Simultaneous detection of Flavobacterium columnare and Saprolegnia ferax during disease outbreak in cultured pacu, Piaractus brachypomus (Cuvier, 1818) from India

Chandra Bhushan  Kumar; Gaurava  Rai; Manoj Kumar Yadav; Ranjana Sinha; Neeraj Sood; Pravata Kumar Pradhan; Gaurav Rathore

doi:10.21077/ijf.2026.73.1.174690-11

Authors

Chandra Bhushan Kumar ICAR-NBFGR, Lucknow
Gaurava Rai ICAR-NBFGR, Lucknow
Manoj Kumar Yadav ICAR-NBFGR, Lucknow
Ranjana Sinha ICAR-NBFGR, Lucknow
Neeraj Sood ICAR-NBFGR, Lucknow
Pravata Kumar Pradhan ICAR-NBFGR, Lucknow
Gaurav Rathore ICAR-NBFGR, Lucknow

https://doi.org/10.21077/ijf.2026.73.1.174690-11

Keywords:

Pacu, Piaractus brachypomus, Flavobacterium columnare, Saprolegnia ferax, Molecular detection

Abstract

The present study reports the simultaneous detection of Flavobacterium columnare and Saprolegnia ferax in cultured pacu (Piaractus brachypomus), during winter. The gross lesions in moribund pacu showed erosion of fins, cotton-wool like growth on the body surface and prominent discolouration on skin and gills. Wet mount from the gill tissue showed haystacklike appearance, presumptive of columnaris disease. F. columnare was detected in gill tissues of diseased pacu by PCR using specific primers. Phylogenetic analysis of 16s rDNA amplified from DNA of diseased pacu gills revealed that the sequence belonged to genetic group 2 of F. columnare. Histopathological findings of the diseased fish showed extensive damage in gills. In addition, wet mount of surface lesions revealed long thin aseptate hyphae presumptive of oomycete infection. Skin and fin tissue from diseased pacu inoculated on GPY agar plates showed cotton wool like growth. The isolated oomycete was purified and identified as S. ferax through amplification and sequencing of ITS region. These results attribute the mortality of pacu to infection with F. columnare and S. ferax. Considering the increase of pacu culture in many countries, and specially its polyculture with major carps in India, the findings of the present study necessitate early detection of these pathogens for health management of pacu.

Keywords: Discolouration, Haystack-like, Histopathology, Oomycete infection, Molecular detection, ITS gene, Wet mount

Downloads

Download data is not yet available.

References

Abraham, T. J, Sarker, S., Dash, G., Patra, A., and Adikesavalu, H. 2017. Piaractus brachypomus (Cuvier, 1817) fries cultured in West Bengal , India. Aquaculture. 473, 223–227. https://doi.org/10.1016/j.aquaculture. 2017.02.016

Bader, J. A., Shoemaker, C. A. and Klesius, P. H. 2003. Rapid detection of columnaris disease in channel catfish (Ictalurus punctatus) with a new species-specific 16-S rRNA gene-based PCR primer for Flavobacterium columnare. J. Microbiol. Methods. 52(2), 209-220. https://doi.org/10.1016/S0167-7012(02)00208-7

Bandilla, M., Valtonen, E. T., Suomalainen, L. R., Aphalo, P. J. and Hakalahti, T. 2006. A link between ectoparasite infection and susceptibility to bacterial disease in rainbow trout. Int. J. Parasitol., 36 (9), 987–991. https://doi.org/10.1016/j.ijpara.2006.05.001

Baldissera, M. D., Souza, C. F., Dias, J. B., Da Silva, T. O., Tavares, G. C., Valladão, G. M. R., da Silva, A. S., Verdi, C. M., Santos, R. C. V., Vencato, M., Da Veiga, M. L., da Rocha, M. I. U. M., Cunha, M. A. and Baldisserotto, B. 2020. Branchial bioenergetics dysfunction as a relevant pathophysiological mechanism in freshwater silver catfish (Rhamdia quelen) experimentally infected with Flavobacterium columnare. Microb. Pathog., 138, 103817. https://doi.org/10.1016/j.micpath.2019.103817

Bernardet, J. F. 1997. Immunization with bacterial antigens: Flavobacterium and Flexibacter infections. Dev. Biol. Stand., 90, 179–188.

Bly, J. E., Lawson, L. A., Szalai, A. J. and Clem, L. W. 1993. Environmental factors affecting outbreaks of winter saprolegniosis in channel catfish, Ictalurus punctatus (Rafinesque). J. Fish Dis., 16(6), 541–549. https://doi.org/10.1111/j.1365-2761.1993.tb00890.x

Bruno, D. W., VanWest, P. and Beakes, G. W. 2011. Saprolegnia and other oomycetes. In: P. Woo, T. K. and Bruno, D. W. (Eds.) Fish Diseases and Disorders. Volume 3: Viral, Bacterial and Fungal Infections, 2nd edn. Wallingford/Oxon: CABI Publishing (pp 669–720).

Cao, H., Zheng, W., Xu, J., Ou, R., He, S. and Yang, X. 2012. Identification of an isolate of Saprolegnia ferax as the causal agent of saprolegniosis of Yellow catfish (Pelteobagrus fulvidraco) eggs. Vet. Res. Commun., 36(4), 239-244. https://doi.org/10.1007/s11259-012-9536-8.

Carraschi, S. P., Garlich, N., Souza-Pollo, A., Pereira, D. I. B., da Cruz, C. and Ranzani-Paiva, M. J. 2018. Isolation of Saprolegnia aenigmatica oomycetes and protocol for experimental infection of pacu (Piaractus mesopotamicus). Acta Sci. Biol. Sci., 40. https://doi.org/10.4025/actascibiolsci.v40i1.38186

Chattarjee, N. R. and Mazumdar, B. 2009. Induced breeding of pacu ( Piaractus brachypomus ) in captivity with pituitary extract. Aquac. Asia. 14, 2-23.

Cox, F. E. G. 2001. Concomitant infections, parasites and immune responses. Parasitol., 122, S23-S38.

Cutuli, M. T., Gibello, A., Rodriguez-Bertos, A., Blanco, M. M., Villarroel, M., Giraldo, A. and Guarro, J. 2015. Skin and subcutaneous mycoses in tilapia (Oreochromis niloticus) caused by Fusarium oxysporum in coinfection with Aeromonas hydrophila. Med. Mycol. Case Rep., 9, 7–11. https://doi.org/10.1016/j.mmcr.2015.06.002

Darwish, A. M., Ismaiel, A. A., Newton, J. C. and Tang, J. 2004. Identification of Flavobacterium columnare by a species-specific polymerase chain reaction and renaming of ATCC43622 strain to Flavobacterium johnsoniae. Mol. Cell. Probes, 18(6), 421-427. https://doi.org/10.1016/j.mcp.2004.07.002

Declercq, A. M., Haesebrouck, F., Van Den Broeck, W., Bossier, P. and Decostere, A. 2013. Columnaris disease in fish: A review with emphasis on bacterium-host interactions. Vet. Res., 44(1), 1-17. https://doi.org/10.1186/1297-9716-44-27

Decostere, A., Haesebrouck, F,, Charlier, G. and Ducatelle, R. 1999. The association of Flavobacterium columnare strains of high and low virulence with gill tissue of black mollies (Poecilia sphenops). Vet. Microbiol., 67(4), 287-298.

Dong, H. T., Nguyen, V. V., Le, H. D., Sangsuriya, P., Jitrakorn, S., Saksmerprome, V., Senapin, S. and Rodkhum, C. 2015a. Naturally concurrent infections of bacterial and viral pathogens in disease outbreaks in cultured Nile tilapia (Oreochromis niloticus) farms. Aquaculture, 448, 427–435. https://doi.org/10.1016/j.aquaculture.2015.06.027

Dong, H. T., Nguyen, V. V., Phiwsaiya, K., Gangnonngiw, W., Withyachumnarnkul, B., Rodkhum, C. and Senapin, S. 2015b. Concurrent infections of Flavobacterium columnare and Edwardsiella ictaluri in striped catfish, Pangasianodon hypophthalmus in Thailand. Aquaculture, 448, 142–150. https://doi.org/10.1016/j.aquaculture.2015.05.046

Durborow, R. M., Thune, R. L., Hawke, J. P. and Camus, A. C. 1998. Columnaris disease: a bacterial infection caused by Flavobacterium columnare. South. Reg. Aquac. Cent., 479, 31-44.

Egusa, S. and Nishikawa, T. 1965. Studies of a primary infectious disease in the so-called fungus disease of eels. Bull. Jpn. Soc. Sci. Fish., 31:804–813. https://doi.org/10.2331/suisan.31.804

Farmer, B. D. 2004. Improved methods for the isolation and characterization of Flavobacterium columnare. Thesis. Baton Rouge, LA: Department of Pathobiological Sciences, Louisiana State University

Figueiredo, H. C. P., Klesius, P. H., Arias, C. R., Evans, J., Shoemaker, C. A., Pereira Jr, D. J. and Peixoto, M. T. D. 2005. Isolation and characterization of strains of Flavobacterium columnare from Brazil. J. Fish Dis., 28(4), 199-204. https://doi.org/10.1111/j.1365-2761.2005.00616.x

Hawke, J. P. and Thune, R. L. 1992. Systemic isolation and antimicrobial susceptibility of cytophaga columnaris from commercially reared channel catfish. J. Aquat. Anim. Health, 4(2), 109-113.

Holt, R. A. 1987. Cythophaga psychrophila the causative agent of bacterial cold water disease in salmonids fish. PhD thesis, Oregon State University, Corvallis, OR

Hussein, M. M. A., Hatai, K. and Nomura, T. 2001. Saprolegniosis in salmonids and their eggs in Japan. J. Wildl. Dis., 37(1), 204-207. https://doi.org/10.7589/0090-3558-37.1.204

Johnsen, B. O. 1978. The effect of an attack by the parasite Gyrodactylus salaris on the population of salmon parr in the River Lakselva, Misvaer in northern Norway. J. Arct. Biol., 11(1), 7-9.

Ke, X. L., Wang, J. G., Gu, Z. M., Li, M. and Gong, X. N. 2009. Morphological and molecular phylogenetic analysis of two Saprolegnia sp. (Oomycetes) isolated from silver crucian carp and zebra fish. Mycol. Res., 113(5), 637-644. https://doi.org/10.1016/j.mycres.2009.01.008

Kiesecker, J. M., Blaustein, A. R. and Miller, C. L. 2001. Transfer of a pathogen from fish to amphibians. Conserv. Biol., 15(4), 1064-1070. https://doi.org/10.1046/j.1523-1739.2001.0150041064.x

Kotob, M. H., Menanteau-Ledouble, S., Kumar, G., Abdelzaher, M. and El-Matbouli, M. 2017. The impact of co-infections on fish: a review. Vet. Res., 47(1), 1-12. https://doi.org/10.1186/s13567-016-0383-4

Kumar, A., Pradhan, P. K., Das, P. C., Srivastava, S. M., Lal, K. K. and Jena, J. K. 2018. Growth performance and compatibility of pacu, Piaractus brachypomus with Indian major carps in polyculture system. Aquaculture, 490, 236–239. https://doi.org/10.1016/j.aquaculture.2018.02.052

Kumar, S., Stecher, G. and Tamura, K. 2016. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol. Biol. Evol., 33(7), 1870-1874. https://doi.org/10.1093/molbev/msw054

LaFrentz, B. R., García, J. C., Waldbieser, G. C., Evenhuis, J. P., Loch, T. P., Liles, M. R., Wong, F. S. and Chang, S. F. 2018. Identification of Four Distinct Phylogenetic Groups in Flavobacterium columnare With Fish Host Associations. Front. Microbiol., 9, 452. https://doi.org/10.3389/fmicb.2018.00452

LaFrentz, B. R., Waldbieser, G. C., Welch, T. J. and Shoemaker, C. A. 2014. Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and standard protocol for genomovar assignment. J. Fish Dis., 37(7), 657-669. https://doi.org/10.1111/jfd.12166

Lilley, J. H., Callinan, R. B., Chinabut, S., Kanchanakhan, S., Macrae, I. H. and Phillips, M. J. 1998. Epizootic Ulcerative Syndrome (EUS) Handbook In: Epizootic Ulcerative Syndrome (EUS) Technical Handbook. The Aquatic Animal Health Research Institute, Bangkok, pp 88.

Li, N., Zhu, Y., LaFrentz, B. R., Evenhuis, J. P., Hunnicutt, D. W., Conrad, R. A., Barbier, P., Gullstrand, C. W., Roets, J. E., Powers, J. L., Kulkarni, S. S., Erbes, D. H., García, J. C., Nie, P. and McBride, M. J. 2017. The type IX secretion system is required for virulence of the fish pathogen Flavobacterium columnare. Appl. Environ. Microbiol., 83(23). https://doi.org/10.1128/AEM.01769-17

Lochmann, R., Chen, R., C hu-Koo, F. W., Camargo, W. N., Kohler, C. C. and Kasper, C. 2009. Effects of carbohydrate-rich alternative feedstuffs on growth, survival, body composition, hematology, and nonspecific immune response of black pacu, Colossoma macropomum, and red pacu, Piaractus brachypomus. J. World Aquac. Soc., 40(1), 33-44. https://doi.org/10.1111/j.1749-7345.2008.00232.x

Mastan, S. A., Radha, M., Reddy, K. and Lakshmi, D. S. 2012. Oomycete infections in freshwater fishes. Int. J. Fish. Aquac., 4(9), 186-190. https://doi.org/10.5897/IJFA11.054

Medina-Morillo, M., Sotil, G., Cordero, G., Arteaga, C., Vásquez, M., Rengifo, G., Murrieta, G. and Yunis-Aguinaga, J. 2023. Pathogenic Aeromonas spp in Amazonian fish: Virulence genes and susceptibility in Piaractus brachypomus, the main native aquaculture species in Peru. Aquac. Rep., 32, 10181. https://doi.org/10.1016/j.aqrep.2023.101811

Morley, N. J. and Lewis, J. W. 2010. Consequences of an outbreak of columnaris disease (Flavobacterium columnare) to the helminth fauna of perch (Perca fluviatilis) in the Queen Mary reservoir, south-east England. J. Helminthol., 84(2), 186. https://doi.org/10.1017/S0022149X09990459

Pilarski, F., Rossini, A. and Ceccarelli, P. 2008. Isolation and characterization of Flavobacterium columnare (Bernardet et al., 2002) from four tropical fish species in Brazil. Braz. J. Biol., 68(2), 409-414. https://doi.org/10.1590/s1519-69842008000200025

Pickering, A. D. and Willoughby, L. G. 1982. Saprolegnia, infection of salmonid fish. In: Roberts, R. J. (Ed.), Microbial Diseases of Fish London, UK: Academic Press (pp. 271–297).

Ravindra, Pradhan, P. K., Pande, V., Yadav, M. K., Verma, D. K. and Sood, N. 2019. Modulation of the innate immune responses in Indian major carp, Catla catla following experimental infection with Flavobacterium columnare. Aquaculture, 510, 22–31. https://doi.org/10.1016/j.aquaculture.2019.05.015

Ravindra, Kumar, R., Ahmad, N., Verma, D.K., Kantharajan, G., Kumar, C.B., Paria, A., Swaminathan, T.R., Rathore, G., Sood, N., Pradhan, P.K. and Lal, K.K., 2022. Mortalities in cultured Pangasianodon hypophthalmus due to oomycete Saprolegnia parasitica infection in Uttar Pradesh, India. Aquac. Rep., 23, p.101047.

Richards, R. H. and Pickering, A. D. 1978. Frequency and distribution patterns of Saprolegnia infection in wild and hatchery‐reared brown trout Salmo trutta L. and char Salvelinus alpinus (L.). J. Fish Dis., 1(1), 69-82. https://doi.org/10.1111/j.1365-2761.1978.tb00006.x

Oliveira, M.S.B. and Tavares-Dias, M. 2025. Mutilation on gill filaments of Piaractus brachypomus (Characiformes: Serrasalmidae) caused by Braga patagonica (Crustacea: Cymothoidae), in the Brazilian Amazon. Rev. Bras. de Parasito., 34(4), e012725. https://doi.org/10.1590/s1984-29612025068

Rintamäki-Kinnunen, P., Bernardet, J. F. and Bloigu, A. 1997. Yellow pigmented filamentous bacteria connected with farmed salmonid fish mortality. Aquaculture 149, 1–14. https://doi.org/10.1016/S0044-8486(96)01431-7

Roberts, R. J. 2001. The Mycology of Teleosts, Fish Pathology (3rd edn.) W.B. Saunders, London, (pp 334)

Rowland, S. J. and Ingram, B. A. 1991. Diseases of Australian native freshwater fishes with particular emphasis on the ectoparasitic and fungal diseases of Murray cod (Maccullochella peeli), golden perch (Macquarie ambigua) and silver perch (Bidyanus bidyanus). Fish. Bull., 4, 1–33.

Sandoval-Sierra, J. V., Latif-Eugenin, F., Martín, M. P., Zaror, L. and Diéguez-Uribeondo, J. 2014. Saprolegnia species affecting the salmonid aquaculture in Chile and their associations with fish developmental stage. Aquaculture, 434, 462–469. https://doi.org/10.1016/j.aquaculture.2014.09.005

Sarker, S., Abraham, T. J., Banerjee, S., Adikesavalu, H. and Patra, A. 2017. Characterization, virulence and pathology of Flavobacterium sp. KG3 associated with gill rot in carp, Catla catla (Ham.). Aquaculture, 468, 579–584. https://doi.org/10.1016/j.aquaculture.2016.11.024

Sahoo, P. K., Swaminathan, T. R., Abraham, T. J., Kumar, R., Pattanayak, S., Mohapatra, A., Rath, S. S., Patra, A., Adikesavalu, H., Sood, N., Pradhan, P. K., Das, B. K., Jayasankar, P. and Jena, J. K. 2016. Detection of goldfish haematopoietic necrosis herpes virus (Cyprinid herpesvirus-2) with multi-drug resistant Aeromonas hydrophila infection in goldfish: First evidence of any viral disease outbreak in ornamental freshwater aquaculture farms in India. Acta Trop., 161, 8-17. http://dx.doi.org/doi:10.1016/j.actatropica.2016.05.004

Shoemaker, C. A., LaFrentz, B. R., Peatman, E. and Beck, B. H. 2018. Influence of native catfish mucus on Flavobacterium columnare growth and proteolytic activity. J. Fish Dis., 41(9) 1395–1402. https://doi.org/10.1111/jfd.12833

Shoemaker, C. A., Olivares-Fuster, O., Arias, C. R. and Klesius, P. H. 2008. Flavobacterium columnare genomovar influences mortality in channel catfish (Ictalurus punctatus). Vet. Microbiol., 127(3-4), 353-359. https://doi.org/10.1016/j.vetmic.2007.09.003

Singh, A. K. 2014. Emerging alien species in Indian aquaculture prospects and threats. J. Aquat. Biol. Fish, 2(1), 32-41.

Stringer-Roth, K. M., Yunghans, W. and Caslake, L. F. 2002. Differences in chondroitin AC lyase activity of Flavobacterium columnare isolates. J. Fish Dis., 25(11), 687-691. https://doi.org/10.1046/j.1365-2761.2002.00421.x

Suomalainen, L. R., Bandilla, M. and Valtonen, E. T. 2009. Immunostimulants in prevention of columnaris disease of rainbow trout, Oncorhynchus mykiss (Walbaum). J. Fish Dis, 32(8), 723-726. https://doi.org/10.1111/j.1365-2761.2009.01026.x

Suomalainen, L. R., Tiirola, M. and Valtonen, E. T. 2006. Chondroitin AC lyase activity is related to virulence of fish pathogenic Flavobacterium columnare. J. Fish Dis., 29(12), 757-763. https://doi.org/10.1111/j.1365-2761.2006.00771.x

Tien, N. T., Dung, T. T., Tuan, N. A. and Crumlish, M. 2012. First identification of Flavobacterium columnare infection in farmed freshwater striped catfish Pangasianodon hypophthalmus. Dis. Aquat. Organ., 100(1), 83-88. https://doi.org/10.3354/dao02478

Tiirola, M., Valtonen, E. T., Rintamäki-Kinnunen, P. and Kulomaa, M. S. 2002. Diagnosis of flavobacteriosis by direct amplification of rRNA genes. Dis. Aquat. Organ., 51(2), 93-100. https://doi.org/10.3354/dao051093

Verma, D. K. and Rathore, G. 2013. Molecular characterization of Flavobacterium columnare isolated from a natural outbreak of columnaris disease in farmed fish, Catla catla from India. J. Gen. Appl. Microbiol., 59(6), 417-424. https://doi.org/10.2323/jgam.59.417

Verma, D. K., Rathore, G., Pradhan, P. K., Sood, N. and Punia, P. 2015. Isolation and characterization of Flavobacterium columnare from freshwater ornamental goldfish Carassius auratus. J. Environ. Biol., 36(2), 433.

White, T. J., Bruns, T., Lee, S. and Taylor, J. W. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J. (Eds.). PCR protocols: a guide to methods and applications. New York, N.Y: Academic Press, Inc. 315–322.

Wood, S. E. and Willoughby, L. G. 1986. Ecological observations on the Fungal Colonization of Fish by Saprolegniaceae in Windemere. J. Appl. Ecol., 737-749. https://doi.org/10.2307/2403930

Zahran, E. and Risha, E. 2013. Protective role of adjuvant and potassium permanganate on oxidative stress response of Nile tilapia (Oreochromis niloticus) challenged with Saprolegnia ferax. SpringerPlus, 2:1–22. https://doi.org/10.1186/2193-1801-2-94

Zhang, C., Li, D. L., Chi, C., Ling, F. and Wang, G. X. 2015. Dactylogyrus intermedius parasitism enhances Flavobacterium columnare invasion and alters immune-related gene expression in Carassius auratus. Dis. Aquat. Organ., 116(1), 11-21. https://doi.org/10.3354/dao02902