Isolation, Identification and Pathogenicity of Flavobacterium columnare SGM4 in Catfish Clarias batrachus (Linnaeus 1758).
Columnaris disease is the most common disease of cultured catfish globally. It is caused by a Gram-negative bacillus Flavobacterium columnare, an acute to chronic bacterial infection, that affects virtually all species of warm water fish (1,2). F. columnare is ubiquitous in the freshwater environment and can cause tragic mortalities in both wild as well as cultured species. The catfish are especially vulnerable to F. columnare infections (3). Mortalities in farmed catfish from columnaris can be as high as 50-60% (2). It is one of the important bacterial pathogens of freshwater fish and can be of economic importance in catfish farming, particularly in the intensive channel catfish Ictalurus punctatus farming. In the United States catfish industry, it caused an estimated annual loss of US$ 30 million (2,4). The gills, skin and fins of fish are normally affected by F. columnare with varying degrees of clinical manifestation and virulence (2,4,5). The disease is often initiated as an external infection on the body surface, fins or gills, and subsequently developed into yellow-orange lesions along the dorsal midline leading to a condition called saddleback (2).
F. columnare is usually of low pathogenicity and infects fish under stressful conditions. Several authors demonstrated divergence in the virulence of F. columnare (2,6-8). The columnaris disease outbreaks are heavily dependent on the environmental factors such as the temperature, pH and hardness of water (3). In aquaculture, the risk for the columnaris is associated with environmental stress. The risk increases with temperature fluctuation, higher feeding rates, more organic loads as well as the increasing stocking densities (2,8). Some strains of this bacterium are highly pathogenic and may cause disease in the absence of documented stress (2). Columnaris disease has been confirmed by molecular characterization of F. columnare in carps such as Catla catla (9), Labeo rohita and Ctenopharyngodon idella (10), goldfish Carassius auratus (11) and climbing perch Anabas testudineus (10) cultured in Indian conditions. The present study aimed at the characterization of F. columnare associated with the gill-rot of diseased Clarias batrachus by phenotypic and molecular means, and the pathogenic potential in C. batrachus fingerlings.
MATERIALS AND METHODS
Isolation and Phenotypic Characterization of Bacteria
In February 2015, extensive mortalities in catfish, C. batrachus (Linnaeus, 1758) of 13-14 cm in length and 40-50 g weight were reported from a nursery in Ramchandrapur (Lat. 22[degrees]52' N; Long. 88[degrees]28' E), North 24 Parganas district, West Bengal, India. About 1000 catfish juveniles were stocked in cemented tanks (10 m x 10 m x 0.5 m, water depth). The nursery experienced chronic mortalities with 10-20 catfish dying daily. The water temperature fluctuated from 16 to 23[degrees]C during the mortality period. The morbidity and cumulative mortality were about 65 and 43%, respectively. Water exchange and benzalkonium chloride (50%) application (0.5 ppm) were attempted thrice. The examination of diseased catfish for gross and clinical signs was done at the site as per Heil (1). The morbid catfish with clinical manifestation of columnaris disease (n=15) were transported to the laboratory in oxygen-filled polythene bags and within 3 hours of collection. Following rinsing in sterile saline and wiping with sterile paper towels, inocula from the catfish gill-rot (n=5) were aseptically streaked onto selective cytophaga agar [SCA] (12) and incubated at 30 [degrees]C for 48 h. Yellow-pigmented rhizoid colonies of 2 mm size were predominant in all the plates. From all diseased fish, one each of representative yellow-pigmented colony was arbitrarily picked (n=5), purified and characterized on the basis of cell morphology, phenotypic (13,14) and genotypic (14) characters. The genotypic characterization was done for only one rhizoid strain SGM4 (Figure 1).
Molecular and Phylogenetic Characterization
The 16S rRNA of the rhizoid strain SGM4 was amplified as per the conditions and protocols described in our earlier study (14) using the universal prokaryotic forward (8F) and reverse (1492R) primers (15). The phylogenetic tree was constructed using 30 16S rRNA gene sequences that included the consensus sequence of the present study (strain SGM4), one type strain and seven non-type strains of F. columnare, 14 type strains of Flavobacterium spp., one each of Chryseobacterium indologenes and Tenacibaculum maritimum. As out-group, the type strains Flectobacillus roseus (n=1), Sphingobacterium thalpophilum (n=1) and Pseudomonas spp. (n=3) were included. The 16S rRNA gene sequences for phylogenetic analysis were collected from the NCBI GenBank and EzBioCloud database (Figure 2). ClustalW 1.6 was followed for the data analysis and multiple alignments. Evolutionary analyses were as per MEGA6 (16).
Pathogenicity of Flavobacterium columnare SGM4
F. columnare SGM4 cell suspension was prepared and the cell counts determined as described in Sarker et al. (14). Healthy hatchery-raised C. batrachus fingerlings (n=125) of weight 3.83[+ or -]0.28 g were procured from a reputed hatchery and transported to the laboratory in oxygen-filled polythene bags. The fingerlings were disinfected in 5 ppm potassium permanganate solution for 5 min and transferred to 500-L capacity fibreglass reinforced plastic (FRP) tanks containing 300-L borewell water. The fingerlings were acclimatized for 20 days under optimal conditions and fed twice daily with pellet feed at 4% of body weight. On alternate days, the faecal matter and other wastes were removed by siphoning and 40-50% water exchanged.
Pathogenicity of F. columnare SGM4 was tested in C. batrachus fingerlings by the abrasion-immersion method as described in Sarker et al. (14). In brief, healthy fingerlings were stocked at 10 fish/tank (58 x 45 x 45 cm) and acclimatized in the aerated tank for 3 days at 24-30 [degrees]C. The catfish were divided into five groups, viz., A, B, C, D and E in duplicate. Prior to the challenge, the scales of catfish from each tank of groups A, B and D were scrapped off gently on one side with a scalpel 1.0 cm from caudal peduncle towards the pectoral fin (abraded). The abraded catfish of groups A and B were then immersed in F. columnare SGM4 suspensions containing 7.2x[10.sup.6] cells/mL, 7.2x[10.sup.5] cells/mL for 30 min, respectively. The group C was non-abraded and immersed in F. columnare SGM4 suspensions containing 7.2x[10.sup.6] cells/mL for 30 min. The group D abraded fish was served as a positive control after immersion in 0.85% saline for 30 min. The fish of all groups were then transferred to the respective tanks. No abrasion or challenge was done to the catfish of group E, which served as a negative control. The challenged and control fish groups were observed for the behavioural abnormalities, external signs of infection and mortality for 28 days. Re-isolation of the challenged bacterium from freshly dead catfish was on SCA followed by phenotypic confirmation.
Antibiotic Sensitivity of Flavobacterium columnare
F. columnare strains (n=5) were screened for their sensitivity to ten broad-spectrum antibiotics, viz., amoxyclav (30 [micro]g), chloramphenicol (30 [micro]g), ciprofloxacin (5 [micro]g), co-trimoxazole (25 [micro]g), erythromycin (15 [micro]g), gatifloxacin (5 [micro]g), gentamicin (10 [micro]g), nitrofurantoin (300 [micro]g), oxytetracycline (30 [micro]g) and sulphafurazole (300 [micro]g) by Kirby Bauer agar-disc diffusion technique (17) on cytophaga agar at 30 [degrees]C. The antibiotic impregnated discs were procured from HiMedia, India. Interpretation of sensitivity was based on the zone size interpretation chart for Gram-negative bacteria (18).
Isolation and Phenotypic Characterization of Bacteria
Inocula from the catfish gill-rot on SCA yielded yellow-pigmented rhizoidal growth within 48 h. The rhizoid colonies were Gram-negative long rods. Five rhizoid strains were characterized phenotypically by conventional biochemical tests, which identified them as F. columnare (Table 1). The strains were, however, identified as Aeromonas salmonicida by the Vitek 2 compact system with a 93% probability. Minor variation in L-Proline arylamidase activity was noted in one of the strains.
Molecular and Phylogenetic Characterization
The amplified 16S rRNA gene ([approximately equal to]1500 bp) of the strain SGM4 was edited to a sequence length of 1432 bp. Phylogenetic analysis confirmed the strain as a member of the family Flavobacteriaceae. Phylogenetically, the members of the genus Flavobacterium clustered together as a separate clade, distinctly different from other bacteria. The strain SGM4 branched with the type strain F. columnare IFO 15943(T) [NCBI accession number AB078047] and the non-type strains of F. columnare with high node value. The 16S rRNA gene sequence of F. columnare SGM4 (accession number KU851952) was deposited in NCBI GenBank.
Pathogenicity of Flavobacterium columnare SGM4
The abraded and immersion challenged C. batrachus fingerlings were sluggish, erratic, hanging and anorectic. In challenged catfish, white patches on the gills, excessive mucus secretion, saddleback, caudal peduncle lesions, tail rot, cutaneous haemorrhages, ulceration in the abraded area, skin discolouration, skin peeling, pale and discoloured kidney and liver, and haemorrhages in the internal organs were observed. About 45[+ or -]5% and 10[+ or -]0% mortalities were noted in abraded groups when challenged at 7.2x[10.sup.6] cells/mL and 7.2x[10.sup.5] cells/mL levels, respectively. The internal organs of challenged catfish were haemorrhagic in the later stage (Table 2).
Antibiotic Sensitivity of Flavobacterium columnare
All F. columnare strains were highly sensitive to amoxyclav, chloramphenicol, ciprofloxacin, gatifloxacin, gentamicin and oxytetracycline. Few strains were resistant to co-trimoxazole (n=2), erythromycin (n=2), nitrofurantoin (n=1) and sulphafurazole (n=4) (Table 3).
The emaciated and diseased catfish had white hues on the gills, gill-rot, tail rot, discoloured skin, saddleback, peeled skin and swollen kidney. Gross and clinical signs observed in diseased catfish signified columnaris disease (2,5,13). The isolation of F. columnare from the gill-rot of diseased C. batrachus indicated the opportunistic potential of this bacterium in immunosuppressed catfish during the winter season. The rhizoid type colonies were reportedly virulent to fish (7,19). The low levels of mortalities with 10-20 fish dying daily suggested chronic columnaris disease in catfish at temperatures in the range of 16-23 [degrees]C. Vitek 2 compact system identified the tested strains as A. salmonicida as the software contained only the database on clinical isolates. Alike our earlier study (14), this identity also contradicted with the conventional and molecular diagnosis. The Vitek-2 records were, therefore, used for characterizing the strains phenotypically. The phenotypic characteristics of all the F. columnare strains, as shown in Table 1, were almost the same. The Vitek-2 results indicated only minor phenotypic variations among the F. columnare strains of diseased catfish. The phylogeny of the strain SGM4 confirmed the bacterium as F. columnare, a member of the family Flavobacteriaceae. It branched with the type strain F. columnare IFO 15943(T) [NCBI accession number AB078047] along with other F. columnare strains with high node value.
Though various challenge routes to induce columnaris disease are available, we chose to follow abrasion and immersion challenge as it gave consistently
better results in our earlier study (14). About 45[+ or -]5% and 10[+ or -]0% mortalities were noted in abraded groups when challenged at 7.2x[10.sup.6] cells/mL and 7.2x[10.sup.5] cells/mL levels, respectively. No mortalities were noted in non-abraded and immersion challenged catfish and other groups at 24-30 [degrees]C. While in the naturally infected population about 65% morbidity and 43% cumulative mortalities were recorded at 16-23[degrees]C in tropical Indian condition. Likewise, in temperate condition, Durborow et al. (20) found that columnaris disease commonly occurs in channel catfish when water temperatures are in the range of 25-32 [degrees]C. Columnaris epidemics reportedly occur in water temperatures below 25 [degrees]C; even as low as 15 [degrees]C, but mortalities and acuteness of disease are significantly less than in higher temperatures (2,3,8). Further, the experiments of Holt et al. (21) revealed that temperatures in excess of 12.2 [degrees]C are required for F. columnare induced mortalities in trout and salmon.
With the increase in water temperature, the mortalities reduced and fish became almost normal. These findings indicated that temperature can have a strong effect on the virulence of F. columnare in the culture environment and presumably on disease progression. In other fish pathogens also the growth of bacteria at higher-than-optimal temperature resulted in decreased virulence (8). Abrasion and low temperature exacerbated the rate of infection, which endorse the findings of Moyer and Hunnicutt (22) in zebrafish Danio rerio. In abrasion-bath treatment with F. columnare, they recorded [LD.sub.50] values in the range of 1.1x[10.sup.6]-1.1x[10.sup.7] cfu/mL. In another study, Nayak et al. (23) recorded the [LD.sub.50] values of F. columnare in Cirrhinus mrigala fry in the range of 3.0x[10.sup.5] - 9.0x[10.sup.6] cfu/mL by immersion assay. In contrast, Swain et al. (24) recorded no mortalities in non-abraded L. rohita fingerlings at a challenge dose of [10.sup.6]-[10.sup.8] cfu/mL in immersion challenge study with F. branchiophilum. The experimental challenge results of this study displayed that the gill associated F. columnare SGM4 can induce mortalities in catfish in conjunction with skin damages. In later stages, the development of haemorrhages in the internal organs of challenged catfish indicated a septicemic condition, which supported the study of Decostere et al. (6).
All F. columnare strains were highly sensitive to most of the tested antibiotics. Likewise, Sarker et al. (25) recorded highly sensitive Flavobacterium spp. in carps of sewage-fed farms in West Bengal, India. They also recorded Flavobacterium spp. resistant to erythromycin, co-trimoxazole, oxytetracycline and nitrofurantoin with multidrug resistance index ranging from 0.000 to 0.667. Contrarily, 97 and 100% of the F. columnare strains were regarded as susceptible to sulfadimethoxine and ormetoprim (5:1) and oxytetracycline, respectively (12). Many earlier studies also reported no exceptional resistance to antimicrobial drugs among the environmental Flavobacterium strains (23,26,27).
In tropical Indian condition, water temperature and challenge mode induced variations in mortalities due to F. columnare infection were noted. Though the F. columnare strains were highly sensitive to antibiotics, with the emergence of antibacterial resistance, the effective preventive measures are warranted. Management measures such as maintenance of optimal stocking densities and water quality parameters, physical removal of biofilm on tank surfaces, adoption of good nursery hygiene, sanitation, and other health management measures would help to minimize the development and spread of columnaris disease.
Peer-review: Externally peer-reviewed.
Author Contributions: Concept: T.J.A; Design: T.J.A; Supervision: T.J.A.; Resource: T.J.A., S.S.; Materials: T.J.A., S.S.; Data Collection and/or Processing: S.S.; Analysis and/or Interpretation: S.S., T.J.A.; Literature Search: S.S.; Writing: S.S., T.J.A.; Critical Reviews: T.J.A.
Ethics Committee Approval: All the experimental protocols with catfish as an experimental animal were approved by the Ethical Committee, WBUAFS, Kolkata, India.
Conflict of Interest: The authors have no conflict of interest to declare.
Financial Disclosure: The research work was supported by the Indian Council of Agricultural Research (Grant F. 10(12)/2012-EPD dated 23.03.2012), Government of India, New Delhi under the Niche Area of Excellence program.
Acknowledgement: The authors thank the Vice-Chancellor, West Bengal University of Animal and Fishery Sciences, Kolkata for providing necessary facilities to carry out the work.
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Sudeshna Sarker (1) (iD), Thangapalam Jawahar Abraham (1) (iD)
(1) West Bengal University of Animal and Fishery Sciences, Faculty of Fishery Sciences, Department of Aquatic Animal Health, West Bengal, India
ORCID IDs of the authors: S.S. 0000-0002-0984-9671; TJ.A. 0000-0003-0581-1307
Please cite this article as: Abraham T.J., Sarker S. Isolation, Identifcation and Pathogenicity of Flavobacterium columnare SGM4 in Catfsh Clarias batrachus (Linnaeus 1758). Eur J Biol 2019; 78(2): 139-146. DOI: 10.26650/EurJBiol.2019.0027
Address for Correspondence: Thangapalam Jawahar Abraham
Revision Requested: 11.10.2019
Last Revision Received: 02.11.2019
Table 1. Phenotypic characteristics of Flavobacterium columnare strains (n=5) as assessed by conventional biochemical tests and VITEK 2 compact system (bioMerieux, France) Biochemical characteristics Reaction Conventional biochemical tests Colony colour Yellow Rhizoid colony + Gram reaction - Cell morphology, Long rod + Gliding motility + Oxidase - Oxidative/Fermentative reaction -/- Casein hydrolysis + Chondroitin sulphate degradation - Congo red reaction + Fibrinogen hydrolysis - Flexirubin pigment presence - Gelatin hydrolysis - Growth in selective cytophaga agar# + Vitek 2 Compact system 5-Keto D-gluconate (5KG) - Adonitol (ADO) - Ala-Phe-Pro-arylamidase (APPA) - Alpha-galactosidase (AGAL) - Alpha-glucosidase (AGLU) - Beta-alanine arylamidase pNA (BAlap) - Beta-galactosidase (BGAL) - Beta-glucuronidase (BGUR) - Beta-glucosidase (BGLU) - Beta-xylosidase (BXYL) - Citrate (sodium) (CIT) - Coumarate (CMT) + D-Cellobiose (dCEL) - D-Glucose (dGLU) - D-Maltose (dMAL) - D-Mannitol (dMAN) - Biochemical characteristics Biochemical characteristics Conventional biochemical tests Vitek 2-Compact system Colony colour D-Mannose (dMNE) Rhizoid colony D-Sorbitol (dSOR) Gram reaction D-Tagatose (dTAG) Cell morphology, Long rod D-Trehalose (dTRE) Gliding motility Ellman (ELLM) Oxidase Fermentation/ glucose (OFF) Oxidative/Fermentative reaction Gamma-glutamyl transferase (GGT) Casein hydrolysis Glu-Gly-Arg-arylamidase (GGAA) Chondroitin sulphate degradation Glutamyl arylamidase pNA (AGLTp) Congo red reaction Glycine arylamidase (GlyA) Fibrinogen hydrolysis H2S production (H2S) Flexirubin pigment presence L Pyrrolydonyl-arylamidase (PyrA) Gelatin hydrolysis L-Arabitol (IARL) Growth in selective cytophaga agar# L-Histidine assimilation (IHISa) Vitek 2 Compact system Lipase (LIP) 5-Keto D-gluconate (5KG) L-Lactate alkalinisation (ILATk) Adonitol (ADO) L-Lactate assimilation (ILATa) Ala-Phe-Pro-arylamidase (APPA) L-Malate assimilation (IMLTa) Alpha-galactosidase (AGAL) L-Proline arylamidase (ProA) Alpha-glucosidase (AGLU) Lysine decarboxylase (LDC) Beta-alanine arylamidase pNA (BAlap) Malonate (MNT) Beta-galactosidase (BGAL) O/129 Resistance (O129R) Beta-glucuronidase (BGUR) Ornithine decarboxylase (ODC) Beta-glucosidase (BGLU) Palatinose (PLE) Beta-xylosidase (BXYL) Phosphatase (PHOS) Citrate (sodium) (CIT) Saccharose/Sucrose (SAC) Coumarate (CMT) Succinate alkalinisation (SUCT) D-Cellobiose (dCEL) Tyrosine arylamidase (TyrA) D-Glucose (dGLU) Urease (URE) D-Maltose (dMAL) [beta]-N-acetyl-galactosaminidase (NAGA) D-Mannitol (dMAN) [beta]-N-Acetyl-glucosaminidase (BNAG) Biochemical characteristics Reaction Conventional biochemical tests Colony colour - Rhizoid colony - Gram reaction - Cell morphology, Long rod - Gliding motility - Oxidase - Oxidative/Fermentative reaction - Casein hydrolysis - Chondroitin sulphate degradation - Congo red reaction - Fibrinogen hydrolysis - Flexirubin pigment presence - Gelatin hydrolysis - Growth in selective cytophaga agar# - Vitek 2 Compact system - 5-Keto D-gluconate (5KG) - Adonitol (ADO) - Ala-Phe-Pro-arylamidase (APPA) - Alpha-galactosidase (AGAL) (-) (*) Alpha-glucosidase (AGLU) - Beta-alanine arylamidase pNA (BAlap) - Beta-galactosidase (BGAL) - Beta-glucuronidase (BGUR) - Beta-glucosidase (BGLU) - Beta-xylosidase (BXYL) - Citrate (sodium) (CIT) - Coumarate (CMT) - D-Cellobiose (dCEL) - D-Glucose (dGLU) - D-Maltose (dMAL) - D-Mannitol (dMAN) - VITEK 2 compact system identified the strains as Aeromonas salmonicida. (*): One strain exhibited a weak reaction. Table 2. Pathogenicity of Flavobacterium columnare SGM4 by abrasion and immersion challenge Treatment group and the challenge dose Mortality (%) Group A: Abraded and immersion challenged at 7.2x[10.sup.6] cells/mL 45.00[+ or -]5.00 Group B: Abraded and immersion challenged at 7.2x[10.sup.5] cells/mL 10.00[+ or -]0.00 Group C: Non-abraded and immersion challenged at 7.2x[10.sup.6] cells/mL 0.00[+ or -]0.00 Group D: Abraded and immersion in 0.85% saline 0.00[+ or -]0.00 Group E: Neither abraded nor challenged 0.00[+ or -]0.00 Table 3. Antibiotic-resistance in Flavobacterium columnare strains (n=5) isolated from columnaris diseased catfish Clarias batrachus Antibiotic ([micro]g/disc) Interpretation of zone size (in mm) Resistant ([less than or equal to]) Amoxyclav (30) 13 Chloramphenicol (30) 12 Ciprofloxacin (5) 15 Co-trimoxazole (25) 10 Erythromycin (15) 13 Gatifloxacin (5) 14 Gentamicin (10) 12 Nitrofurantoin (300) 14 Oxytetracycline (30) 11 Sulphafurazole (300) 12 Antibiotic ([micro]g/disc) Interpretation of zone size (in mm) Sensitive ([greater than or equal to]) Amoxyclav (30) 18 Chloramphenicol (30) 18 Ciprofloxacin (5) 21 Co-trimoxazole (25) 16 Erythromycin (15) 23 Gatifloxacin (5) 18 Gentamicin (10) 15 Nitrofurantoin (300) 17 Oxytetracycline (30) 15 Sulphafurazole (300) 17 Antibiotic ([micro]g/disc) Numbers Numbers resistant sensitive Amoxyclav (30) 0 5 Chloramphenicol (30) 0 5 Ciprofloxacin (5) 0 5 Co-trimoxazole (25) 1 4 Erythromycin (15) 1 4 Gatifloxacin (5) 0 5 Gentamicin (10) 0 5 Nitrofurantoin (300) 1 4 Oxytetracycline (30) 0 5 Sulphafurazole (300) 4 1
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|Author:||Sarker, Sudeshna; Abraham, Thangapalam Jawahar|
|Publication:||IUFS Journal of Biology|
|Date:||Dec 1, 2019|
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