Detecting Aphanomyces Invadans in Pure Cultures and EUS-infected Fish Lesions by Applying PCR

Authors

  • Fatemeh Afzali UPM
  • Hassan Hj Mohd Daud UPM
  • Shiv Shankar Mokpo National University
  • M Shuaib Khan UPM
  • Samanesadat Afzali Institute of Standards and Industrial Research of Iran

DOI:

https://doi.org/10.18034/mjmbr.v3i2.410

Keywords:

PCR, EUS, Aphanomyces invadans, DNA extraction

Abstract

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.

 

Downloads

Download data is not yet available.

Author Biographies

  • Fatemeh Afzali, UPM

    Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor D.E, MALAYSIA

  • Hassan Hj Mohd Daud, UPM

    Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor D.E, MALAYSIA

  • Shiv Shankar, Mokpo National University

    Department of Food Engineering, Mokpo National University, 61 Dorimri, Chungkyemyon, Muangun, REPUBLIC OF KOREA

  • M Shuaib Khan, UPM

    Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor D.E, MALAYSIA

  • Samanesadat Afzali, Institute of Standards and Industrial Research of Iran

    Institute of Standards and Industrial Research of Iran, Sari, Mazandaran, IRAN

References

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

-- 0 --

Published

2016-12-31

Issue

Vol. 3 No. 2 (2016): July - December Issue

Section

Peer-reviewed Article

License

The CC BY-NC 4.0 license lets others remix, tweak, and build upon your work non-commercially, and although their new works must also acknowledge & be non-commercial, they don’t have to license their derivative works on the same terms.
|| View License Deed ||
|| View Legal Code ||
 
Publishing paper with the "Malaysian Journal of Medical and Biological Research" means that the author or authors retain the copyright in the paper.
This journal is granted an exclusive noncommercial reuse license by the author(s), but the author(s) are able to put the paper onto a website, distribute it to colleagues, give it to students, use it in your thesis, etc, so long as the use is not directed at a commercial advantage or toward private monetary gain.
The author(s) can reuse the figures and tables and other information contained in their paper published by Malaysian Journal of Medical and Biological Research in future papers or work without having to ask anyone for permission, provided that the figures, tables or other information that is included in the new paper or work properly references the published paper as the source of the figures, tables or other information, and the new paper or work is not direct at a private monetary gain or commercial advantage.

How to Cite

Afzali, F. ., Daud, H. H. M. ., Shankar, S. ., Khan, M. S. ., & Afzali, S. . (2016). Detecting Aphanomyces Invadans in Pure Cultures and EUS-infected Fish Lesions by Applying PCR . Malaysian Journal of Medical and Biological Research, 3(2), 75-84. https://doi.org/10.18034/mjmbr.v3i2.410
Crossref
Scopus
Google Scholar
Europe PMC