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\begin{document}

\title{Laboratory diagnosis of a new outbreak of acute African swine fever in
smallholder pig farms in Jos, Nigeria, in 2019}



\author[1]{Emmanuel TIZHE}%
\author[2]{Pam Luka}%
\author[3]{Adeyinka Adedeji}%
\author[4]{Polycarp Tanko}%
\author[4]{George Gurumyen}%
\author[4]{Deborah Buba}%
\author[5]{Ussa Tizhe}%
\author[4]{Asinamai Bitrus}%
\author[4]{Arthur Oragwa}%
\author[4]{Samson Shaibu}%
\author[4]{Essieniffiok Unanam}%
\author[6]{Ikechukwu Igbokwe}%
\author[7]{Stephen Akpavie}%
\author[4]{Celestine Njoku}%
\affil[1]{University of Jos}%
\affil[2]{National Veterinary Research Institute Vom}%
\affil[3]{National Veterinary Research Institute, Vom, Nigeria}%
\affil[4]{University of Jos, Nigeria}%
\affil[5]{Ahmadu Bello University}%
\affil[6]{University of Maiduguri, Nigeria}%
\affil[7]{University of Ibadan}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
African swine fever is a highly contagious fatal infectious disease of
pigs with a worldwide occurrence and economic importance. Two adult
large white boars from two farms in Jos North, Plateau State-Nigeria
kept under intensive management system were diagnosed of African swine
fever between the last week of July and the first week of August 2019 at
the Veterinary Teaching Hospital University of Jos. In both cases the
farmers complained of sudden deaths of pigs. At post mortem examination
carcasses grossly showed splenomegaly, hemorrhagic lymphadenitis, and
hepatomegaly with severe congestion. The kidneys were enlarged and had
generalized petechiae and blood clot in the pelvis.The heart was
moderately enlarged.On microscopy, the spleen and lymph nodes, showed
severe lymphocytic depletion, haemorrhage and severe haemosiderosis. The
liver was severely congested with focal coagulative necrosis of the
hepatocytes. The kidneys were severely congested and showed renal
tubular necrosis with few tubular protein casts. Tissue samples were
confirmed to be positive for ASFV by polymerase chain reaction and
phylogenetic analysis revealed that the isolate belonged to genotype-I.
Sequences obtained were compared and deposited in the GenBank and were
accessioned MN888693 and MN888694. Keywords: African swine fever;
laboratory; diagnosis; smallholder farms; pig; Nigeria%
\end{abstract}%



\sloppy


\textbf{Introduction}

African swine fever (ASF) is a highly contagious, fatal, economically
important, transboundary, viral disease of pigs caused by a DNA virus
belonging to the genus Asfavirus and in the family Asfaviridae
(Murphy\emph{et al} ., 1999; FAO, 2013; Igbokwe and Maduka, 2018). The
first ASF outbreak in Nigeria was reported in 1998 at a farm located in
Lagos (FAO, 1998; Odemuyiwa \emph{et al} ., 2000). The disease has
spread widely within the country and has become endemic resulting in
huge economic losses in the pig industry (Majiyagbe, 1999; Luther, 2001;
Costard \emph{et al} ., 2009; Fadiga \emph{et al} ., 2013; Igbokwe and
Maduka, 2018). It is a very fatal disease that can cause up to 100\%
mortality in a na\selectlanguage{ngerman}ïve pig population (Costard \emph{et al} ., 2009). Poor
biosecurity, bad abattoir practices and extensive or free range pig
farming systems led to extensive spread of the disease in the country
(Owolodun \emph{et al} ., 2010). African swine fever was previously
thought to exist only within domestic pig population with humans and
other formites potentiating its spread, however, Luther \emph{et al} .
(2007a,b) reported the existence of ASF virus in the bush pig
(\emph{Potamochoerus porcus} ) and warthog (\emph{Phacochoerus
aethiopicus} ). It is therefore now a known fact that both domestic and
wild pigs are susceptible to ASFV but the suids are hitherto said to be
asymptomatic. The soft tick (Ornithodoros moubata) has been established
as the primary reservoir of the virus (Luka \emph{et al} ., 2016;
Luka\emph{et al} ., 2017a). Areas with high pig-related activities such
as marketing, consumption, and farming, have higher prevalence compared
with areas with less pig-related activities. Farm-gate buyers, marketing
systems and transport of untested pigs within the country has greatly
aided the circulation of the virus in the country (Fasina \emph{et al}
., 2010) The disease is characterized by a febrile syndrome, erythema
and cyanosis of the skin, anorexia, bloody diarrhea, abortion in
pregnant sows, meningitis, interstitial pneumonia, high morbidity and
mortality (Fasina \emph{et al.} 2010; Igbokwe and Maduka, 2018).
Diagnosis of ASF is based on history, clinical signs, lesions and
laboratory confirmation by viral isolation, and molecular
characterization. The report represents the investigation of an acute
ASF outbreak (July-August, 2019) in pig farms in Jos, Nigeria.

\textbf{Materials and Methods}

\textbf{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.

\textbf{Postmortem examination (PM):} The PM was carried out on two
submitted carcasses and gross postmortem lesions observed were
identified and recorded.

\textbf{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
60\textsuperscript{o}C, embedded in plastic embedding rings, cut into 5
\selectlanguage{greek}µ\selectlanguage{english}m 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
Baker\emph{et al} . (2000).

\textbf{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 37\textsuperscript{O}C in a CO\textsubscript{2} incubator as
outlined by the standard operating procedure for the isolation of ASFV
by the European Union Reference Laboratory for ASF (EURL-ASF, 2013).

\textbf{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
MgCl\textsubscript{2}, 0.4 \selectlanguage{greek}µ\selectlanguage{english}m concentration of primers, 0.2 mM
dinucleotide triphosphates (dNTPs) and 2.5 U Taq polymerase in a total
volume of 25 \selectlanguage{greek}µ\selectlanguage{english}l. The PCRs were performed in an Applied BioSystems\selectlanguage{ngerman}®
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 \selectlanguage{english}final extension step at 72degC 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.

\textbf{Results}

Postmortem examination revealed splenomegaly and severe congestion 2/2
(figure 1), enlarged and haemorrhagic lymphnodes 2/2 (figure 2),
hepatomegaly and severe congestion 1/2 (figure 3a), white multi-focal
patches on the liver 1/2 (figure 3b), globosed, severely congested,
generalized cortical petechiae and severe blood clot in the pelvis 2/2
(figures 4a and b). Histopathological findings were thus: spleen: severe
lymphocytes depletion (figure 5). Lymph node: severe lymphocytes
depletion and severe hemosiderosis (figure 6). Liver: severe congestion
with focal areas of coagulation necrosis (figure 7). Kidney: severe
congestion and renal tubular and glomerular necrosis with few tubular
proteins cast (figures 8a and b).

Viral isolation on the culture plate was revealed by haemadsorption. The
presence of the virus typified by the attachment of large numbers of pig
erythrocytes to the surface of infected cells thereby forming the
rosette appearance (figure 9) as opposed to the negative wells which
were not inoculated with the infected tissues (figure 10).

Gel picture (Figure 11) shows that DNA fragments of 278bp (confirming
the presence of ASFV) were successfully extracted and amplified from the
spleen, lymph node and kidney of the infected animals. Note that the
size of the PCR products in lanes 1, 2, and 3, which were ASFV positive
amplicons presented similar DNA fragment base pairs to the product of
the positive control of ASFV in lane 4. The nucleotide sequences of ASFV
generated from this study were deposited in the Genbank with accession
numbers MN888963 and MN888964.

The result of phylogenetic analysis based on the p72 (B646L) gene is
presented in figure 12. It shows the ancestral relatedness of the
sequence from this study to other 53 sequences downloaded from the
Genbank database, including those from Nigeria, different parts of
Africa and beyond. Apparently, the sequence from this study failed to
cluster with any other, including the ones from Nigeria. However, the
closest relatives based on the phylogenetic analysis are sequences that
make up the genotype-I ASFV group. These sequences were generated mainly
from Nigeria, other countries (especially Western and Central Africa),
and Europe. Consequently, the sequence from this study was considered to
also belong to genotype-I ASFV.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
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\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
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\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
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\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
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\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
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\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
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\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

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\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

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\includegraphics[width=0.70\columnwidth]{figures/image9/image9}

\end{center}
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\includegraphics[width=0.70\columnwidth]{figures/image10/image10}

\end{center}
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\includegraphics[width=0.70\columnwidth]{figures/image11/image11}

\end{center}
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\includegraphics[width=0.70\columnwidth]{figures/image12/image12}

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\includegraphics[width=0.70\columnwidth]{figures/image13/image13}

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\includegraphics[width=0.70\columnwidth]{figures/image14/image14}

\end{center}
\end{figure}

\textbf{Discussion}

Gross pathological lesions observed in the spleen were severe
congestion, splenomegaly and severe depletion of lymphocytes. The
lesions may be because of leucopoenia and lymphopoenia that were said to
be occasioned by apoptosis, necrosis of the lymphocytes or due to
activation and mobilization of the lymphocytes to fight the virus
(Otesile \emph{et al} ., 2005; Junt \emph{et al} ., 2008; Ganowiak,
2012; Liao and Padera, 2013; Jubb \emph{et al} ., 2016).

Similarly, the lymph nodes were severely enlarged and haemorrhagic
likely due to severe haemorrhages and necrosis as reported previously by
Jubb \emph{et al} . (2016). Severe lymphocytes depletion and severe
hemosiderosis was recorded in the lymph nodes histologically, which may
be due to apoptosis, necrosis or severe erythrolysis (Ganowiak, 2012;
Jubb \emph{et al} ., 2016). Although moderate multifocal white patches
on the liver has not been previously reported in ASF, liver in this
study showed severe congestion and moderate enlargement with severe
necrosis on histopathological evaluation. Previous reports on ASF
documented congestion and necrosis as lesions associated with the acute
form of the disease and adduced it to marked thrombocytopenia or
disruption in blood clotting factors. (Oura , 2005; Otesile \emph{et al}
., 2005; Jubb\emph{et al} ., 2016). The kidney of pigs was severely
congested with severe cortical petechiae which is a consistent finding
previously reported in ASF and severe ecchymotic haemorrhages in the
renal medulla which we observed for the first time in ASF.
Histopathological examination revealed severe necrosis largely
characterized by severe pyknosis and moderate renal tubular protein fat.
Similar lesions were also observed in earlier reports on ASF and were
attributed to be likely due to severe thrombocytopenia or disruption in
blood clotting factors (Oura, 2005; Ganowiak, 2012; Jubb \emph{et al} .,
2016). The heart on the other hand was observed to have mild focal
congestion grossly with severe vascular congestion histomorphologically.
The disease was reported by earlier researchers to cause cardiopathology
ranging from hydropericardium, hemopericardium and fibrinous
pericarditis whichwas said to be associated with the chronic form of the
disease. The lesions so recorded in the heart were said to be likely
caused by oxidative stress and ischaemia (Oura, 2005; Otesile \emph{et
al} ., 2005; Ganowiak, 2012; Jubb \emph{et al} ., 2016; Semerjyan
\emph{et al} ., 2018).

The aetiological diagnosis of the disease was by virus culture and
indentification via haemadsorption test characterized by erythrocytes
attachment to the surface of the infected cells several hours post
inoculation. This has been documented to be a reliable and effective
method of ASFV isolation (Oura, 2005; Jubb \emph{et al} ., 2016).
Furthermore, the aetiological diagnosis was achieved by amplification of
viral glycoprotein VP72 from the tissues of the infected pigs which
among others, plays an important role in attaching to and entering
target cells. Successful amplification of a 278bp of the glycoprotein by
PCR has earlier been reported to be confirmatory of ASFV in a given
clinical sample (Otesile \emph{et al} ., 2005; Fernandez-pinero \emph{et
al} ., 2012; Jubb, 2016; Mwiine \emph{et al} ., 2019).

Phylogenetic analysis based on the VP72 (b646L) gene sequences showed
that the isolate from this study failed to cluster with others
downloaded from the Genbank database, including the Nigerian isolates.
This observation is inconsistent with earlier finding that Nigerian
isolates always cluster together on the tree (Luka \emph{et al} .,
2017b). The reason for the observed difference is not known for certain,
however, it could be an outcome of continued evolution of the virus. It
could also be possible that there are unreported strains of the virus
circulating in the country, and this, therefore, highlights the need for
more extensive studies to characterize the circulating strains of ASFV
in the country as previously recommended (Luka \emph{et al} ., 2017b).
This, if done, would serve as baseline for formulating adequate and
appropriate control strategies against ASF outbreaks, including vaccine
production. Furthermore, the phylogenetic analysis revealed that the
virus associated with the disease reported in this study belonged to
genotype-I. This further corroborates the previous reorts
(Owolodun\emph{et al} ., 2020; Luka \emph{et al} ., 2017b) on
circulating viruses in Nigeria.

\textbf{Data availability statement:} The data that support the findings
of this study as presented in the manuscript are available from the
corresponding author, Dr. Tizhe E.V., upon reasonable and/or genuine
request.

\textbf{Ethical statement:} Ethical clearance is not applicable in this
study as the data were generated from the swine carcasses submitted to
the Veterinary Teaching hospital, University of Jos, 2019 for diagnostic
purpose.

\textbf{Acknowledgement:} The author's hereby acknowledge that the PCR
and sequencing were sponsored by the ASF research grant of Dr. P.D. Luka
and his team to whom the authors remain eternally grateful.

\textbf{Competing interest:} The authors declare that there is no
competing interest.

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