Detecting Aphanomyces Invadans in Pure Cultures and EUS-infected Fish Lesions by Applying PCR
DOI:
https://doi.org/10.18034/mjmbr.v3i2.410Keywords:
PCR, EUS, Aphanomyces invadans, DNA extractionAbstract
Aphanomyces invadans is an oomycete fungus which causes Epizootic Ulcerative Syndrome (EUS) disease in wide range of fresh and brackish water fish worldwide imposing serious economic losses. A diagnostic procedure, based on a polymerase chain reaction method (PCR) was developed to detect infection of fish with the A.invadans. A set of primers (1APM 1F and 1APM 6R) was used to specifically amplify A. invadans DNA. The PCR amplifies a 400 bp amplicon. A protocol for the extraction of A. invadans DNA from infected fish tissue and pure fungal cultures was developed. The method was tested on seven EUS-susceptible fish species (snakehead, snakeskin gourami, moonlight gourami, koi carp, catfish, gold fish, climbing perch) and one EUS-resistant fish (tilapia), artificially infected with A. invadans and pure cultures of Aphanomyces spp., Saprolegnia spp., Achlya spp., and Allomyces sp. Detection of A. invadans was possible at the early stage of sampling, which was 24 hours post injection in both EUS-susceptible and resistant fish. Resistant fish was found to be PCR-negative after 6 days of inoculation but in susceptible fish PCR-positive results obtained even after day 28 or in dead fish. Therefore PCR may be a useful method for detection EUS infection in fish from early stage of disease onset.
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Afzali, S.F., Hassan, M.D., Mutalib A.R, A.M., Sharifpour, I. and Sabri, J. (2013). Isolation and Identification of Aphanomyces Species from Natural Water Bodies and Fish Farms in Selangor, Malaysia. Malaysian Applied Biology Journal. 42(2): 1-11.
Baldock, F.C., V. Blazer, R. Callinan, K. Hatai, I. Karunasagar, C.V. Mohan and M.G. Bondad-Reantaso. 2005.Outcomes of a short expert consultation on epizootic ulcerative syndrome EUS): Re-examination of causal factors, case definition and nomenclature. In P. Walker, R. Lester and M.G. Bondad-Reantaso (eds). Diseases in Asian Aquaculture V, pp. 555-585. Fish Health Section, Asian Fisheries Society, Manila.
Ballesteros, I., Martín, M.P., and Diéguez-Uribeondo, J. (2006). First isolation of Aphanomyces frigidophilus (Saprolegniales) in Europe. Mycotaxon. 95: 335–340.
Blazer, V.S., Lilley, J.H., Schill, W.B., Kiryu, Y., Densmore, C.L., Panyawachira, V., and Chinabut, S. (2002). Aphanomyces invadans in Atlantic Menhaden along the East Coast of the United States. Journal of Aquatic Animal Health. 14 (1): 1-10. DOI: https://doi.org/10.1577/1548-8667(2002)014<0001:AIIAMA>2.0.CO;2
Hutson, K. S. (2013). Infectious Diseases of Asian Seabass and Health Management. Biology and Culture of Asian Seabass Lates Calcarifer, 102. DOI: https://doi.org/10.1201/b15974-7
Jerry, D. R. (Ed.). (2013). Biology and Culture of Asian Seabass Lates Calcarifer. CRC Press. DOI: https://doi.org/10.1201/b15974
Kurata, O., Sanpei, K., Hikiji, K., and Hatai, K. (2002). A Galactose-Binding Protein Revealed as a Hemagglutinin in Aphanomyces piscicida. Fish Pathology. 37(1): 1–6. DOI: https://doi.org/10.3147/jsfp.37.1
Lilley, J., Hart, D., Panyawachira, V., Kanchanakhan, S., Chinabut, S., Soderhall, K., and Cerenius, L. (2003). Molecular characterization of the fish pathogenic fungus “Aphanomyces invadans”. Journal of Fish Diseases. 26(5): 263–275. DOI: https://doi.org/10.1046/j.1365-2761.2003.00448.x
Oidtmann, B., Schaefers, N., Cerenius, L., Soderhall, K., and Hoffmann, R. W. (2004). Detection of genomic DNA of the crayfish plague fungus Aphanomyces astaci (Oomycete) in clinical samples by PCR. Veterinary microbiology. 100(3): 269–282. DOI: https://doi.org/10.1016/j.vetmic.2004.01.019
Oidtmann, B., Steinbauer, P., Geiger, S., and Hoffmann, R. W. (2008). Experimental infection and detection of Aphanomyces invadans in European catfish, rainbow trout and European eel. Diseases of Aquatic Organisms. 82(3): 195– 207. DOI: https://doi.org/10.3354/dao01973
OIE. (2013). Infection with Aphanomyces invadans (Epizootic Ulcerative Syndrome). OIE Aquatic Animal Health Standards Commission. www.oie.int/international-standard-setting/aquatic-manual/access-online.
Phadee, P., Kurata, O., Hatai, K., Hirono, I., and Aoki, T. (2004). Detection and identification of fish-pathogenic Aphanomyces piscicida using polymerase chain reaction (PCR) with species-specific primers. Journal of Aquatic Animal Health. 16(4): 220–230. DOI: https://doi.org/10.1577/H03-047.1
Shariff, M., Soon, S., Lee, K.L., and Tan, L.T. (2000). Practical problems with PCR detection in Asia: the importance of standardization. DNA-Based Molecular Diagnostic Techniques: Research Needs for Standardisation and Validation of the Detection of Aquatic Animal Pathogens and Diseases. 45-51.
Tang, Y.W., Procop, G.W., and Persing, D.H. (1997). Molecular diagnostics of infectious diseases. Clinical chemistry. 43(11): 2021–2038. DOI: https://doi.org/10.1093/clinchem/43.11.2021
Vandersea, M.W., Litaker, R.W., Yonnish, B., Sosa, E., Landsberg, J.H., Pullinger, C., Moon-Butzin, P. (2006). Molecular assays for detecting Aphanomyces invadans in ulcerative mycotic fish lesions. Applied and environmental microbiology. 72(2): 1551. DOI: https://doi.org/10.1128/AEM.72.2.1551-1557.2006
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