Aquaculture 439 (2015) 60–65

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Phenotypic and molecular characterization and pathology ofFlectobacillus roseus causing flectobacillosis in captive held carpLabeo rohita (Ham.) fingerlings

Harresh Adikesavalu, Avijit Patra, Sayani Banerjee, Agniswar Sarkar, T. Jawahar Abraham ⁎

Department of Aquatic Animal Health, Faculty of Fishery Sciences, West Bengal University of Animal and Fishery Sciences, 5 Budherhat Road, Chakgaria, Kolkata 700 094, West Bengal, India

⁎ Corresponding author. Tel.: +91 94333 68328 (mobifax: +91 33 2432 8763.

E-mail address: abrahamtj1@gmail.com (T.J. Abraham

http://dx.doi.org/10.1016/j.aquaculture.2014.12.0360044-8486/© 2015 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 27 November 2014Received in revised form 20 December 2014Accepted 23 December 2014Available online 30 December 2014

Keywords:Flectobacillus roseusLabeo rohitaFlectobacillosisColumnaris-likePhylogenetic analysis

A new bacterial disease (flectobacillosis) caused by Gram negative, long rod shaped Flectobacillus roseus incaptive held carp, rohu (Labeo rohita) in West Bengal, India is described. Besides the phenotypic and molecularcharacterization, histopathology of F. roseus of naturally infected and challenged rohuwere studied. The affectedrohu had loss of mucus and scale, discoloured body, peeled skin and rotten tail, but no signs of gill damage. Onselective cytophaga agar, F. roseus produced pink to rose pigmented colonies. Phylogenetically, F. roseus RF1formed a monophyletic branch with F. roseus EU921645 (96% sequence similarity) and Flectobacillus sp.KC588923 (94% sequence similarity). It caused significant mortalities in rohu when challenged. The naturallyinfected rohu showed epithelial disruption, inflammation and necrosis of skeletal muscle tissue accompaniedby haemocyte infiltration. The kidney of challenged rohu showed proximal and distal tubule degeneration,necrosis, thickening of epithelial layer of proximal and distal tubules, necrotised haematopoietic tissue, nephritictubule with widened lumen, vacuolation of tubular epithelium, glomerular congestion, desquamation, glomeru-lopathy with dilated Bowman's space, cellular and nuclear hyperprophy, pycnotic nuclei, melano-macrophageaggregates and granuloma-like inflammatory response. These results suggest that F. roseus can produce systemicpathology in kidney similar to many other known fish pathogens.

© 2015 Elsevier B.V. All rights reserved.

1. Introduction

Columnaris, since its first report (Davis, 1922), is one of the mostfrequently encountered anddevastating bacterial diseases of freshwaterfish (Tripathi et al., 2003; Declercq et al., 2013). The etiologic agent ofcolumnaris disease is a Gram-negative, long, thin, gliding rod that hasbeen reclassified as Flavobacterium columnare (Bernardet et al., 1996).Strains of F. columnare vary in virulence and infection of fish may berelated to stressful events. Natural infections with F. columnare mayoccur frequently at water temperature of 20 °C. Such disease outbreaksare associatedwith highmortality thatmay reach 100%. Columnaris dis-ease has also been reported in coldwater fish at normal environmentaltemperatures ranging from 6 to 12 °C (Tripathi et al., 2003). Severalother species of Gramnegative, long, thin rod shaped bacteria belongingto the genera Flavobacterium, Flectobacillus, Sphingobacterium andTenacibaculum in aquatic environment have also been described

le), +91 33 2478 0126 (office);

).

(Madetoja et al., 2000; Avendaño-Herrera et al., 2006; Saha andChakrabarti, 2006; Starliper, 2011; Kolygas et al., 2012; Subhash et al.,2013). Few of them are reportedly causing diseases in fish (Madetojaet al., 2000; Avendaño-Herrera et al., 2006; Austin and Austin, 2007;Starliper, 2011).

The Gram negative, long rod shaped bacterium of the genusFlectobacillus, proposed by Larkin et al. (1977), was isolated mainlyfrom aquatic environment. Two species namely Flectobacillusmarinus and Flectobacillus glomeratus were initially assigned tothis genus (Borrall and Larkin, 1978; McGuire et al., 1987) whichwas later, reclassified as Cyclobacterium marinum (Raj and Maloy,1990) and Polaribacter glomeratus (Gosink et al., 1998), respective-ly. F. major was the first accepted Flectobacillus species (Larkin et al.,1977), followed by F. lacus isolated from a highly eutrophic pond(Hwang and Cho, 2006) and Flectobacillus roseus isolated fromfreshwater ecosystem (Sheu et al., 2009). None of the recognizedspecies of the genus Flectobacillus were, so far, known to cause dis-eases in fish. In this paper, a new bacterial disease (flectobacillosis)caused by Gram negative, long rod shaped bacterium, F. roseus incaptive held carp (Labeo rohita) with signs and symptoms similarto columnaris is described.

Table 1Biochemical characteristics of Flectobacillus roseus RF1 strain and recognizedFlectobacillus spp.

Characteristics Flectobacillusroseus RF1

Flectobacillusroseusa

Flectobacilluslacusb

Flectobacillusmajorb

Gram reaction − − − −Morphology, long rod + + + +Motility − − − −Cytochrome oxidase + + + +Catalase + + + +Urease − − − VNitrate reduction (+) − V −Enzyme activity

Beta-galactosidase + + + VAlpha-galactosidase + + + +Lipase − − − NDBeta-glucoronidase + − − NDBeta-glucosidase + + + NDAlpha-glucosidase + + + ND5 keto-gluconate − + − +

(+): Weak reaction; V: variable; ND: no data.a Sheu et al. (2009).b Hwang and Cho (2006).

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2. Material and methods

2.1. Isolation and phenotypic characterization of F. roseus

Captive held Indian major carp, rohu (L. rohita Ham.) fingerlingsweighing 20–30 g showing loss of mucus, loss of scale in caudal pedun-cle region, body discolouration, peeled skin and tail rot in a series of500 l capacity fibreglass reinforced plastic tanks in Kolkata (Lat 22°28′45.53″ N; Long 88°24′07.49″ E), West Bengal, India were examinedduring May 2013 as per Heil (2009). Prior to sample collection, the

Table 2Additional biochemical characteristics of Flectobacillus roseusRF1 strain as assessed byRapidHiA

Characteristics Reaction

Rapid HiAssorted™Citrate utilization −Lysine decarboxylase −Orinithine decarboxylase −Urease −Phenylalanine deamination −Nitrate reduction +

VITEK 2 — compact

D-Glucose (dGLU) +

Saccharose/sucrose (SAC) +D-Cellobiose (dCEL) −Adonitol (ADO) −L-Arabitol (IARL) −D-Mannitol (dMAN) −D-Sorbitol (dSOR) −D-Maltose (dMAL) −D-Mannose (dMNE) −D-Tagatose (dTAG) −D-Trehalose (dTRE) −H2S production (H2S) −Lysine decarboxylase (LDC) −Orinithine decarboxylase (ODC) −L-Malate assimilation (IMLTa) −Citrate (sodium) (CIT) −Urease (URE) −Fermentation/glucose (OFF) −Phosphatase (PHOS) +Beta-glucoronidase (BGUR) +Lipase (LIP) −Alpha-glucosidase (AGLU) +Beta-xylosidase (BXYL) −Beta-galactosidase (BGAL) +

fish were rinsed in sterile saline and wiped with sterile paper towel.Inocula from the caudal peduncle of infected rohu (n = 5) werestreaked on to selective cytophaga agar supplemented with neomycin5 μg/ml and polymyxin B 200 IU/ml (SCA; Hawke and Thune, 1992)and incubated at 30 °C for 48 h. The SCA plates invariably yielded palepink to rose pigmented colonies of 1–2 mm size. Representativecolonies from all samples were randomly picked and Gram stainedfor preliminary observation. For further studies, a rose pigmented,round, convex and smooth with entire margined colony on SCA waspicked aseptically, purified by subculturing on cytophaga agar withoutantibiotics (CA) and maintained on CA slants at 30 °C and also inglycerol stock at −20 °C. Biochemical characterization was done byRapid HiAssorted™ biochemical test kit (HiMedia, India) and also byan automated bacterial identification system (VITEK 2 compact,BioMerieux, France).

2.2. Molecular characterization

The genomic DNA of F. roseus RF1 was isolated using Genomic DNAisolation kit (Macherey-Nagel, Germany) as per manufacturer's proto-col. The 16S small subunit ribosomal RNA (16S rRNA) was amplifiedby Eppendorf Master cycler Pro S using a set of universal prokaryoticprimers 8F, 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1492R, 5′-GGTTACCTTGTTACGACTT-3′ (Eden et al., 1991). The PCR master-mix contained50 ng of genomic DNA, 10 μM of each primer and 2× PCR TaqMixture(HiMedia, India). Amplification was done by initial denaturation at95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for30 s, annealing of primers at 44 °C for 30 s and extension at 72 °C for60 s. The final extension was at 72 °C for 5 min. The PCR productswere analysed on a 1.5% agarose gel containing 0.5 μg/ml ethidiumbromide in 1× Tris-acetate-EDTA (TAE) buffer.

ssorted™ biochemical test kit (HiMedia, India) andVITEK 2 compact (Biomerieux, France).

Characteristics Reaction

H2S production −Glucose +Adonitol −Lactose +Arabinose −Sorbitol −

Ala–Phe–Pro–arylamidase (APPA) +

L pyrrolydonyl–arylamidase (PyrA) −Beta-N-acetyl-glucosaminidase (BNAG) +

Glutamyl arylamidase pNA (AGLTp) +Gamma-glutamyl transferase (GGT) −Beta-alanine arylamidase pNA (BAlap) −L-Proline arylamidase (ProA) −Tyrosine arylamidase (TyrA) +

Beta-N-acetyl-galactosaminidase (NAGA) +

Glycine arylamidase (GlyA) +

Glu–Gly–Arg–arylamidase (GGAA) −Ellman (ELLM) −L-Lactate assimilation (ILATa) −L-Histidine assimilation (IHISa) −5-Keto D-gluconate (5KG) −Palatinose (PLE) −Malonate (MNT) −L-Lactate alkalinisation (ILATk) −Succinate alkalinisation (SUCT) −Alpha-galactosidase (AGAL) +Beta-glucosidase (BGLU) +Coumarate (CMT) −O/129 resistance (O129R) −

Fig. 1.Agarose gel (1.5%) showing 16S rRNA geneamplification of Flectobacillus roseus fromLabeo rohita. Lane L: 1 kb DNA Ladder (Takara Bio Inc., Japan); Lane S: Flectobacillus roseus.

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2.3. Sequencing and phylogenetic analyses

The PCR amplicon was first purified using EXO-SAP treatment. Theconcentration of purified DNA was determined and subjected to auto-mated DNA sequencing on ABI 3730xl Genetic Analyzer. Sequencingwas carried out using BigDye® Terminator v3.1 Cycle sequencing kit(Applied Biosystems, USA) following manufacturers' instructions.Electrophoresis and data analysis were carried out on the ABI 3730xlGenetic Analyzer. The two forward and reverse sequences were assem-bled by DNA Baser Sequence Assembler v3.5.3 (2012), to form consen-sus sequence. Phylogenetic analysiswas performed on a selection of 16SrRNA gene sequences that comprised the new consensus sequence,

Fig. 2. Molecular phylogenetic tree produced by maximum likelihood (ML) analysis. Numbersnumber is provided for each species. Units of evolutionary distance are in the number of base

closely related sequences determined by Basic Local Alignment SearchTool (BLAST) and other representatives of the family Flexibacteriaceae.Phylogenetic tree was constructed based on the alignment of twenty1300–1400 bp long 16S rRNA gene sequences comprising 13Flectobacillus spp. along with seven other species of the familiesSphingobacteriaceae, Flexibacteraceae and Flavobacteriaceae usingphyML v3.1.2 from http://www.phylogeny.fr/ (Dereeper et al., 2008).The evolutionary history was inferred using maximum likelihood(ML)method with 1000 bootstrap replicates and clustering of the asso-ciated taxa as per bootstrap test was given in terms of percentage nextto the branches (Felsenstein, 1985). The nucleotide sequence informa-tion of F. roseus RF1 has been deposited in NCBI GenBank under the ac-cession number KF481925.

2.4. Pathogenicity

2.4.1. Preparation of cell suspension of F. roseus RF1F. roseus RF1 maintained on CA slant was streaked on to a CA plate

and incubated at 30 °C for 24 h to get young discrete colonies. Twoyoung colonies were aseptically picked, transferred to 10 ml cytophagabroth (CB; Song et al., 1988) and incubated at 30 °C for 24 h. This 24 hold culture was then transferred to 300 ml CB and reincubated at30 °C for 48 h. The cells were harvested by centrifugation at 7500 rpmfor 20 min at 25 °C in a refrigerated centrifuge. The cell pellets werewashed twice by centrifugation with sterile physiological saline andfinally resuspended in 10 ml sterile saline and used immediately. Aportion of the cell suspension was suitably diluted up to 10−9 in sterilesaline and the number of cells/ml of suspension was determined bydrop plate method (Miles et al., 1938) on CA after incubation at 30 °Cfor 48 h.

2.4.2. Experimental challengePathogenicity of F. roseus RF1was tested by twomethods, viz., intra-

muscular injection (Iqbal et al., 1999) and immersion of skin woundedrohu in F. roseus cell suspension (Madetoja et al., 2000) atpredetermined doses in duplicate. Six glass aquaria (60 × 45 × 30 cm)were used for injected, skin wounded and control groups. All glassaquaria were filled with clean bore-well water to a volume of 30 leach and conditioned for three days. All the experimental rohu finger-lings (25–30 g size) were first disinfected by placing in 5 ppm KMnO4

at nodes indicate bootstrap confidence values (1000 replicates). The GenBank accessionsubstitutions per site.

Fig. 4.Histopathology of naturally infected Labeo rohita showing skin epithelial disruption(ED) (H & E staining; 200×).

Fig. 3. Pathogenicity of Flectobacillus roseus RF1 on Labeo rohita fingerlings. Bars sharingcommon superscripts are significantly different (a: P = 0.0509; b: P b 0.01).

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solution for 15 min. The weak fish were removed immediately. Thehealthy ones were stocked at the rate of 10 fish/aquaria and acclima-tized for 3 days with continuous aeration. All fish were fed a balancedbasal dry pellet feed twice daily at the rate of 2% of their body weightand maintained under optimal condition. The wastes and faecal matterwere siphoned out and 50%water exchangewas done on alternate days.The fish of first group were injected with 0.1 ml of bacterial suspensionat 2.0 × 107 cells/fish (injected group). In the second group, fish scaleswere scrapped off gently with a scalpel from caudal peduncle to thepectoral fin, i.e., in the opposite direction (skin wounded group). Theskin wounded rohu were then transferred to the respective aquariacontaining 30 l water and to which added was 3 ml of F. roseus cellsuspension containing 2.0 × 109 cells/ml. The final concentration ofF. roseus in water was ≈2.0 × 106 cells/ml. The control group wasneither skin-wounded nor injected with F. roseus. The challenged andcontrol groups were maintained in the respective aquaria for 28 days.The external signs of infection, behavioural abnormalities andmortalitywere recorded daily. The bacterium F. roseuswas reisolated from freshlydead fish and confirmed phenotypically. Significant differences inmortalities among the fish groups were determined by the Chi-squaretest (Snedecor and Cochran, 1967).

2.5. Histopathology

The muscle of normal and naturally infected rohu and the kidney ofchallenged rohu were fixed in Bouin's solution for 24 h. The fixedmuscle and kidney samples were processed by standard techniquesand embedded in paraffin wax. Thin (5 μm) sections were preparedand stained with haematoxylin and eosin (Presnell and Schreibman,1997).

3. Results

During captivity, the rohu were stocked at a density of 75 numbers/tank. The initial mortality was about 4–6 fish/tank/day, which increasedwith increasing days of captivity and within 2 weeks about 90% of thefish died. Inocula from infected rohu on SCA yielded predominantlypink to rose pigmented colonies within 48 h. The bacterium was aGram negative long, thin and rod shaped. The biochemical characteris-tics of the present stain F. roseus RF1 and few recognized Flectobacillusspp. are presented in Table 1. The results of additional biochemicaltests of this bacterium as assessed by Rapid HiAssorted™ biochemicaltest kit and by VITEK 2 compact are presented in Table 2.

The universal prokaryotic primer 8F, 5′-AGAGTTTGATCCTGGCTCAG-3′and 1492R, 5′-GGTTACCTTGTTACGACTT-3′ successfully amplified≈1500 bp sequence of the 16S rRNA gene from F. roseus RF1 (Fig. 1).The edited 16S rRNA gene sequence of F. roseus RF1 was 1258 bp. Onthe basis of 16S rRNA gene sequence similarity, it was related mostclosely to F. roseus EU921645 (96%) and F. roseus EU420062 (96%).The levels of 16S rRNA gene sequence similarity between the presentstrain F. roseus RF1 and the type strains of all other recognizedFlectobacillus species were not more than 91%. The evolutionary phylo-genetic analyses (Fig. 2) based on the alignment of twenty sequences,clustered F. roseus RF1within the genus Flectobacillus. It formed trichot-omy with F. roseus EU921645 and Flectobacillus sp. KC588923 with lownode support (71%).

In injected rohu, the signs of tail erosion developed 36 h post-injection and mortalities occurred a day after. In skin wounded rohu,mortality started within 24 h. All the dead rohu displayed gross andclinical signs similar to that of natural infection, but with no evidenceof gill damage. The mortality was high in skin wounded group (65 ±5%) than intramuscular injected (50 ± 10%) and control (10 ± 0%)groups (Fig. 3). Significant differences in mortality existed betweenthe control and skin wounded groups (P b 0.01) and control andinjected groups (P = 0.0509). The difference in mortality between theinjected and skin wounded groups was insignificant (P N 0.05).

Histopathology of naturally infected rohu showed epithelial disruption(Fig. 4), inflammation and necrosis of skeletal muscle tissue, whichwas accompanied by haemocyte infiltration (figure not shown). Noobvious abnormities were observed in the liver, spleen and anteriorkidney of naturally infected carp. The kidney of experimentallychallenged rohu showed nephritic tubule with widened lumen,

64 H. Adikesavalu et al. / Aquaculture 439 (2015) 60–65

thickening of epithelial layer of proximal and distal tubules, necrotisedhaematopoietic tissue, pycnotic nuclei, increased tubular lumen, vacuo-lation of tubular epithelium, glomerular congestion and desquamation,glomerulopathy with dilated Bowman's space, proximal and distaltubule degeneration and necrosis and cellular and nuclear hyperprophy,pycnotic nuclei, melanomacrophage aggregates and granuloma-likeinflammatory response (Fig. 5A–D).

4. Discussion

A perusal of literature on the distribution of Flectobacillus species in-dicated that it is common in freshwater ecosystem (Larkin et al., 1977;Hwang and Cho, 2006; Sheu et al., 2009). Recently, Sophia et al.(2013) described a Flectobacillus sp. derived from the effluent treatmentplant activated sludge in India, which possibly suggested its saprophyticmode of life. The isolation of F. roseus in captive held and diseased rohushowing tail rot, scale loss, peeled skin and body discoloration indicatedthat it can be an opportunistic fish pathogen. Due to its colonialmorphology and phenotypic reaction, it was considered to be one ofthe species of the genus Flectobacillus. VITEK 2 compact (Biomerieux,France) identified this bacterium as Sphingobacterium thalpophilumwith 99% probability. As this identification contradictedwith themolec-ular identification, the VITEK 2 data was used only to characterize thebacterial isolate phenotypically. As pointed out by Peix et al. (2003),the identification of non-clinical isolates is often wrong with VITEK 2system. Phylogenetic analysis based on 16S rRNA gene sequences indi-cated that the novel sequence belonged to the family Flexibacteraceae,phylum Bacteroidetes, and fell within the evolutionary radiation of thegenus Flectobacillus. The 16S rRNA gene sequence of this bacteriumwas closely related to F. roseus EU921645 with 96% similarity (Sheu

Fig. 5.A–D. Photomicrograph of kidney of Flectobacillus roseus RF1 challenged Labeo rohita showand thickening of epithelial layer (TE) of proximal and distal tubules (H& E staining; 100×); [B]glomerular congestion (GC) and desquamation (D) (H& E staining; 200×); [C]: glomerulopathyE staining; 200×); [D]: cellular (CH) and nuclear hyperprophy (NH), pycnotic nuclei (PN), melastaining; 400×).

et al., 2009) as well as Flectobacillus sp. KC588923 with 94% similarity(Sophia et al., 2013). Nevertheless, the present stain exhibited slightvariation in biochemical reactions when compared to other species ofthe genus Flectobacillus and also within the same species, which mightbe because of the geographical distribution.

In fish, the cutaneous mucus layer works like a protective coatingthat resists bacterial infection. Previous studies on columnaris diseasedemonstrated that columnaris is primarily a skin disease on fish withdamaged mucus layer and it can also affect fish gills (Declercq et al.,2013). The naturally infected rohu of the present study exhibited asimilar pathobiology as a result of confinement, crowding and environ-mental stress, which suggested that F. roseus primarily affects skin withthe loss of mucus, scale and fins. Unlike columnaris, the gills of the af-fected rohu showed noevidence of damage, thusmakingflectobacillosisdifferent from columnaris. During the experimental infection, diseasesymptoms and mortality appeared quickly in skin wounded rohu thanthe injected rohu. Themortality was significantly high in skin woundedrohu (65±5%) compared to control,might be due to compromised pro-tective mucus layer in skin wounded group. Infiltration of haemocytesas seen in histologic section of muscle has probably occurred inresponse to tissue damage caused by the bacterium, which corroboratethe findings of Tripathi et al. (2005) on columnaris disease. The histo-pathological alterations such as disruption of epithelial layer, inflamma-tion, necrosis of skeletal muscle and haemocyte infiltration suggestedthat this bacterium has the potential to cause pathological symptomssimilar to many other known fish pathogens including Flavobacterium,Aeromonas spp., Edwardsiella spp. and Pseudomonas spp. (Austin andAustin, 2007; Heil, 2009). The systemic pathological alterations seenin the kidney of experimentally challenged rohu (Fig. 5A–D) suggestedthe presence of some yet to be known factors that help either in

ing [A]: necrotised haematopoietic tissue (N), nephritic tubule with widened lumen (WL): pycnotic nuclei (PN), increased tubular lumen (IL), vacuolation (V) of tubular epithelium,(G)with dilated Bowman's space (DB), proximal anddistal tubule degeneration (TD) (H&nomacrophage aggregates (MM) and granuloma-like inflammatory response (GR) (H & E

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increasing the aggressiveness of the bacterium or suppressing the hostdefence as suggested by Mudarris and Austin (1988). It appears fromthe histopathological evidences that the inflammation type followinginduced flectobacillosis in rohu was granuloma-like.

Earlier studies on Flectobacillus have focused on the isolation, identi-fication and characterization (Mcguire et al., 1987; Raj andMaloy, 1990;Hwang and Cho, 2006; Sheu et al., 2009), but none have established itspathogenicity towards fish. The results of the present study on F. roseuscausing flectobacillosis (columnaris-like disease) in rohu are worthyand significant, because no reports on pathogenic Flectobacillus speciesare available yet. There can be little doubt whether or not F. roseuswas responsible for the infection. However, it is reasonable to believethat in experimental challenge this bacterium produced a systemicpathology in rohu kidney similar to many other known fish pathogens.Further studies are warranted to explore various domains like the host-pathogen interaction, mode of infection, rapid identification of bacteri-um in the environment, the role of stressors and effective preventivemeasures through efficient molecular manipulation systems.

Acknowledgements

The researchworkwas supported by the Indian Council of AgriculturalResearch (10(12)/2012-EPD dtd 23.3.2012), Government of India, NewDelhi under the Niche Area of Excellence programme. The authorsthank the Vice Chancellor,West Bengal University of Animal and FisherySciences, Kolkata for providingnecessary infrastructure facilities to carryout the work.

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