogenic effectof Aeromonas C03 was significantly different fromAeromonas C24 and Acinetobacter C17 (P < 0.05).Corynebacterium C16 revealed a lack of patho-genic effect in fairy shrimp throughout the exper-imental period. Descriptive statistics of theexperiment are given in Table 3. The comparisonsof pathogenic effects of all strains are presented inFig. 6.

(a)

(c)

(b)

Figure 1 Infected fairy shrimps showing

signs of black disease; (a) infection on the

dorsal surface, (b) infected thoracopods,

(c) healthy fairy shrimp, Streptocephalussirindhornae male [1] and female [2].

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Molecular identification

Figure 7 shows the genomic DNA extracted from 5bacterial strains. The selected strains, AeromonasC03, Aeromonas C24, Aeromonas C26, AeromonasC29 and Acinetobacter C17, were identified basedon DNA sequencing. Approximately 1500 bsp of apartial 16S rDNA gene was analysed. Thesequences of Aeromonas C29 and Aeromonas C26matched completely with A. sobria Popoff & Veron

under accession number AB473004. The sequencesof Aeromonas C03, Aeromonas C24 and Acinetobac-ter C17 matched (99%) with A. hydrophila (Ches-ter) (AB473030), A. caviae Eddy (AY987728) andAcinetobacter sp. SH-94B (FN377701), respec-tively.

Discussion

The present study revealed that black disease mostlyoccurred during the cold and early rainy seasons. Inthe rainy season, water quality fluctuated consider-ably because of flooding, acidic water and eutro-phication; thus, fairy shrimp became weak and wereeasily infected. In this study, a high prevalence ofinfection was observed in June 2009 because ofheavy rainfall which affected the water quality andcaused long-term flooding, particularly in theearthen ponds containing S. sirindhornae. In addi-tion, the severity of disease was observed to besignificantly greater in winter (November 2009–January 2010) probably because the low tempera-tures (20–26 �C) are unfavourable for fairy shrimp.Low temperature is also recognized as a stresscondition in fish (Snieszko 1974). Stress conditionsinhibit the immune systems of aquatic animalsresulting in increasing susceptibility to infections(Roberts 1975; Li, Guo, Zhao, Qian, Sun, Xiao,Liang & Wang 2006). Moreover, overcrowding anddietary deficiency may cause disease in crustaceans(Lavens & Sorgeloos 1991; Dhont, Lavens &Sorgeloos 1993). S. sirindhornae was reared inearthen ponds containing organic matter, clay andother particles. The prevalence of infection washigher than that of B. thailandensis which wasreared in more favourable conditions. The resultsstrongly suggest that the cultivation of fairy shrimpin winter should be avoided to minimize the risk ofinfection and that proper management is strictlyrequired during this period.

The study showed that the clinical signs of blackdisease were characterized by the appearance ofblack spots. Mortality occurred after the appearanceof the dark pigment. Scanning electron micrographsshowed that bacteria were localized on the black-ened parts; it is conceivable that bacteria are able tocolonize the tissue surface of fairy shrimp. Lee, Yin,Ge & Sin (1997) showed that A. hydrophila invadesthe host cell by adhesion and that bacterial adhesionis the initial stage of the infection (Beachey 1981;Verschuere 2000). In the present study, histopa-thology revealed that bacteria were found in

(a)

(b)

(c)

Figure 2 Scanning electron micrographs of the cuticles of

infected and healthy fairy shrimps. (a) The cuticle of thoraco-

pods from diseased Streptocephalus sirindhornae (bar = 10 lm),

(b) the cuticle of thoracopods from diseased Branchinellathailandensis (bar = 2 lm), (c) the cuticle of healthy fairy shrimp

(bar = 2 lm).

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scattered masses throughout the tissue and haem-ocytic encapsulation was present in the lesion.Encapsulation would occur in order to ingest andeliminate the invading particles. Furthermore, the

dark pigment appears because of the action ofphenoloxidase enzyme and melanization. Therefore,the black spots are a typical consequence ofcrustacean defence mechanisms to pathogenic bac-teria (Lightner 1993; Vargas-Albores & Yepiz-Plascencia 2000). A haemocytic response to thebacterial infection was also found in the black partsof the infected shrimp, Litopenaeus vannamei(Boone) (Krol, Hawkins, Vogelbein & Overstreet1989). Liu, Jiravanichpaisal, Cerenius, Lee,Soderhall & Soderhall (2007) reported that mela-nin formation plays a key role in the crayfish,Pacifastacus leniusculus (Dana), against Aeromonashydrophila infection. Similarly, dark pigment wasalso observed in the damaged organs of deadkuruma prawn, Penaeus japonicus (Bate), infectedwith fungi (Duc, Wada, Kurata & Hatai 2010).The histopathological results also demonstrated thatgeneralized septicaemia was clearly observed in thebody of affected fairy shrimp.

Aeromonas spp. were predominantly isolatedfrom affected fairy shrimp. Muangsan & Senamon-tee (2008) found that one of the bacteria isolated

(a) (b)

Figure 3 Histological features of fairy

shrimp with black spots and bacteria in the

tissue (arrowheads). (a) Haemocytic

encapsulation in tissue (H&E, bar = 10 l-m). (b) Generalized septicaemia in the

tissue (H&E, scale bar = 30 lm).

Table 1 The prevalence of infection by black disease naturally

found in fairy shrimps, Streptocephalus sirindhornae and Branchi-nella thailandensis

Collection period

Rate of infection (%)

S. sirindhornae B. thailandensis

June 2009 64.61 35

July 2009 26.67 10

August 2009 36.67 10

September 2009 30 30

October 2009 28 20

November 2009 46.67 50

December 2009 70 65.71

January 2010 72.22 67.86

February 2010 0 5

March 2010 0 0

April 2010 0 5

May 2010 0 5

Total 41.06 (74*/243**) 30.45 (108*/263**)

*Number of infected fairy shrimp; **total number of fairy shrimp

collected.

Figure 4 Seasonal prevalence of black

disease in fairy shrimps, Streptocephalussirindhornae and Branchinella thailandensis,from June 2009 to May 2010.

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Table 2 Biological and biochemical characteristics of Aeromonas spp. isolated from Streptocephalus sirindhornae and Branchinellathailandensis with black disease

Reaction

Bacterial isolates

Aeromonas spp.aC02 C03 C06 C07 C11 C12 C13 C14 C15 C21

Gram ) ) ) ) ) ) ) ) ) ) )Cell morphology Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod

Motility + + + + + + + + + + +

Oxidase + + + + + + + + + + +

Catalase + + + + + + + + + + +

Nitrate reduction + + + + + + + + + + +

Urease ) ) ) ) ) ) ) ) ) ) )Gelatinase + + + + + + + + + + +

OF test F F F F F F F F F F F

H2S production ) ) ) ) ) ) ) ) ) ) )Simmon�s citrate + + + + + + + + + ) V

Indole ) + + + + + + + + + V

MR ) ) ) ) ) ) ) ) ) ) V

VP + ) + + ) + + + + ) V

Acid from

Sucrose + + + + + + + + + + V

Maltose + + + + + + + + + + +

Galactose + + + + + + + + + + +d

Rhamnose ) ) ) ) ) ) ) ) ) ) )Inositol ) ) ) ) ) ) ) ) ) ) V

Xylose ) ) ) ) ) ) ) ) ) ) )Rhaffinose ) ) ) ) ) ) ) ) ) ) )Sorbital ) ) ) ) ) ) ) ) ) ) V

Lactose ) ) ) ) ) ) ) ) ) ) V

Arabinose ) ) ) ) ) ) ) ) ) ) V

Mannitol + + + + + + + + + + +

Mannose + + + + + + + + + + +

Trehalose + + ) + ) + + + + + +

Dulcitol ) ) ) ) ) ) ) ) ) ) )Dextrin + + + + + + ) + ) + ND

Reaction

Bacterial isolates

Aeromonas spp.aC22 C23 C24 C25 C26 C27 C28 C29 C30 C31

Gram ) ) ) ) ) ) ) ) ) ) )Cell morphology Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod

Motility + + + + + + + + + + +

Oxidase + + + + + + + + + + +

Catalase + + + + + + + + + + +

Nitrate reduction + + + + + + + + + + +

Urease ) ) ) ) ) ) ) ) ) ) )Gelatinase + + + + + + + + + + +

OF test F F F F F F F F F F F

H2S production ) ) ) ) ) ) ) ) ) ) )Simmon�s citrate + + + + + + + ) ) + V

Indole ) ) ) ) + + ) ) ) + V

MR ) ) ) ) + + ) ) ) ) V

VP + + ) + ) ) + + + ) V

Acid from

Sucrose + + + + + + + + + + V

Maltose + + + + + + + + + + +

Galactose + + ) + + + + + + ) +

Rhamnose ) ) ) ) ) ) ) ) ) ) )Inositol ) ) ) ) ) ) ) ) ) ) V

Xylose ) ) ) ) ) ) ) ) ) ) )Rhaffinose ) ) ) ) ) ) ) ) ) ) )Sorbital ) ) ) ) ) ) ) ) ) ) V

Lactose ) ) ) ) ) ) ) ) ) ) V

Arabinose ) ) ) ) ) ) ) ) ) ) V

Mannitol + + ) + + + + + + ) +

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from the fairy shrimp, B. thailandensis with blackdisease, was A. sobria.

The results of experimental infections indicatedthat the effect of infection was observed rapidly andmortality occurred in all diseased animals. Theimmune system of the shrimp, although stimulatedby the infections, was unable to prevent death ofaffected animals (Soltanian, Thai, Dhont, Sorgeloos& Bossier 2007; Garcia-Carreno, Cota & NavarreteDel Toro 2008). Abnormal swimming behaviour

was found in the diseased fairy shrimp as a result ofdamage to the thoracopods. Most of the infectedareas were localized in this organ. The thoracopodsserve as a gill for respiratory activity and extractingdissolved oxygen similar to the gills of othercrustaceans. Therefore, this organ is very vulnerableto disease. Infection on the gills was also observed inother crustaceans including northern shrimp, Pand-alus borealis Krøyer (Rinaldo & Yevich 1974) andmantis shrimp, Oratosquilla oratoria (De Haan)(Duc & Hatai 2009).

In this study, A. hydrophila had the highestpathogenicity of the bacteria tested. The resultswere similar to the observations of Dierckens et al.(1998), in Europe, which showed the pathogen ofblack disease in fairy shrimps [Branchipus schaefferiFischer, Chirocephalus diaphanus Prevost and Strep-tocephalus torvicornis (Waga)] was also A. hydrophil-a. Although Aeromonas spp. are known as aquaticbacteria and part of the normal flora of aquaticanimals, infections caused by these bacteria havebeen reported in many aquatic species including

Table 2 (Continued)

Reaction

Bacterial isolates

Aeromonas spp.aC22 C23 C24 C25 C26 C27 C28 C29 C30 C31

Mannose + + + + + + + ) + + +

Trehalose ) + + + + + + ) + + +

Dulcitol ) ) ) ) ) ) ) ) ) ) )Dextrin + + + + + + + + + + ND

+, positive reaction; ), negative reaction; F, fermentation; V, variable; ND, no data available; OF, oxidation and fermentation test; MR, Methyl Red test;

VP, Voges-Proskauer test; d, differs among species or strains.aData from Holt et al. (1994) and Graevenitz (1985).

Figure 5 Melanin (arrowhead) on infected thoracopods of fairy

shrimp.

Table 3 Descriptive statistics of the cumulative number of

infected Streptocephalus sirindhornae and Branchinella thailand-ensis exposed to bacterial concentrations of 104 and

106 cfu mL)1 (Means � SE)

Bacterial strain

Percentage of infected fairy shrimp

104 cfu mL)1 106 cfu mL)1

Aeromonas C03 73.33 � 6.67 93.33 � 6.67

Aeromonas C29 73.33 � 6.67 73.33 � 6.67

Aeromonas C26 53.33 � 17.64 73.33 � 6.67

Aeromonas C24 53.33 � 6.67 66.67 � 17.64

Acinetobacter C17 53.33 � 17.64 60.00 � 11.55

Corynebacterium C16 0 6.67 � 6.67

Figure 6 Comparisons of pathogenic effects of all bacterial

strains on fairy shrimp at concentrations of 104 and

106 cfu mL)1.

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fish (Lee et al. 1997; Rahman, Colque-Navarro,Kuhn, Huys, Swings & Mollby 2002; Garvin,Merino & Tomas 2004) and also amphibians(Mauel, Miller, Frazier & Hines 2002), especiallywhen animals are raised under stressful environ-ments (Smith, Brown & Hauton 2003). Fairyshrimp are highly sensitive crustaceans, so thepossibility that they will be infected is greatlyenhanced.

Acknowledgements

This work was supported by the Commission onHigher Education, Thailand, under the programmeStrategic Scholarships for Frontier Research Net-work. We thank the Applied Taxonomic ResearchCenter, Faculty of Science, Khon Kaen University,Thailand and the Laboratory of Fish Disease,School of Veterinary Medicine, Nippon Veterinaryand Life Science University, Japan for providingfacilities for this research.

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Received: 3 December 2010Revision received: 26 March 2011Accepted: 30 March 2011

920� 2011

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Journal of Fish Diseases 2011, 34, 911–920 C Saejung et al. Black disease in fairy shrimps