History and clinical presentations
The two cases reported in this outbreak were presented to the Veterinary Teaching Hospital, University of Jos, two weeks apart i.e last week of July and first week of August, 2019. The chief complaint from both smallholder farmers was sudden high mortalities in their farms. The first farmer had about sixty pigs of varying ages and six of the pigs died before he was advised to dispose the rest of the pigs for slaughter, disinfect the farm and fallow it before restocking. The second farmer had four adult pigs and all died of the ASF. He was also advised to disinfect the farm and allow it to fallow before restocking. The clinical sings observed in both farms were sudden deaths with pigs in good body conditions, moderate multifocal areas of hyperemia on the skin and weakness in the infected pigs.
Postmortem examination (PM): The PM was carried out on two submitted carcasses and gross postmortem lesions observed were identified and recorded.
Histopathology: Tissue samples of the affected organs (spleen, lymph nodes, liver, kidney and heart) were collected and fixed in 10% neutral buffered formalin. The samples were dehydrated in graded concentrations of alcohol, cleared in xylene, and impregnated in paraffin wax. Samples were subsequently incubated in vacuum oven at 60oC, embedded in plastic embedding rings, cut into 5 µm sections using a microtome, deparaffinized with xylene, rehydrated in graded concentrations of alcohol, stained with haematoxylin and eosin, and viewed under light microscope at X40 objective as outlined by Bakeret al . (2000).
Virus isolation: African swine fever virus (ASFV), was recovered from pooled tissues cultured on porcine leucocytes primary cell line and presumptively identified by the haemadsorption test, with pig erythrocytes on the inoculated cell culture plate incubated for one day at 37OC in a CO2 incubator as outlined by the standard operating procedure for the isolation of ASFV by the European Union Reference Laboratory for ASF (EURL-ASF, 2013).
Polymerase Chain Reaction (PCR) confirmation of ASFV: Genomic DNA was extracted from tissue samples (spleen, lymph node and kidney) using QIAamp DNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s specifications. Lyophilized freeze-dried E70 from the reference laboratory for ASF (CISA-INIA, Madrid, Spain) was used as control of this study. The presence of ASFV DNA was confirmed by the amplification of 278bp fragment of the VP72 gene (OIE, 2018) using the following primer pairs (i) p72 D [GTACTGTAACGCAGCACAG (forward)] and p72-U [GGCACAAGTTCGGACATGT (reverse)] and (ii) CVR-FL1 [TCG GCC TGA AGC TCA TTA G (forward)] and [CVR-FL2 CAG GAA ACT AAT GAT GTT CC (reverse)]. (OIE, 2008). The conditions for the PCR assay were as follows: 19 PCR buffer (50 mM KCl, 10 mM Tris–HCl), 2 mM MgCl2, 0.4 µm concentration of primers, 0.2 mM dinucleotide triphosphates (dNTPs) and 2.5 U Taq polymerase in a total volume of 25 µl. The PCRs were performed in an Applied BioSystems® thermal cycler 9500 (Applied BioSystems, Waltham, MA, USA) withan initial denaturation at 94°C for 15 s, followed by 30 cycles of denaturation at 94°C for 15 s, annealing at 62°C for 15 s and extensionat 72°C for 15 s; and a final extension step at 72°C for 5 min t. The PCR products were resolved by electrophoresis in a 1.5 % agarose gel and the ladder used was 100 bp. The DNA amplicons were sequenced using Sanger’s sequencing by LGC genomics (GmbH, Berlin Germany). The electropherographs of the sequenced genes were aligned, trimmed and deposited in the Genbank.