Detection of African swine fever virus genomic dna in a Nigerian red river hog (Potamochoerus porcus)
IN sub Saharan Africa, the maintenance and transmission of African swine fever virus (ASFV) involves the cycling of virus between argasid soft ticks (Ornithodoros moubata) and the free living population of warthogs and bush pigs. This association is unlikely ever to be eliminated, making it very difficult to eradicate the disease in Africa, and ensuring that the continent will remain a main reservoir of ASFV for the foreseeable future (Majiyagbe and others 2004) This short communication is the first report of natural ASFV infection detected in a free living bush pig in Nigeria; previous reports have come from eastern and southern African countries only (Scott 1965, Plowright and others 1969) Tissue samples from a red river hog (Potamochoerus porcus) suspected to have died of tuberculosis at the Jos Wildlife Park, Plateau State, Nigeria, were tested for ASFV genomic DNA by PCR, according to a standard protocol (OIE 2000) DNA was extracted from samples of spleen, lymph node, liver and lung using the guanidinium thiocyanate method, as modified by Bloom and others (1990) Reagents and buffers used for both the DNA extraction and PCR were of molecular grade and supplied by the Onderstepoort Veterinary Institute for Exotic Diseases, South Africa. The final reaction volume of 25 ml PCR master mix comprised 2*0 ml extracted DNA template, 17*5 ml highperformance liquid chromatography grade water, 1*0 ml oligonucleotide primers, both upstream (PAS1: 5' ATGGATACCGAGGGAATAGC 3' and downstream (PAS2: 5' CTTACCGATGAAAATGATAC 3' 2*5 ml 10 x buffer solution (50mM potassium chloride, 10mM Tris HCl [pH 8*3] and 1*5mM magnesium chloride) 0*5 ml Taq polymerase (SA TaqMD) and 0*5 ml 100mM dNTPs. Each tube of PCR reaction mix was overlaid with 60 ml mineral oil to prevent evaporation of the reaction mixture during PCR. Each tube was placed in an automated PCR thermal cycler (FPHC3CD; Techne Cyclogene) for amplification for 30 cycles as follows: denaturation at 94degC for 15 seconds, annealing at 62degC for 15 seconds and extension at 72degC for 15 seconds. Gel electrophoresis was performed for 45 minutes at a constant voltage of 80 V on 10 ml of each amplified PCR product, using 1*5 per cent agarose gel in 1 x Tris acetate buffer (TAE buffer) containing ethidium bromide at a final concentration of 0*5 ml/ml and 2 ml of 0*25 per cent orange G in aqueous solution of 30 per cent glycerol as loading buffer. The gel was visualised under UV light and photographed using a Polaroid MP4 Land Camera (D11105B) A single discrete and specific band, of the expected size (278 base pairs [bp] when measured against a 50 bp marker (Roche) was observed from all five tissue samples (Fig 1) A band of the same size was also seen for the known positive control tissue. As expected, the negative control tissue produced no band. This result was reproducible in three repetitions of the assay. The association of ASFV with free living pigs in Africa is an established host parasite relationship, in which prolonged infection occurs with no manifestation of the disease. ASFV has been recovered previously from wild pigs in southern and eastern Africa, mainly from warthogs (Phacochoerus aethiopicus) (Scott 1965, Plowright and others 1969) and less frequently from red river hogs and giant forest hogs (Hylochoerus meinertzhageni) (Heuschele and Coggins 1965, Detray 1967) The virus has also been isolated from hippopotamuses, porcupines and hyenas (Cox 1963) The previous attempt to isolate ASFV in warthogs and bush pigs in Nigeria took place in Yankari National Park in the 1970s (Taylor and others 1977) no virus or antibodies to ASFV were found. Since that study, many changes are likely to have occurred in the epidemiology of the disease in Nigeria. In this preliminary study, ASFV DNA was detected in a Nigerian bush pig for the first time, using PCR. Although the study involved only one bush pig, this is an important finding, and suggests that widespread surveys of Nigerian bush pigs and warthogs should be undertaken to determine their involvement in the epidemiology of ASF in Nigeria. This should open up a rich field for investigation, as the disease status and degree of parasitism by soft ticks of the large numbers of warthogs and bush pigs in Nigeria is, at present, unknown. Further studies are in progress in an attempt to isolate the virus. Only tissue samples were submitted to the laboratory for PCR analysis; no serological test was carried out to detect antibodies to the virus. However, in southern and eastern Africa, extensive serological work has been carried out (Thomson and others 1983, Thomson 1985, Plowright and others 1994) Seroconversion rates in warthogs have been shown to vary from one area to another, and high rates of seropositivity have not always been associated with the presence of O moubata ticks. Although Taylor and others (1977) failed to find virus in the tissues of Nigerian warthogs or bush pigs, it is not safe to assume that these animals are less important in maintaining ASFV. The present study indicates that more wild pigs in Nigeria should be examined before they are ruled out as a possible reservoir of the virus. This is particularly important in Nigeria, where the role of these animal species in the epidemiology of ASF is still to be established. Such studies are extremely important, as they may reveal the presence of other ASF endemic foci. The existence of new foci would then modify the approach to the investigation, prevention and control of outbreaks. Veterinary Record (2007) 160, 58 59
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