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.