Granulomatous Necrotizing Myositis in Swine Affected by Porcine Circovirus Disease

Porcine circovirus type 2 (PCV2) is a nonenveloped, circular, single-stranded DNA virus. ²⁰ There are 4 PCV2 genotypes: PCV2a, PCV2b, PCV2c, ^3,9,16 and PCV2d. ^6,21 The virus is associated with multiple clinical syndromes in pigs, collectively known as porcine circovirus diseases (PCVD). ¹² Clinically, PCVD may vary, and affected pigs usually display progressive weight loss, dyspnea, diarrhea, and lymphadenopathy. Histologically, lymphoid depletion and infiltration of histiocytes and multinucleated giant cells are observed in lymphoid organs. ⁷ In addition to these organs, PCV2 lesions have been described in the kidneys, small and large intestines, stomach, liver, lungs, central nervous system (CNS), and heart. ^2,10,12,15 The aim of this study is to describe 4 individual cases of severe skeletal muscle lesions associated with PCV2 infection in 3 outbreaks of PCVD in pigs.

The outbreaks occurred in 3 swine growing–finishing sites (1, 2, and 3), located in the state of Rio Grande do Sul, Brazil, that received pigs from the same sow farm, which contained 1500 breeding females. All piglets were weaned at 21 days old, vaccinated for PCV2 with a single-dose protocol (inactivated chimeric vaccine) and for Mycoplasma hyopneumoniae with a double-dose protocol (inactivated vaccine), and sent to the 3 sites at 60 days of age. The outbreaks lasted for 60 days (July to August 2016), and the affected pigs were 80 to 120 days old. The 3 sites had a total of 2152 pigs within this age group, of which 92 died during the outbreaks. These 92 pigs had apathy, weight loss, diarrhea, cyanosis, and reddening of the ears, abdomen, and distal parts of the thoracic and hind limbs over a clinical course of 7 to 10 days. Approximately 30 of these 92 pigs presented stiff gait, muscle weakness, hind limb paresis, and recumbency (Fig. 1).

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Figures 1, 2. Granulomatous necrotizing myositis in porcine circovirus disease, pigs. Figure 1. Pig No. 4. Hind limb paresis and sternal recumbency. Figure 2. Pig No. 3. Hind limb. The cut surface shows locally extensive pallor involving large areas of muscle. Inset: gracilis muscle with locally extensive hemorrhages.

Twelve pigs were subjected to euthanasia followed by necropsy (5 pigs from site 1, 2 pigs from site 2, and 5 pigs from site 3). Of these 12 pigs, 4 presented skeletal muscle lesions (pig No. 1 from site 1, pig No. 2 from site 2, and pig Nos. 3 and 4 from site 3). Samples of the hind limb, thoracic limb, and lumbar dorsal skeletal muscles as well as the heart, skin, kidneys, lungs, liver, brain, spinal cord, lymph nodes, spleen, tonsils, and small and large intestines were collected during necropsy, fixed in 10% formalin, routinely processed for histology, and stained with hematoxylin and eosin. Lymph node, tonsil, spleen, kidney, liver, CNS, and skeletal muscle sections were also subjected to immunohistochemistry with a polyclonal anti-PCV2 antibody, as previously described. ²

All necropsied pigs (12) had PCVD, as confirmed by the clinical features, histologic lesions, and detection of the virus by immunohistochemistry. Grossly, the muscular lesions of the 4 pigs consisted of multifocal to coalescent pale discoloration of the skeletal muscles, which was severe for the hind limbs (Fig. 2), moderate for the thoracic limbs, and mild for the dorsal lumbar skeletal muscles. Pig Nos. 2 and 4 also had multifocal petechiae and suffusions on the muscles. Other lesions included generalized lymphadenomegaly with whitish and reddish intermixed areas on the cut surface; enlarged kidneys with multifocal to coalescing pinpoint to round whitish areas; and splenomegaly with infarcts and multifocal white pulp hyperplasia. The liver was enlarged with diffuse orange-reddish discoloration (pig Nos. 2 and 4); the skin had ulcers and irregular reddened lesions on the limbs and ear cyanosis (pig Nos. 2, 3, and 4); and there was extensive ulceration and fibrin deposition over tonsillar surfaces and small intestinal mucosa along the Peyer’s patches (pig Nos. 2 and 4). The remaining 8 necropsied pigs did not present any skeletal muscle lesions, whereas similar lesions affected the lymph nodes (8/8), kidneys (6/8), spleen (5/8), liver (5/8), and small intestine (1/8).

Microscopically, the skeletal muscle lesions were characterized by granulomatous necrotizing myositis. This predominantly affected the hind and thoracic limbs and mildly affected the dorsal lumbar skeletal muscles. Microscopically, myofiber lesions were multifocal, with an intense hyaline and floccular necrosis in all cases, characterized by swollen and hypereosinophilic myofibers with loss of striations, and myofiber fragmentation with intense infiltration of macrophages and multinucleated giant cells in the sarcoplasm, sometimes occupying the whole myofiber sarcoplasm (Fig. 3). There was also multifocal moderate vacuolization of the myofiber sarcoplasm. Pig Nos. 1 and 3 presented mostly acute lesions, which consisted of a moderate inflammatory infiltrate of macrophages, multinucleated giant cells, lymphocytes, and plasma cells distributed in interstitial and perivascular spaces as well as multifocal mild hemorrhage, endothelial cell hypertrophy, rare myofiber regeneration, and mild multifocal thrombosis. Conversely, pig Nos. 2 and 4 presented mostly subacute to chronic lesions, which were composed of an intense multifocal infiltrate of macrophages, multinucleated giant cells and lymphoid aggregates of lymphocytes and plasma cells in interstitial spaces, in addition to an intense myofiber regeneration (Fig. 4). These pigs also exhibited mild lymphohistiocytic vasculitis affecting blood vessels within the skeletal muscle, characterized by fibrinoid degeneration and necrosis of blood vessels and severe multifocal hemorrhage. The cardiac and smooth muscles did not present any microscopic lesions.

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Figures 3–6. Granulomatous necrotizing myositis in porcine circovirus disease, skeletal muscle, pig. Figure 3. Pig No. 1. Multifocal necrosis and fragmentation of muscle fibers with macrophages and multinucleated giant cells (inset). Hematoxylin and eosin (HE). Figure 4. Pig No. 2. Multifocal necrosis of muscle fibers, with lymphocytes, macrophages, and giant cells (inset), and multifocal hemorrhage. HE. Figures 5, 6. Pig Nos. 1 and 3. Variable immunostaining in the cytoplasm and nuclei of macrophages and multinucleated giant cells, and mild immunostaining in the sarcoplasm of necrotic fibers (arrow) and satellite cells. Immunohistochemistry for porcine circovirus type 2.

The lymphoid tissues (spleen, tonsils, and lymph nodes) of the 4 pigs showed an intense lymphocyte depletion and infiltrate of epithelioid macrophages and multinucleated giant cells, multifocal lymphohistiocytic vasculitis associated with fibrinoid degeneration of blood vessels (affecting tissues other than skeletal muscle), and extensive multifocal areas of coagulative necrosis. Similar severe lesions were observed in the interstitial spaces of the kidney and periportal spaces of the liver (4/4), and mild lesions were observed in the neuropil and in leptomeningeal perivascular spaces of the CNS (4/4). The tonsils exhibited extensive areas of mucosal ulceration (pig Nos. 2 and 4). Mild endothelial cell degeneration in the CNS was also observed (4/4). The cutaneous lesions of pig Nos. 2, 3, and 4 consisted of infarcts, with multifocal thrombosis in the superficial dermis and a moderate perivascular lymphoplasmacytic infiltrate. Pig Nos. 2 and 4 exhibited intense granulomatous enteritis associated with areas of necrosis and mucosal ulceration, distributed along the Peyer’s patches. The remaining 8 pigs did not present any microscopic skeletal muscle lesions or skin lesions, whereas similar lesions were observed in the lymphoid tissues (8/8), kidneys (6/8), liver (6/8), brain (7/8), spinal cord (2/8), and small intestine (1/8) and thus were characterized as PCV2 systemic disease.

Immunohistochemistry on lymphoid tissues of all 12 pigs was characteristic of PCVD, with intense immunostaining in the cytoplasm of macrophages and multinucleated giant cells and moderate immunostaining of endothelial cells. Immunohistochemistry analysis of skeletal muscles from the 4 pigs showed intense multifocal immunostaining mainly in the cytoplasm and nuclei of macrophages, lymphocytes, and multinucleated giant cells, in addition to mild immunostaining in the cytoplasm of endothelial cells, necrotic fibers, and satellite cells of the skeletal muscle (Figs. 5, 6). The kidneys, liver, and CNS exhibited immunostaining of macrophages, multinucleated giant cells, and lymphocytes, in addition to endothelial cells in the CNS.

DNA was obtained from the lymph nodes, spleen, and skeletal muscle of the 4 pigs that presented muscle lesions, from which a polymerase chain reaction (PCR) product of 481 bp was amplified. DNA extraction and PCR for PCV2 were performed as described previously. ⁴ The PCR products were sequenced, which indicated 99% identity with sequences in GenBank from the PCV2b genotype.

The diagnosis of granulomatous necrotizing myositis in swine affected by PCV2 systemic disease was based on the clinical, epidemiologic, pathologic, immunohistochemical, and molecular findings. The pathologic findings in the lymphoid organs, kidneys, skin, and liver are identical to those previously described in swine infected by PCV2. ^11,13,14 However, skeletal muscle lesions caused by PCV2 have not yet been reported in natural infections, and in the present study, these were multifocal polyphasic reactions suggesting continued damage over a prolonged period of time, culminating in granulomatous necrotizing myositis that varied according to the clinical course. Pig Nos. 1 and 3 presented mostly acute lesions, whereas pig Nos. 2 and 4 exhibited mostly subacute to chronic lesions. However, pig Nos. 1 and 3 were necropsied within 1 week of each other, as was the case for pig Nos. 2 and 4. Thus, the infection probably occurred in distinct periods, and the lesion pattern variations were attributed to the different stages of muscle necrosis, leukocyte infiltration, and muscle fiber regeneration that may be observed in inflammatory myopathies. ¹

Porcine circovirus disease is caused by several PCV2 genotypes, and some cases may include vascular lesions. ^8,12,13 However, the vascular lesions within skeletal muscle of the pigs in the present study were mild, whereas the necrotic lesions of the myofibers were severe. Thus, we propose that the skeletal muscle lesions observed are most likely a direct effect of the viral infection, since positive immunostaining was observed in the sarcoplasm of myocytes and in macrophages within the muscle. ^8,18

The development of neurologic clinical signs, such as hind limb paresis and paralysis, has been described in PCV2 systemic disease due to lesions in the spinal cord. ²² The 4 pigs that displayed skeletal muscle lesions also presented spinal cord lesions, yet these were mild, which strengthens the hypothesis that the locomotor clinical signs were directly related to muscle damage. In addition, 2 of the 8 remaining pigs presented spinal cord lesions but no neurologic clinical signs, which supports that locomotor clinical signs resulted from the PCV2-induced muscle damage.

Porcine circovirus disease primarily affects 2- to 4-month-old pigs with a mortality rate of 4% to 20%, as in the present outbreaks. ¹⁵ Infectious and noninfectious cofactors, as well as risk factors that predispose the animals to stress, exacerbate the clinical signs and severity of the disease. ¹⁵ Commercially available vaccines against PCV2 have been an important tool for both controlling and reducing the prevalence of PCVD. ⁵ Experimentally, the administration of a single-dose subunit PCV2 vaccine in 21-day-old piglets elicited PCV2-specific neutralizing antibodies. ⁵ However, the use of a single dose in weaning piglets in the present study did not appear to provide effective immunity against PCV2b infection. Many factors may influence the efficacy of the vaccine against natural infections, such as herd sanitary conditions or a mix of pigs at different ages and vaccine protocols. ¹⁷ Although the reason for this apparent lack of adequate immunity and resultant clinical outbreak of PCVD remains undetermined, it ended with the slaughter of the pigs from the affected sites. The disease did not recur in subsequent groups of pigs.

Ionophore toxicity and vitamin E/selenium deficiency are the main differential diagnoses based on the gross findings, which are similar to those observed in the present work. ^1,19 Vitamin E/selenium deficiency most commonly affects weaned pigs and, microscopically, causes mineralization of degenerate fibers, whereas ionophore poisoning causes a monophasic and multifocal degeneration of myofibers. ¹ However, the affected pigs did not consume ionophore antibiotics, and the skeletal muscle microscopic lesions were primarily characterized by granulomatous necrotizing myositis, which causes distinct lesions from the above-mentioned toxic and metabolic myopathies. ^1,19

Therefore, locomotor clinical signs and granulomatous necrotizing myositis should be considered as another expression of PCV2 infection in pigs.