MICROBIOLOGY STUDIES ON THE AFFECTIONS OF SKIN IN SHARP TOOTH CATFISH (CLARIAS GARIEPINUS)

MICROBIOLOGY STUDIES ON THE AFFECTIONS OF SKIN IN SHARP TOOTH CATFISH (CLARIAS GARIEPINUS)

ISMAIL S. MOHAMAD^*; MOHAMED W. ABD AL-AZEEM^**; MAHMOUD M. MAHMOUD^*** and AML MOKHTAR M. ABD-EL AAL^****

^* Department of Microbiology and Immunology, Faculty of Medicine, Assiut University

^** Department of Microbiology and Immunology, Faculty of Veterinary Medicine, South Valley University

^*** Department of Fish Disease and Management, Faculty of Veterinary Medicine, Assiut University

^**** Veterinary Practitioner

Email: amlMokhtar 2011@hotmail.com

INTRODUCTION

African Sharptooth cat fish is widely accepted by consumers in Upper Egypt as a relatively cheap source of fish protein. Commercial farming of African sharptooth catfish Clarias gariepinus, has significantly increased in Upper Egypt over the past few years.

Marzouk, et al. (2003) concluded that in Egypt, the mycotic diseases constitute one of the most important diseases causing troubles in fresh and culture fish with several economic losses. Isolated Aspergillus flavus, Aspergillus, niger, Penicillium, Alternaria, Cladosporum, Fusarium, Mucor and Saprolegnia from naturally infected fish, this revealed the presence of clinical abnormalities in the form of skin darkening, necrotic foci with sloughing of tail and body fins with peticheal haemorrhages and cotton wool like growth on various parts of the skin with sloughing of the uppermost layers of skin.

Refai et al. (2010) reported that mycologyical examination of Clarias gariepinus catfish revealed that isolated moulds belonged to the following genera: Saprolegnia. Aspergillus. Fusarium, Mucor, Penicillium, Rhizopus and other genera.

Burgess et al. (2000) reported that ulcers and other bacterial lesions are a common fish disease problem.  They are one of the most difficult problems to deal with, especially if large numbers of fish are affected.

  * This Ms has bean extracted from Ph.D. thesis entitled {studies on Microbial skin Affections in Sharp Tooth Catfish (Clarias gariepinus)} by Ismail S. Mohamad; Mohamed W. Abd-El Azeem; Mahmoud M. Mahmoud and AmlMokhtar M. Abd-El Aal

Corresponding Author: AmlMokhtar, M.Abd -El Aal, Vet. Practitioner Egypt.

Eltallawy, (2008)mentioned that skin abrasion or gill damage act as the main portal of entry to fish and was essential to induce infection. Skin is the target organs for isolation of Flavobacterium spp.

Nguyen et al. (2012)concluded that the bacterium Flavobacterium columnare was recovered and identified as the aetiological agent causing freshwater columnaris infection in catfish that had suffered high mortality rates.

Analia Murias, (2012) recorded that bacteria of the genera Aeromonas and Pseudomonas were present in infected fish in Amazonin ponds caused mortality rate 100%. Hayes & Jon (2007): Fulton& MacDonald, (2008) detected that Aeromonas hydrophila is widely considered a major fish and amphibian pathogen, as will as pathogenic for humans. Rao et al. (2001), and Mathew et al. (2001) reported that Edwardsiella tarda is a responsible pathogen for Edwardsiella septicemia of catfish, also cause diseases in higher vertebrates including humans.

Huber et al. (2004) isolated and identified Pseudomonas and Proteus from external and internal lesions in naturally infected fresh water fish.   

Kar, (1999) stated that bacteriological examination of the surface lesions and other organs of fishes showing signs of bacterial infection result in isolation of haemolytic strains of Escherichia coli, Aeromonas hydrophila, Pseudomonas aeruginosa, Staphylococcus epidermitis and Klebsiella sp.

Also Bakeer et al. (1991) reported that Flavobacterium columnare, Pseudomonas sp. Aeromonas sp., Staphylococcus sp. And Proteus sp. caused ulceration in skin.

From the above mentioned data, it was important from the bacteriological and mycological point of view to investigate the role of different bacteria and fungi as disease etiology among the sharp tooth catfish in Assuit Governorate and it's around cities. 

MATERIALS and METHODS

Tow hundred forty fish samples (catfish Clarias gariepinus) collected from The River Nile and El-Ebrahemia canal and examined for determination of clinical abnormalitis and postmortem lesions according to Stoskopf (1993).

Isolation and Identification of Fungi

Samples of the collected fish  (skin, fins, tails and barbel)  were squashed and incubated at 25C for 5-10 days on Sabouraud dextrose agar and Czapek Dox agar (Biolife, Italy) during which the developing fungal colonies were counted and morphologicaly identified by lactophenol methylene blue. Wet mount preparation of the samples were commonly made in 10% KOH (Adwic). Fungal identification based on macro and microscopic characteristics following the key of  Moubasher, (1993), Noga, (1993) and Ellis D, (2007).

Isolation and Identification of bacteria

Samples of skin surface (skin, fins, tails and barble) were collected from C. gariepinus and streaked on Tryptone soya agar and Brain heart infusion agar (Biolife, Italy), Cytophaga agar medium according to Dian G Ellitt, (2003), Salmonella Shigella broth and Blood agar (Biolife, Italy) which are the most commonly used mediums for isolation and identification of the different bacterial strains in our study. Suspected bacterial colonies were picked up and subcultured for purification and further study. A single colony was inoculated in slant tube of BHI or TSA agar medium for identification by biochemical tests Also, isolates were kept in BHI orTSA broth with 20% (Vol./Vol.) glycerol at -20 °C for further investigations.

Antimicrobial susceptibility testing of isolated strains:  

The most frequent bacterial isolates were investigated against 12 antimicrobial agents using the disc agar diffusion technique according to Finegold and Martin, (1982).

The discs of the following antimicrobial agents Neomycin 30µg, Erythromycin 15µg, Enerofloxacin 5µg, Streptomycin 10µg, Oxolinic acid 30µg, Amoxicillin 25µg, Oxytetracycline 30µg, Ampicillin 10µg, Gentamycin 10µg, Chloramphenicol 30µg,, Sulfamethoxazole 100µg, and Nalidixic acid 30µg,   (Bioanalyse, Turkey) were used. Interpretation of the zones of inhibition were estimated according to the limits of NCCLS (2011).

RESULTS

Mycological examination revealed the isolation of 1200 fungal isolates from 240 Clarias gariepinus fish. The percentage of isolated moulds from skin of Clarias gariepinus fish were detected as Fusarium solani 210(17.5%) Aspergillus flavus 184(15.2%), Aspergillus niger 170(14.3%), Mucor hiemlis 162(13.5%) isolates, Penicillium chrysogenum 97(8.1%), Penicillium aurantiogriseum 95(7.9%), Chladosporium herbarum 85(7.1%), Saprolegnia 60 (5.0%) isolates, Rhizopus 54(4.5%) isolates, Chladosporium sphaerospermum 53(4.4%) Acremonium strictum  18(1.5%) isolates and Alternaria alternata 12 (1.0%) isolates (Fig.1).

Bacteriological examination of fish samples resulted in isolation of 370 isolates. According to cultural, morphological and biochemical characteristics. *Percentage of strains isolated were Flavobacterium columnare (115) 31.1%, Aeromonas hydrophila (75)20.3%, Edwardsiella tarda (57)15.4% Pseudomonas sp. (43)11.6%, Echerichia coli (21)5.7%, Proteus sp. (19)5.1%, Klebsiella (12) 3.2%, Streptococus sp. (15)4.1% and Staphylococcus sp. (13)3.5% (Fig.2).

Antibiotic sensitivity test reveled that F. columnarewas highly sensitive to Enerofloxacin, Oxytetracycline, and Chloramephenicol, while was highly resistant to Erythromycin, Neomycin and Amoxicillin A.hydrophilawas highly sensitive to Enerofloxacin, Oxytetracycline, Chloramephenicol and Neomycin while was highly resistant to Erythromycin, Ampcillin and Amoxicillin E.tardawas highly sensitive to Oxytetracycline, Chloramepheniol and Nalidixic acid, while was highly resistant to Erythromycin, Amoxicillin and Sulfamethazole, Pseudomonas sp.was highly sensitive to Enerofloxacin, Oxytetracycline, Chloramephenical and Nalidixic while highly  resistant to Erythromycin, Amoxicillin and Neomycin Table (1).

Table 1: Antibiotic sensitivity test for the prevelant isolated gram –ve bacteria

Photo. (1): Non septated broad hyphae of Saprolegnia sp., Photo. (2): Characteristic cotton –wool like growth of Saprolegnia, Photo. (3): P.aurantoigriseum on (SDA), Photo. (4): P.aueantiogriseum showing brush-like arrangement of fruiting head, Photo. (5): Uni and biseriate conidophores with conidia of Aspergillus flavus by lactophenol cotton blue stain, Photo. (6): Colonies of Aspergillus flavus on (SDA), Photo. (7): Colonies of Aspergillus niger on (SDA), Photo. (8): Aspergillus niger showing characteristic round head with black conidia, Photo. (9): Conidiophores and smooth-walled, ellipsoidal conidia, Photo. (10): Penicillium chrysogenum with different colour and texture on (SDA), Photo. (11): Fusaruim solani on (SDA) with the reverse, Photo. (12): Fusaruim solani with characteristic slender, multicelled conidia, Photo. (13): Rhizopus sp. colony on SDA showing dens wooly mycelia, Sporangia was seen as small black dots, Photo. (14): Rhizopus sp. showing long branched sporangiophores and terminate with rhizoids, Photo. (15): Conidiophores, part of a conidial chain, and liberated conidia of Alternaria alternate, Photo. (16): Grey, felty and powdery colonies of Alternaria alternate, Photo. (17): Pink colonies of Acremonium strictum on (SDA), Photo. (18): Conidiophores and conidia of Acremonium strictum, Photo. (19): Conidiophores and conidia of Cladosporium herbarum, Photo. (20): Charactarestic velvety, olive-green to olivaceous brown colonies of Cladosporium sphaerospermum on (SDA)

Photo. (1): Motility test +ve , Photo. (2): Gram –ve short rod bacilli E. tarda, Photo. (3): Gm –ve short rod of Flavobacterium columnare, Photo. (4): Gram –ve Areomonas hydrophila, Photo. (5): Sever heamorrhage on head Columnaris infection, Photo. (6): f. columnare infection, Photo. (7): haemorrhage and ulceration oftail, Photo. (8): Fin rot columnaris infection, Photo. (9): Pink colonies of Klebsiella on MacConcy agar, Photo. (10, 11): Swarming with irregular edges of F.columnare on (cytophaga agar), Photo. (12): Blue –black colonies with greenish metallic sheen of E.coli on (EMB),

DISCUSSION

Mycological incidence and distribution:

Mycological examination revealed the isolation of moulds belonged to the following genera: Aspergillus flavus, Aspergillus niger, Fusarium sp., Penicillium aurantiogriseum, Penicillium chrysogenum, Mucor hiemalis, Chladosporium sphaerospermum, Chladosporium herbarum, Saprolegnia, Rhizopus sp., Acremonium strictum and Alternaria sp. are isolated. The nearly same fungal isolates were reported by Abd El- Alim (1992); khalil (1993); Mohamed (1994); Marzouk et al. (2003) and Refai et al. (2010). The Clinical findings of Clarias gariepinus normally infected  with these fungi were exophthalmia, skin darkening on various parts of the body, moderate abdominal distention and haemorrhages  all over the body surface and white to brown hyphal growths on skin and fins.

Our study revealed that the main isolates from Catfish on both Czapek Dox agar and Sabouraud dextrose agar media at 25°C. were Fusarium solani 17.5%, Aspergillus flavus 15.2% and Aspergillus niger 14.3% the obtained results agree with Manal (1988); Salem et al. (1989); but Mohamed, N. (1994); Marzouk et al. (2003) and Refai et al. (2010) reported that Aspergillus flavus was the first  fungi isolated and Fusarium came  after that in their incidenecs that result are nearly similar to our result. El-Hissy et al. (1989) found that A. flavus and A.niger were the dominant aspergi­lli in Clarias gariepinus, Also Salem et al. (1989) and Bagy et al.  (1993) found the prevalent aspergilli that were isolated from skin and gills of Nile fishes A.flavus and A.niger. El-Hissy et al. (1989) isolated Penicillium, Fusarium and Mucor from Catfish.  Also, members of Mucor including M. hiemalis isolated from skin of catfish by (Bagy et al., 1993). In the current work, some fungi were isolated from skin of catfish Cladosporium sphaerospermum, Cladosporium herbarum, Alternaria alternata and Acremonium strictum dominant in fins and skin of fish, They were also isolated by Badran, (1989) and Mohamed, N (1994) Rhizopus isolated from samples of fins and skin. This was encountered from fish and Nile water by El-Zayat, (1988) and Badran, (1989) isolated Rhizopus species from fish in high incidence 100% from skin, fins and gills.

Alternaria alternate and Acremonium strictum was isolated in low percent  in our study but Alternaria alternate  appeared in high frequency of occurrence in both fins and skin (62- 80%) . Saprolegnia species were isolated from skin 5.0% in combination with other fungal and bacterial infection, in agree­ment with that reported by Robert, (1989) and Marzouk et al. (2003)who recorded that saprolegnia is a secondary fungal infection after any predisposing stress factors such as traumatic injury, cold stress and hormonal changes in the body of fish. Also, Refai et al. (2010)concluded that Aspergillius is the first isolate (43.0%) and Saprolegnia (4.2%) was the secondary fungal infecton.

Bacterial inciedance and distribution:         

The most frequent bacterial isolates was identified as Flavobacterium columnare (31.1%), Aeromonas hydrophila (20.3%), Edwardsiella tarda (15.4%) and Pseudomonas sp. (11.6%). They arenearly similar findings to that reported by Huber et al. (2004); Saad El-Deen, (2005); Abd El-Rahman and Elkamel, (2007); Hayes, John. (2007) and Eltallawy (2008).

Intensity of the skin lesions caused by infections depended on the fish species and causative agent(s) involved. The main clinical signs observed on naturally infected samples were erosions and ulceration of skin at the base of the dorsal fin and on the head, paleness, sloughing, erosion, petechial haemorrhages and loss of the tips of the fins and fin rot with separation of fin rays as previously reported by Durborow et al. (1998) and Tripathi et al. (2005).

Our result in the present study concluded that F. columnare was the first major isolates with incidence of 31.1%, in accordance with those reported by Eltallawy (2008)who concluded that F. columnareis the main cause of skin lesions and fin rot with incidence of 32.1% in sharptooth catfish.

Although in some cases,Bader et al. (2003) and Welker et al. (2005) can be isolated F. columnare from inner organs but skin and gills are the tissues of choice for isolation Abd EL-Rahman and Elkamel., (2007) concluded that the first bacterial isolate from skin of sharptooth catfish was F. columnare and the second cause was A. hydrophila which were in accordance with our result. But Atallah et al. (1997)reported that A. hydrophila was the first bacteria isolated from skin and fins of sharptooth catfish followed by Pseudomonas fluorescens. Also Plumb (1994) recorded that A. hydrophila was the most predominant bacteria isolated from fish species suffering from fin and tail rot. Some researchers believe that A. hydrophila is a primary fish pathogen while others consider it only a secondary invader of already weakened fish, this reported by Hayes John. (2007). In our result all A. hydrophila infected fish already infected with fungi. Eissa and Yassien (1994) reported that E. tarda one of the main causative agent of skin affections in sharptooth catfish. Moreover Saad El-Deen (2005) reported that E .tarda is the main isolate from skin of infected sharptooth catfish.Abd EL-Rahman and Elkamel (2007) reported that Pseudomonas.sp was the third major isolates from affected skin of sharptooth catfish. In the present study Pseudomonas sp. was the fourth major bacteria isolated so, this lower rate may be due to the fact that the fish had a generalized condition of septicemia rather than a confined case of bacterial  skin infection as the clinical signs of diseased fish described support this suggestion.  

Antibiotic test:

Antibiotic Disc agar diffusion  reveled that F. columnarewas sensitive to Enerofloxacin, Oxytetracycline, and Chloramephenical, while was  resistant to Erythromycin, Neomycin, Amoxicillin A. hydrophilawas sensitive to Enerofloxacin, Oxytetracycline, Chloramephenical and Neomycin, while was resistant to Erythromycin, Ampcillin and Amoxicillin E. Tardawas sensitive to Oxytetracycline, Chloramephenicol, Oxolinic acid and Nalidixic acid and was resistant to Erythromycin,Amoxicillin and Sulfamethazole Pseudomonas sp.was sensitive to Enerofloxacin, Oxytetracycline, Chloramephenical and Nalidixic acid while resistant to Erythromycin, Ampcillin, Amoxicillin and Neomycin These results are nearly similar to those reported by Kar, (1999); Dalsgaard and Madsen, (2000); Abd El-Rahman, (2002); Saad El-Deen,(2005); Abd EL-Rahman and Ahmad Elkamel (2007) and Eltallawy (2008).

REFERENCES

Abd El-Rahman A.A. and Elkamel, A. (2007): Bacterial causes of skin lesions in some freshwater fish. Alex. Jor. Vet. Med. Vol 25, No. 1.

Abdel-alim, K. (1992): The role of fish in transmitting some bacterial and fungal diseases to man. M.V.Sc. Thesis, Faculty of Vet. Med., Alexandria University.

Abd-El-Rahman, A.A. (2002): Bacteriological studies on the causative agent of bacterial gills disease (Flavobacterium columnare)in fresh water fish. 10^th Scientific Congress, Faculty of Veterinary Medicine, Assiut Univ. Egypt.

Analia Murias (2012): Amazonin fish mortality http://fis.com/fis/Worldnews/search brief.asp.  

Atallah, O.; Easa, M.; Abd-Elmonem, I.; Diab, A. and Saker, S. (1997):Pathologic changes associated with the fin-rot- inducing bacterial diseases in fresh-water fish. Alex. J. Vet. Science, vol. 13, No. 6: 629-644.

Aya, G. Saad El-Deen, (2005): Some Studies on Edwardsiellosis in Catfish (Clarias Gariepinus) in Assiut Governorate. Ph.D. Thesis Fac. Vet. Med. Assiut University.

Bader, J.A.; Nusbaum, K.E. and Shoemaker, C.A. (2003):Comparative challenge model of Flavobacterium columnare using abraded and unabraded channel catfish, Ictalurus punctatus (Rafinesque). J. Fish Dis. 26: 461-467.

Badran, R.A.M. (1989): Studies on fungi associated with Tilapia fish in River Nile water. Ph.D. Thesis, Botany Dept., Faculty of Science, Assiut University, Egypt.

Bagy, M.M.K.; Hemida, S.K. and Mahmoud, U.M. (1993): Terrestrial fungi inhabiting certain species of Nile fishes in Egypt. Zentralbl. Mikrobiol., 148: 289-297.

Bakeer, A.M.; Marzouk, M.S.M.; Abozid, A.; Moustafa, M. and Husean, M. (1991):Experimental morphopathological studies on tilapia infected by F. columnrae.Beni-Suef. Vet. Med. Vol.1: 53-65.

Burgess, P.; Bailey, M. and Exell, A.A-Z of Tropical Fish. Howell Books 2 ed. New York, NY. (2000): www.peteducation.com/article.cfm 

Dalsgaard, I. and Madsen, L. (2000):Bacterial pathogens in rainbow trout, Oncorhynchus mykiss (Walbaum), reared at Danish freshwater farms. J. Fish Dis. 23: 199-209.

Durborow, R.M.; Thune, R.L.; Hawke, J.P. and Camus, A.C. (1998):Columnaris Disease: a Bacterial Infection Caused by Flavobacterium columnare. SRAC (Southern Regional Aquaculture Center), Stoneville, MI, USA. Publication No. 479.

Diane G. Elliott (2003): General procedures for bacteriology U.S. Geological Survey, Biological Resources Discipline Western Fisheries Research Center Fish Health Section BLUE BOOK, 2007 EDITION. 

Eissa, I.A.M. and Yassieu, M.A. (1994): Some studies on Emphysematus putrefactive disease among catfish, Clarias lozera, in Lake Manzala. Alexandria Journal of Veterinary Science. 10 (1): 41-48.

El-Hissy, F.T.; Khallil, A.M. and El-Nagdy, M.A. (1989): Aquatic fungi associated with seven species of Nile fishes (Egypt). Zentralbl.  Mikrobiol., 144: 305-314.                                                                     

Eltallawy Hitham, H.M. (2008): Studies on Flavobacterial infection in African Sharptooth Catfish (Clarias Gariepinus). M.V.Sc Thesis, Fac. Vet. Med. Assiut University.

Ellis, D.; Davis, S.; Alexiou, H.; Handke, R. and Bartley, R. (2007):Description of medical fungi 2^nded,The National library of Australia.

El-Zayat, S.A.M. (1988):Studies on freshwater fungi of Aswan high Dam Lake Ph.D. Thesis, Botany dept. Faculty of Science Aswan University, Egypt.

Finegold, S.M. and Martin, W.J. (1982):Bailey and scott's Diagnostic Microbiology. 6 Ed. The C.V. Mosby Co., St. Lowis, Tornoto, London.

Fulton and MacDonald. (2008): "The Bacterium Aeromonas hydrophila from Lizards of the genus Anolis in Puerto Rico". Louisiana State University Medical Center, New Orleans.

Hayes, John. (2007): "Aeromonas hydrophila." Oregon State University. NewYork. http://hmsc.oregonstate.edu/classes/MB492/hydrophilahayes.

Huber, I.; Spanggand, B.; Appel, K.F.; Rossen, L.; Nielsen, T. and Gram, L. (2004):Phylogenetic analysis and in situ identification of the intestinal microbial community of rain bow trout {Oncorhynchus mykiss walbaum). Journal applied microbiology. 96 (1): 117-132.

Kar, D. (1999):Microbiological and Environmental Studies in relation to Fishes of India. Gordon Research Conference, Connecticut, USA.

Khalil, R.H. (1993): Some studies on mycotic infection in some freshwater fish with special reference to its control. Thesis; M.Sc.; Fish Diseases & Hygiene Alex. Univ. Fac. of Vet. Med.

Manal, A. (1988):Studies on mycotic infections in freshwater fish. M.V.Sc. Thesis, Zagazig University.

Marzouk, M.S.; Samira, S.R. and El-Gamal, M.H. (2003): Mycological investigations on cultured Tilapia in Kafcr El- Sheikh Governorate. Kafer El-Sheikh Vet.Med. J.,1 (2): 97-114.

Mathew, J.A.; Tan, Y.P.; Srinivasa Rao, P.S.; Lim, T.M. and Leug, K.Y. (2001): Edwardsiella tarda mutants defective in siderophore production, motility, serum resistance and catalase activity. Pathogenicity and medical microbiology Jor. 147: 449-457.

Moubasher, A.H. (1993): Soil Fungi in Qatar and other Arab countries. The center for Scientific and Applied Research University of Qatar, Doha, Qater (pp. 566).

Mohamed Nagla, A. (1994): Some studies on mycoflora of freshwater fish with special reference to Aspergiliosis. Ph.D. Thesis, Fac. Vet. Med. Assiut Univ.

Nguyen Thi Tien, Tu Thanh Dung, Nguyen Anh Tuan and Mags Crumlish (2012): First identification of Flavobacterium columnare infection in farmed freshwater striped catfish, Pangasianodon hypophthalmus DAO Vol. 100, No. 1 Print ISSN: 0171-8630.

Noga, E.J. (1993): Fungal and algal disease of temperate freshwater and estuarine fishes, in Stoskopf MK (Ed). Fish Medicine. Philadelphia, PA, Saunders, 278-283.

Plumb, J.A. (2011):Health Maintenance and Principal Microbial Diseases of Cultured Fished. Iowa State University Press, Ames, Iowa. 328 pp.

Rao, P.S.; Lim, T.M. and Leung, K.Y. (2001):Opsonized virulent Edwardsiella tarda strains are able to adhere to and survive and replicate within fish phagocytes but fail to stimulate reactive oxygen intermediates. Infectious and immunity. 69 (9): 5689-5697.

Refai, M.K.; Laila, A. Mohamed; Amany, M. Kenawy, and Shimaa, El.S.M.A. (2010): The assessment of mycotic settlement of fresh water fishes in Egypt. Jor. of American Science. 2010; 6(11).   

Roberts, R.J. (1989): The mycology of telcosts, in Roberts RJ (ed).Fish Pathology, 2nd edition. London, England, Baillere Tindall, pp       320-336.

Salem, A.A.; Refai, M.K.; Eissa, I.A.M.; Marzouk M.S.; Moustafa, M. and Manal, A. (1989): Mycological investigations on cultured Tilapia in Egypt. Alex. J. Vet. Sci, 5(2): 625-636.

Stoskopf, S.K. (1993):Bacterial diseases of freshwater fish. In fish medicine (ed. Stoskopf). W. B. Saunders, Philadelphia, London.

Tripathi, N.K.; Latimer, K.S.; Gregory, C.R.; Ritchie, B.W.; Wooley, R.E. and Walker, R.L. (2005): Development and evaluation of an experimental model of cutaneous columnaris disease in koi Cyprinus carpio. J. Vet. Diagnostic Investigation. 17: 45-54.

Welker, T.L.; Shoemaker, C.A.; Arias, C.R. and Klesius, P.H. (2005):Transmission and detection of Flavobacterium columnare in channel catfish Ictalurus punctatus. Dis. Aquat. Org. 63: 129-138.