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.