False positive results using PCR detection method for African swinefever virus in wild boars from northern Romanian hunting zones
African swine fever virus (ASFV) is an acute virus with a tropism for the pig macrophage and the ability to persist. In the absence of a vaccine, a good understanding of the ecology and epidemiology of the disease is fundamental to implement effective control measures. The recent occurrence and spread of ASFV in East Europe is perceived as a serious risk for the pig industry in the European Union. The aim of this study was to evaluate the classic detection method using polymerase chain reaction (PCR). A total number of 107 wild boar blood, tissues, and organs samples were collected from hunting areas of Romania's northern counties, from which 24 samples were positive by conventional PCR using the OIE manual for ASFV diagnostics. The positive samples were analyzed by sequencing techniques and the results were negative. Furthermore, we obtained only sequences that corresponded with a predicted uncharacterized mRNA from the Sus scrofa genome, leading to false positive results. Due to these results, an improvement in the detection method should be made, diagnostics should be based on multiple molecular tests, and a continuous monitoring plan for Romania should be applied to avoid the appearance of outbreaks.
False positive results using PCR detection method for African swinefever virus in wild boars from northern Romanian hunting zones
African swine fever virus (ASFV) is an acute virus with a tropism for the pig macrophage and the ability to persist. In the absence of a vaccine, a good understanding of the ecology and epidemiology of the disease is fundamental to implement effective control measures. The recent occurrence and spread of ASFV in East Europe is perceived as a serious risk for the pig industry in the European Union. The aim of this study was to evaluate the classic detection method using polymerase chain reaction (PCR). A total number of 107 wild boar blood, tissues, and organs samples were collected from hunting areas of Romania's northern counties, from which 24 samples were positive by conventional PCR using the OIE manual for ASFV diagnostics. The positive samples were analyzed by sequencing techniques and the results were negative. Furthermore, we obtained only sequences that corresponded with a predicted uncharacterized mRNA from the Sus scrofa genome, leading to false positive results. Due to these results, an improvement in the detection method should be made, diagnostics should be based on multiple molecular tests, and a continuous monitoring plan for Romania should be applied to avoid the appearance of outbreaks.
___
- Fauquet CM, Mayo MA, Maniloff J, Deselberger U, Ball LA. Virus Taxonomy: VIIIth Report of the ICTV. London, UK: Elsevier Academic Press; 2005.
- Costa JV. African swine fever virus. In: Darai G, editor. Molecular Biology of Iridoviruses. London, UK: Kluwer Academic Publishers; 1990. p. 247.
- Costard S, Mur L, Lubroth J, Sanchez-Vizcaino J, Pfeiffer D. Epidemiology of African swine fever virus. Virus Res 2013; 173: 191–197.
- Wesley R, Tuthil A. Genome relatedness among African swine fever virus filed isolates by restriction endonuclease analysis. Prev Vet Med 1984; 2: 53–62.
- Michaud V, Randriamparany T, Albina E. Comprehensive phylogenetic reconstructions of African swine fever virus: proposal for a new classification and molecular dating of the virus. PLoS One 2013; 8: e69662.
- Blasco R, Agüero M, Almendral J, Viñuela E. Variable and constant region in African swine fever virus DNA. Virology 1989; 168: 330–338.
- Bastos A, Penrith M, Cruciere C, Edrich J, Hutchings G, Roger F. Genotyping field strains of African swine fever virus by partial p72 gene characterisation. Arch Virol 2003; 148: 693– 706.
- Lubisi B, Bastos A, Dwarka R, Vosloo W. Molecular epidemiology of African swine fever in East Africa. Arch Virol 2005; 150: 2439–2452.
- Rowlands R, Michaud V, Heath L, Hutchings G, Oura C, Vosloo W, Dixon L. African swine fever virus isolate, Georgia, 2007. Emerg Infect Dis 2008; 14: 1870–1874.
- Boshoff C, Bastos A, Gerber L, Vosloo W. Genetic characterisation of African swine fever viruses from outbreaks in southern Africa (1973–1999). Vet Microbiol 2007; 121: 45– 55.
- Beltran-Alcrudo D, Lubroth J, Depner K, De La Rocque S. African Swine Fever in the Caucasus. Rome, Italy: FAO EMPRES Watch; 2008.
- Gallardo C, Soler A, Martín E, Pelayo V, Simón A, Arias M. Report on the molecular characterization of African swine virus (ASFV) samples received from Azerbaijan. FAO EMPRES Transboundary Animal Diseases Bulletin 2009; 33: 17–21.
- Beltrán-Alcrudo D, Khomenko S, Dietze K. FAO takes a close look at the threat of African swine fever introduction into Eastern Europe, specifically Ukraine. FAO EMPRES Transboundary Animal Diseases Bulletin 2010; 36: 2–17.
- Mur L, Martínez-López B, Costard S, de la Torre A, Jones B, Martínez M, Wieland B. Modular framework to assess the risk of African swine fever virus entry into the European Union. BMC Vet Res 2014; 10: 145.
- Oura CAL, Arias M. African swine fever. In: OIE Biological Standards Commission, editor. OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. 7th ed. Paris, France: Office International des Epizooties; 2012. pp. 1069–1082.
- Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molec Biol Evol 2011; 28: 2731–2739.
- Hall TA. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT. Nucl Acids Symp Ser 1999; 41: 95–98.
- Gallardo C, Reis A, Kalema-Zikusoka G, Malta J, Soler A, Blanco E, Parkhouse R, Leitao A. Recombinant antigen targets for serodiagnosis of African swine fever. Clin Vaccine Immunol 2009; 16: 1012–1020.
- Gallardo C, Okoth E, Pelayo V, Anchuelo R, Martín E, Simón A, Llorente A, Nieto R, Soler A, Martín R et al. African swine fever viruses with two different genotypes, both of which occur in domestic pigs, are associated with ticks and adult warthogs, respectively, at a single geographical site. J Gen Virol 2011; 92: 432–444.
- Tignon M, Gallardo C, Iscaro C, Hutet E, Van der Stede Y, Kolbasov D, De Mia GM, Le Potier MF, Bishop RP, Arias M et al. Development and inter-laboratory validation study of an improved new real-time PCR assay with internal control for detection and laboratory diagnosis of African swine fever virus. J Virol Methods 2011; 178: 161–170.
- Oura CAL, Edwards L, Batten C.A. Virological diagnosis of African swine fever—comparative study of available tests. Virus Res 2013; 173: 150–158.
- Steiger Y, Ackermann M, Mettraux C, Kihm U. Rapid and biologically safe diagnosis of African swine fever virus infection by using polymerase chain reaction. J Clin Microbiol 1992; 30: 1–8.