Journal of General and Molecular Virology
Subscribe to JGMV
Full Name*
Email Address*

Article Number - 50671A948774


Vol.6(4), pp. 36-45 , November 2014
DOI: 10.5897/JGMV2014.0062
ISSN: 2141-6648



Full Length Research Paper

Comparison of immunoperoxidase monolayer assay, polymerase chain reaction and haemadsorption tests in the detection of African swine fever virus in cell cultures using Ugandan isolates



Mathias Afayoa
  • Mathias Afayoa
  • College of Veterinary Medicine, Animal Resources and Bio-security, Makerere University,P.O.BOX 7062, Kampala, Uganda.
  • Google Scholar
David Kalenzi Atuhaire
  • David Kalenzi Atuhaire
  • College of Veterinary Medicine, Animal Resources and Bio-security, Makerere University,P.O.BOX 7062, Kampala, Uganda.
  • Google Scholar
Sylvester Ochwo
  • Sylvester Ochwo
  • College of Veterinary Medicine, Animal Resources and Bio-security, Makerere University,P.O.BOX 7062, Kampala, Uganda.
  • Google Scholar
Julius Boniface Okuni
  • Julius Boniface Okuni
  • College of Veterinary Medicine, Animal Resources and Bio-security, Makerere University,P.O.BOX 7062, Kampala, Uganda.
  • Google Scholar
Kisekka Majid
  • Kisekka Majid
  • College of Veterinary Medicine, Animal Resources and Bio-security, Makerere University,P.O.BOX 7062, Kampala, Uganda.
  • Google Scholar
Frank Norbert Mwiine
  • Frank Norbert Mwiine
  • College of Veterinary Medicine, Animal Resources and Bio-security, Makerere University,P.O.BOX 7062, Kampala, Uganda.
  • Google Scholar
William Olaho-Mukani
  • William Olaho-Mukani
  • College of Veterinary Medicine, Animal Resources and Bio-security, Makerere University,P.O.BOX 7062, Kampala, Uganda.
  • Google Scholar
Lonzy Ojok
  • Lonzy Ojok
  • College of Veterinary Medicine, Animal Resources and Bio-security, Makerere University,P.O.BOX 7062, Kampala, Uganda.
  • Google Scholar







 Received: 14 October 2014  Accepted: 13 November 2014  Published: 25 November 2014

Copyright © 2014 Author(s) retain the copyright of this article.
This article is published under the terms of the Creative Commons Attribution License 4.0


African swine fever (ASF) is a devastating viral disease of pigs and is among the major hindrances to pig industry in sub-Saharan Africa including Uganda. The aim of this study was to compare immunoperoxidase monolayer assay (IPMA) to PCR in detection of ASF virus in infected macrophage cultures and to categorize ASF viral isolates in Uganda by haemadsorption assay. Field strains of ASF virus were isolated from infected pigs into swine alveolar macrophages culture. The effect of the inocula on the cell culture was monitored daily and the presences of ASF virus in the inoculated macrophages were detected using PCR and IPMA. The isolates were then categorized by haemadsorption assay. 58.8% of the samples had ASF virus DNA and ASF virus was isolated from 27% of the samples. IPMA detected ASF viral antigens in 80% of the inoculated macrophages culture 48 h post infection compared to the 100% by PCR. 95% of the virulent ASF viral isolates from Uganda were haemadsorbing. This study makes the first attempt to use IPMA and haemadsorption assay for the detection of ASF virus and categorization of the African swine fever virus (ASFv) field isolates into haemadsorbing and non-heamadsorbing in Uganda, respectively. The study demonstrates that IPMA is an appropriate option to PCR and could be used to detect ASF virus in cell cultures. It is recommended that the genome of the non-haemadsorbing ASF viral isolates could be sequenced and compared with that of haemadsorption (HAD) isolates to identify molecular peculiarities and markers of these two categories of ASFv. 
 
Key words: African swine fever (ASF), African swine fever virus (ASFv), Immunoperoxidase Monolayer Assay (IPMA), swine alveolar macrophages, haemadsorption (HAD) Polymerase Chain Reaction (PCR).

Aguero M, Fernandez J, Romer L, Sanchez Mascaraque C, Arias M, and Sanchez Vizcaino JM (2003). Highly sensitive PCR assay for routine diagnosis of African Swine fever virus in clinical samples. J. Clin. Microbiol. 41(9):4431-4434.
Crossref
 
Ana LR, Parkhouse R.M.E, Ana RP, Carlos M and, Alexandre L (2007). Systematic analysis of longitudinal serological responses of pigs infected experimentally with African swine fever virus. Portugal. Ave. Univ. Tec. Lisbon.
 
Anderson EC (1986). African swine fever, current concepts on its pathogenesis and immunology. Revised scientific technique for OIE, 5 (2): 477-486. Depat. Experiment. Pathol. animal virus Res. institute, pirbright, Surrey GU 24 ONF, UK.
 
Atuhaire DK, Afayoa M, Ochwo S, Mwesigwa S, Okuni J.B, Olaho-Mukani W, and Ojok L (2013). Molecular characterisation and phylogenetic study of African swine fever virus isolates from recent outbreaks in Uganda (2010 – 2013). J. Virol.10:247-248.
Crossref
 
Boinas FS, Hutchings GH, Dixon LK and, Wilkinson PJ (2004). Characterisation of pathogenic and non –pathogenic African swine fever virus isolated from Ornithodoros eraticus inhabitic pig premises in Portugal. J. Gen. virol. 85(8):2117-2187.
Crossref
 
Boinas FS., Cruz B, Portugal FC, Portugal R, Mendes s, Leitao A, Matin SC and, Rosinha A (2001). Evaluation of the role of ornithodorus erraticus as a reservoir of African swine fever in Alentego – Portugal. Report on the annual meeting of national swine fever laboratories. 97-98.
 
Borca MC, Carrillo C, Zsak L, Laegreid WW, Kutish GF, Neihan JG, Burraje TG and, Rock DL (1998). Deletion of a CD2 like gene, 8 DR from African swine fever virus affects viral infection in domestic swine. J. virol. 72:2881-2889.
 
Carolina urtado, Maria Jose Bustos, Angle L. Carrascosa (2010). The use of Cos – 1 Cells for studies of field and Laboratory African swine Fever Virus samples. Elsevier editorial system (tm) J. Virol. methods.
 
Carrascossa AL, Santeren JF and, Vinuela E (1982). Production and titration of African swine fever virus in porcine alveolar macrophages. J. Virol. methods 3:303-310.
Crossref
 
Casal I, Enjuanes L, Vinuela E, (1984). Porcine leukocytes cellular subsets sensitive to African swine fever virus in vitro. J. Virol. 52:37-46.
PMid:6384555 PMCid:PMC254486
 
Colgrove GS, Haelterman EO and, Coggins L (1969). Pathogenesis of African swine fever in young pigs. American J. Vet. Res. 30: 1343-1359.
 
Direksin K, Joo H and, Goyal MS (2002). An immunoperoxidase monolayer assay for detection of antibodies against swine influenza virus. J. Vet. Diagnos. investig. 14:169.
 
Dixon LK, Escribano JM, Martins C, Rock DL, Salas ML, Wilkinson PJ (2005). Asfarviridae in : Fauquent CM, Mayo MA, Maniloff J and Ball LA (eds). Virus Taxonomy, VIIIth Report of International Committee on Taxonomy of Viruses. London, UK. Elsev. Academ. Press: 135-143.
 
Gallardo C, Ademun AR, Nieto R, Nantima N, Arias M, Martin E, Pelayo V, Bishop RP (2011). Genotyping of African swine fever virus isolates associated with disease outbreaks in Uganda in 200. Afr. J Biotechnol. 10(17):3488-3497.
 
Gallardo C, Solar A, Nieto R, Carraishop RP, Martin C, Fasine FO, Couacy- Hymman E, Heath L, Pelayo V, Martin E, Simon A, Martin R, and Arias M (2012). Comparative evaluation of noval African swine fever virus antibody detection techniques derived from specific African swine fever viral genotypes with the OIE Internationally prescribed serological tests. Vet. microbiol.
 
Go Tag® green Master Mix 2012, Promega Corporation USA. Product information sheet.
 
James HE, Ebert K, Mc Gonigle R, Reid SM, Boonham N, Tomlinson JA, hutchings GH, Denyer M, oura CA, Dukes JP and, King DP (2010). Detection of African swine fever by loop mediated isothermal amplification. J. Virol. Methods. 1640:68-74.
 
Kay- Jackson PC, Goatley LC, Cox L, Miskin JE, Parkhouse RME, Wienands J And, Dixon LK (2004). The CD2v protein of African swine fever virus interact with the action binding adapter protein SH 3P7. J. Gen. Virol. 85:119-130.
Crossref
 
Kleiboecker SB (2002). Swine fever: classical swine fever and African swine fever. Vet. Clin. Food anim. 18:431-451.
 
Liang H, Wang H, Zhang L, Gu H and, Zhang G (2013). Development of a novel Immuno-Peroxidase Monolayer Assay for detection of Swine hepatitis E virus antibodies based on stable cell lines expressing the ORFS protein.
 
Malmquist, WA, and Hay D (1960). Haemadsoption and cytopathic effect produced by African Swine fever Virus in bone marrow and buffy coat cultures. Ame. J. Vet. Research. 21:104-108.
 
Mebus CA (1998). African swine fever In: Foreign animal diseases. United States animal Health Association, Richmound. 52-61.
 
OIE Terrestrial manual (2012). African swine fever; Chapter 2.8.1 section 2.8 SUIDAE. Version adopted by the World assembly of delegates of the OIE in May 2012.
 
Oura CA, powell PP, Parkhouse RM, (1998). Detection of African swine fever virus in infected pig tissues by immunocytochemistry and in situ hybridisation. J. Virol. Methods. 72: 205-217.
Crossref
 
Oura CAL, Edwards L, and balten CA (2012). Virological diagnosis of African swine fever. Comparative study of available tests.
 
Pan IC and Hess WR (1984). Virulence in African swine fever: its measurement and implication. Ame. J. Vet. Res. 45:361-366.
PMid:6711963
 
Ruteberika C and, Nantema (2002), Uganda and neighbouring under serious threat of African swine fever. Emergency prevention system for transboundary Animal Pests and Diseases. 
 
Sajjakambwe P, Okee-Acai J and, Ojok L (2012). Prevalence of African swine fever viral antigens in slaughter pigs at Nalukolongo abattoir, Kampala. ROAVS. www.roavs.com. ISSN 2221-1896.
 
Solenne C, Barbara W, Willian de Glanville, Ferran J, Rebecca R, Wilna V, Francois R, Dirk UP and, Linda KD (2009). African Swine fever, How can Global Spread be Prevented, philosophical transactions. UK, J.Royal soc. Pub. Biol. Sci. 364(1530):2683-2696.
Crossref
 
Strobe (2001). Trypan blue exclusion test for cell viability. Current protocol in immunology. US National Library of Medicine, National institute of Health. Crossref
 
Tejler Emma (2012). Outbreak of African swine fever in domestic pigs in Gulu distric- Uganda. Swedish university of Agricultural sciences (SLU). ISSN 1652-8697.
 
Vigario JD, Terrinha AM and, Moura Nunes JF (1974). Antigenic relationships among strains of African swine fever virus. Arch Gesamte virus forsch 5:272-277.
Crossref
 
Wilkinson PJ (2000). African swine fever manual of standards for diagnostic test and vaccines. 4th Edition, International Office for Epizootics, Paris France. 189 -198. www.qiagen.com/products/dna/qiaamp-dna-mini kit, QIAiamp® DNA and Blood mini handbook (2012)
 
Zsak L, Lu Z, Burrage TG, Neiland JG, kutish GF, Moore DM and, Rock DL (2001). African swine fever virus multigene family 360 and 530 genes are novel macrophage host range determinants. J. Virol. 75: 3066-3076.
Crossref

 


APA Afayoa, M., Atuhaire, D. K., Ochwo, S., Okuni, J. B., Majid, K., Mwiine, F. N., Olaho-Mukani, W., & Lonzy Ojok (2014). Comparison of immunoperoxidase monolayer assay, polymerase chain reaction and haemadsorption tests in the detection of African swine fever virus in cell cultures using Ugandan isolates. Journal of General and Molecular Virology, 6(4), 36-45.
Chicago Mathias Afayoa, David Kalenzi Atuhaire, Sylvester Ochwo, Julius Boniface Okuni, Kisekka Majid, Frank Norbert Mwiine, William Olaho-Mukani and Lonzy Ojok. "Comparison of immunoperoxidase monolayer assay, polymerase chain reaction and haemadsorption tests in the detection of African swine fever virus in cell cultures using Ugandan isolates." Journal of General and Molecular Virology 6, no. 4 (2014): 36-45.
MLA Mathias Afayoa, et al. "Comparison of immunoperoxidase monolayer assay, polymerase chain reaction and haemadsorption tests in the detection of African swine fever virus in cell cultures using Ugandan isolates." Journal of General and Molecular Virology 6.4 (2014): 36-45.
   
DOI 10.5897/JGMV2014.0062
URL http://academicjournals.org/journal/JGMV/article-abstract/50671A948774

Subscription Form