Polymerase Chain Reaction (PCR) Detection Of The Genome Of African Swine Fever Virus (ASFV) From Natural Infection In A Nigerian Baby Warthog (Phacochoereus Aethiopicus)

Abstract

The National Veterinary Research Institute, Vom procured baby warthog (Phacochoreus aethiopicus) caught by hunters in the forest of Adamawa state of Nigeria to be screened for the presence of genome to African swine fever virus (ASFV). The Spleen, lymph nodes and liver tissues obtained from the warthog were accessed for the presence of African swine fever virus genomic DNA by the polymerase chain reaction (PCR). DNA extracted from these tissues were amplified for a region of ASFV DNA genome by the PCR using specific primers of 20bp (base pair) from a highly conserved region of the ASFV DNA genome. The amplified genome was visualized following gel electrophoresis of PCR products using 1.5% agarose gel in 1x tris-acetate buffer (TAE buffer). A single, discrete and specific band of expected size (278bp) when measured against 200bp (base pair) DNA molecular marker (Roche) was observed from all the three tissues tested. The detection of these bands confirms virus presence. This finding therefore, provides current evidence of the presence of ASFV in Nigerian warthogs. African swine fever virus (ASFV), the causative agent of African swine fever (ASF) is a unique, complex, icosahedral DNA arbovirus belonging to the genus asfivirus in the family asfaviridae (Dixon et al., 2000). The virus multiplies in the cytoplasm of the infected cells. In nature, maintenance and transmission of this virus involves the cycling of virus between argasid soft ticks (Ornithodoros moubata complex) and wild pig population (warthogs and bush pigs) in sub-Saharan Africa. This association is unlikely ever to be eliminated, hence making it potentially very difficult to eradicate the disease in Africa. And it appears that this continent will be the main reservoir of ASFV for the foreseeable future (Majiyagbe et al., 2004). The ASFV genome comprised of a linear double stranded DNA molecule which are covalently closed at both ends by a 37 nucleotide-long hair pin loop composed almost entirely of partially paired adenine (A) and thymine (T) residues (Dixon et al., 2000). Depending upon the virus isolate, the size of the DNA molecule oscillates between 170 and 190-kilo base pair (Kbp) (Murphy et al., 1995). ASF, a highly significant disease of domestic swine occurs in several disease forms ranging from per acute to sub-clinical or inapparent forms (Mebus and Dardiri, 1980 and Mebus, 1988). The disease entered Nigeria around 1997 through the southwestern region of the country most probably through cross border contacts with infected pigs/or pig products smuggled through the Nigerian border from neighboring Benin Republic who had earlier reported the outbreak of the disease (Anon, 1998a). Within a short period the disease became widely distributed through pig producing areas of Nigeria (Majiyagbe, 1999 and Luther, 2001) and has been causing a lot of devastation to the national pig herd. The present situations of ASF in Nigeria revealed that virtually all the pig producing centres are affected. Of the 36 states of the Federation, the disease has been confirmed through laboratory test in 18, covering the south-west, south-east and central states of Nigeria (Majiyagbe et al., 2004). Fast and accurate detection of ASF outbreaks is required to limit the spread of the disease because the disease resembles several other haemorrhagic diseases of pigs both clinically and pathologically. The application by PCR for the detection of regions of ASFV genomic DNA presents a sensitive and specific method of identifying the virus. The technique consists essentially of the enzymatic synthesis of millions of copies of a target DNA sequence (Saiki et al., 1985). Using a thermostable DNA polymerase (Saiki et al., 1988), and a succession of cycles that includes denaturation of the template DNA, hybridization of specific DNA primers to the template and extension of the primers, it becomes possible to enzymatically generate several copies of the target region. Using this highly sensitive technique, it became possible to detect the presence of ASFV genomic DNA in a Nigerian warthog. This communication herein reported is the first documented report of the detection of ASFV from a Nigerian warthog reported hitherto only in eastern and southern-African countries (Scott, 1965 and Plowright et al., 1969).

Keywords: PCR, ASF, ASFV, Oligonucleotide primers, Warthog

Nigerian Veterinary Journal Vol. 28 (2) 2007 pp. 63-67