Discussion
The genotype II of ASFV strains in this study showed acute to subacute
forms of ASFV based on the clinical presentation as well as gross and
histopathological findings. A 70% mortality rate was estimated in a
single farm (case 74), whilst the accurate mortality rates in seven
other affected farms were undetermined because of the rapid enforcement
of herd depopulation. The main lesions of acute ASF, including hyperemic
splenomegaly, haemorrhagic lymph nodes, especially in the peripheral
lymphatic sinus, petechial haemorrhage in the renal cortex, pulmonary
oedema, haemorrhages in the epicardium, and subacute ASF, including
hyperemic splenomegaly, severe and more diffuse haemorrhagic lymph nodes
(cortical and medulla), petechial haemorrhages in the cortex and pelvis,
gall bladder wall oedema, haemorrhages in the epicardium, as reported
previously (Sánchez-Vizcaíno et al, 2015), were seen among the eight
cases studied.
There are several known viral pathogens causing porcine haemorrhagic
disease in Vietnam, including highly pathogenic porcine reproductive and
respiratory syndrome virus (HP-PRRSV) and classical swine fever virus
(CSFV), which produced very similar haemorrhagic lesions in the lymph
nodes (Huong Giang et al, 2016; Choe et al, 2020). Antigen detection of
HP-PRRSV and CSFV by the RT-PCR method yielded negative results (data
not shown), confirming that the lesions seen in those tissues were
highly attributable to ASFV infection. Even though ASFV mainly targeted
the macrophages/mononuclear phagocytic system similar to both HP-PRRSV
and CSFV, the histopathological lesions in the lymph node tissues were
found to be different among the viruses. There were marked histiocytic
hyperplasia in CSFV infection, which is not seen in ASFV infection. In
contrast, CSFV infection manifested more marked and severe lymphoid
depletion (disappearance of lymphoid follicles) compared to HP-PRRSV
infection in relation to the strain causing outbreaks in Vietnam from
2010 to 2018 (personal observation). Bacterial diseases causing similar
haemorrhagic lesions, such as in septicemic salmonellosis and swine
erysipelas, were also ruled out based on negative PCR detection (data
not shown) and the lack of histopathological characteristics of both
bacterial infections.
ASFV primary replication site is in the mononuclear cells/macrophages;
therefore, the ASFV antigen was consistently detected in a large number
in the lymphoid organs. In the lymph node, ASFV causes apoptosis of
lymphocytes, mainly in the T cells compared to B cells (Carrasco et al,
1996). The death of the lymphocytes is caused by an indirect mechanism
involving the release of proinflammatory cytokines, mainly tumor
necrosis factor-α, interleukin-1 and interleukin-6 by activated
monocytes and macrophages (Salguero et al, 2005), as the virus had been
shown not to replicate in the lymphocytes (Carrasco et al, 1996).
Similarly, we observed apoptotic bodies more intensely distributed in
the diffuse lymphoid tissue (T cell area) of tonsil and lymph nodes in
this study. IHC also showed frequent ASFV antigen–labelled mononuclear
cells/macrophages in the diffuse lymphoid tissue area compared to the
lymphoid follicles. In the spleen, the degree of haemorrhage was very
severe in several cases, resulting in very few viable cells surrounding
the follicular structures and severe lymphoid depletion. This phenomenon
was described as a consequence of necrotic fixed macrophages of the
splenic cords and subsequent deposition of fibrin (Carrasco et al,
1997). However, we did not observe frequent fibrin deposition in all the
cases, including in the most severely haemorrhagic splenic tissue.
During the outbreak, ASFV-infected pigs showed neurological signs of
paddling, spasm and convulsion in lateral recumbency before the loss of
consciousness, coma or death. In the central nervous system,
histopathological lesion attributed to ASFV was perivascular cuffing in
the brain, which contained more nuclear debris than what is seen in CSFV
infection (Robinson & Robinson, 2016). The detection of ASFV antigen by
IHC in the brain tissue of a domestic pig is rarely reported elsewhere.
In this study, the ASFV antigen was presented in the mononuclear cells
of the meninges (with or without clear haemorrhage) and perivascular
cuffing in the grey matter of the cerebral cortex, which may contribute
to the characteristic lesions seen in the brain tissue. Notably, the
number of ASFV antigen–labelled cells was significantly fewer compared
to other tissues in this study, and a single case of non-suppurative
meningoencephalitis showed negative ASFV antigen. These findings may
support the role of proinflammatory cytokines contributing to the
inflammatory lesion in the brain.
In the lungs, pulmonary intravascular macrophages (PIMs) were easily
identified in the ASFV-infected pigs. Based on the IHC results, lung
tissue is also a primary site of ASFV antigen detection. PIMs play a
significant role in several species of animals, including ruminants,
cats, pigs and horses as the primary cells in defense mechanisms against
hematogenous pathogens (López & Martinson, 2017), and increases in the
size, number and phagocytic activity of PIMs were reported during an ASF
viral infection (Carrasco et al, 2002). In acute ASF cases, the release
of proinflammatory cytokines as mentioned from activated PIMs lead to
increased vascular permeability, resulting in pulmonary oedema (Carrasco
et al, 1996). However, there were no obvious correlations between the
number of ASFV antigens found in the lung tissue and the occurrence of
pulmonary oedema observed in this study.
In the liver, multifocal necrosis and hemadsorption phenomenon were
reported in Kupffer cells, circulating monocytes and hepatocytes
(Fernandez et al, 1992; Sierra et al, 1987). In this study, ASFV caused
multifocal haemorrhages mainly in the midzonal (zone 2) region of the
hepatic lobules with different severity of necrosis. Numerous Kupffer
cells/circulating macrophages in the sinusoids and haemorrhagic areas
were labelled with ASFV antigen, but positively-labelled hepatocytes
were rare, except in a severe case (case 20). In severe cases, more
generalized, coalescing multifocal haemorrhages produce an appearance of
selective midzonal (zone 2) to nearly centrilobular necrosis with
haemorrhage. The lesion observed in this study showed the midzonal
necrosis was frequently accompanied by haemorrhages and mononuclear cell
infiltrations, while most of the hepatocytes in zones 1 and 3 remained
less affected. Only one case showed a negative detection of ASFV despite
the presence of histopathological lesions, which may require further
investigation.
Multifocal haemorrhages were found in the heart, gastrointestinal tract
and kidney tissues. There was a presence of many spindle-shaped cells
labelled with ASFV antigen in the haemorrhagic lesion of the epicardium
and renal pelvis, which were not identified as macrophages by IHC using
Iba-1 antibody (data not shown). The origin of these cells was not
confirmed; however, their morphology may suggest a fibroblastic origin.
The significance of these ASFV infected-cells contributing to the lesion
in the epicardium and renal pelvis is not known, but infected
fibroblasts and smooth muscle cells in the later stage of infection have
been reported (Blome et al, 2013). Multifocal haemorrhages in the
gastrointestinal tracts vary in their location, localized in the lamina
propria of gastric and large intestinal epithelium or only presented in
the outer muscularis layer of the small intestine. There are few ASFV
antigen–labelled cells found in these areas, which may imply the
lesions mainly attributed to the concentration of cytokines reaching and
affecting the area by the blood circulation. In ASFV-infected pigs, the
gastrohepatic, renal and mesenteric lymph nodes were some of the major
lymph nodes with haemorrhagic lesions. The anatomical location of the
gastrointestinal tract in the vicinity of affected lymph nodes may have
contributed to the large concentration of proinflammatory cytokines
supplied to those organs by the blood or lymph, causing the
vascular-related changes in those organs. The factors relating to such
occurrence require further pathological investigation.
Reportedly, renal tubular epithelium is not a leading replication site
of ASFV. Among the eight cases studied, three cases (case 58, 59, 60)
showed the absence of renal epithelium labelled with an ASFV antigen.
Furthermore, the staining pattern of the ASFV-labelled renal epithelium
showed a granular cytoplasmic pattern, instead of diffuse, intense
staining in the cytoplasm of macrophage/mononuclear cells. Instead of an
active viral replication site, it may be inferred that there was a high
load of ASF viral particle reabsorption from the urine in the renal
tubular epithelium.
In the lymphoid tissues, the ASFV positive cells were consistently
observed with no regard for the severity in the apoptotic, haemorrhage
and necrotic lesions in those tissues. However, in many other tissues of
different organs, there were instances where the number of ASFV antigen
was few or negative even though haemorrhagic lesions, as discussed
previously, were observed. On the other hand, there were also instances
where the ASFV antigen was observed without obvious lesions. The amount
of proinflammatory cytokines produced by the infected cells in the main
target organs (lymphoid organs) may have a greater influence on the
production of the pathological lesions at distant organs rather than the
number of infected cells presented in those organs.
In the absence of an effective treatment or vaccination against ASFV,
the depopulation of infected pig herd and all herds surrounding the
affected farm in a high-density pig area resulted in a significant
monetary loss to the Vietnam’s majority smallholder and backyard farmer.
In ASF-endemic countries, it is worth noting that the ASFV may become
less virulent and infected pigs may show less severe clinical
presentation in which case the histopathological lesions may eventually
vary in the future (Robinson & Robinson, 2016). Thus, this study
highlighted the histopathological findings of ASFV genotype II in its
initial epidemic in Vietnam.