Ultrastructural Findings in Lymph Nodes from Pigs Suffering from Naturally Occurring Postweaning Multisystemic Wasting Syndrome

Materials and Methods

A total of 6 pigs were used in the present study. Four of the pigs were diagnosed as PMWS, based on the individual case definition of the disease fulfilling 3 criteria: ²⁰ 1) clinical signs mainly characterized by growth retardation, 2) moderate-to-severe lymphocyte depletion and granulomatous inflammation in lymphoid tissues, and 3) moderate-to-high amount of PCV2 in those tissues detected by in situ hybridization. ¹⁶ The 2 remaining pigs were clinically healthy and had no lymphoid lesions, and they were negative for PCV2 using the mentioned in situ hybridization technique. All pigs were seronegative to porcine reproductive and respiratory syndrome virus (PRRSV) (measured by enzyme-linked immunosorbent assay [ELISA] test) and yielded negative results by means of a PRRSV reverse-transcriptase–polymerase chain reaction (RT-PCR) in serum.

Small blocks (1 mm³) of mediastinal and superficial inguinal lymph nodes of all 6 pigs were taken during necropsy and fixed for ultrastructural studies with 2.5% paraformaldehyde (vol/vol) and 2% glutaraldehyde (vol/vol) in 0.1 M phosphate buffer (PB), pH 7.4, during 24 hours. Subsequently, tissue blocks were washed with PB 0.1 M, pH 7.4, twice and stored in paraformaldehyde 2% (vol/vol) in 0.1 M PB, pH 7.4, at 4°C until further processing. Once histopathologic and in situ hybridization results were available (1 week after necropsy), the above-mentioned lymph nodes of the 6 selected pigs were postfixed with 1% osmium tetroxide at 4°C overnight in 0.5 M sodium acetate, followed by washes with distillated water. After sequential dehydration in acetone, samples were embedded in Eponate 12 resin (Ted Pella, Inc., Redding, CA) and polymerized at 60°C for 48 hours. Semithin, 90-nm-thick sections were obtained with Leica Ultracut UCT Microtome (Leica Microsystems GmbH, Wetzlar, Germany), stained with 1% aqueous toluidine blue solution, and observed with a light microscope to select suitable blocks. Ultrathin sections were cut at 60 to 70 nm thick, placed on electron microscopy (EM) copper grids and, then, contrasted with 2% uranyl acetate and Reynold's lead citrate solution. Sections were observed with a Jeol 1400 (Jeol LTD, Tokyo, Japan) transmission electron microscope and photographed with Gatan Ultrascan ES1000 CCD camera 2048 × 2048 pixels. Ultrastructural changes were semiquantitatively scored from lack of (score 0) to severe or high amount (score 3). Mediastinal lymph node of pig No. 1 was finally not able to be examined by EM.

Results

Ultrastructural changes of inguinal and mediastinal lymph nodes were noted in PMWS-affected pigs (Table 1). Ultrastructural changes were mainly observed in histiocytes and, to a lesser extent, in dendritic cells, lymphocytes, and endothelial cells. No significant subcellular changes were observed in any control, non-PCV2-infected pig.

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Table 1.

Summary of ultrastructural features observed in histiocytes of inguinal and mediastinal lymph nodes of PMWS-affected pigs.∗

		Pig 1		Pig 2		Pig 3		Pig 4
Subcellular Changes		LNi∗	LNm	LNi	LNm	LNi	LNm	LNi	LNm
Alterations in Organelles	Rough endoplasmic reticulum proliferation and dilation	2	NA	2	1	1	2	2	1
	Ribosome separation	2	NA	1	1	1	1	2	1
	Proliferation Golgi complexes	1	NA	2	2	1	2	1	1
	Vacuolar bodies	2	NA	1	0	1	0	0	0
	Lysosome proliferation	2	NA	2	2	1	1	1	1
	Caveolae proliferation	2	NA	2	2	0	0	0	0
	Mitochondria proliferation and megamitochondria	3	NA	3	3	1	0	1	1
	Mitochondrial swelling	3	NA	3	2	1	1	2	1
	Apoptosis	1	NA	2	1	2	0	1	0
Viral Morphology	Paracrystalline arrays	3	NA	2	1	0	0	0	0
	Intracytoplasmic inclusion bodies	3	NA	3	3	2	1	2	1
	Intranuclear inclusion bodies	2	NA	2	1	1	0	1	1
Other	Intranuclear hyaline-like bodies	1	NA	1	0	0	0	0	0
	Increased numbers of myelinoid figures	2	NA	2	2	3	3	3	2

^∗ PMWS = postweaning multisystemic wasting syndrome; LNi = inguinal lymph node; LNm = mediastinal lymph node; 0 = no lesion; 1 = mild lesion; 2 = moderate lesion; 3 = severe lesion; NA = sample not available.

Ultrastructurally, histiocytes showed severe mitochondrial proliferation and enlargement (megamitochondria), with a swollen appearance due to disruption of cisternae and external membrane (Fig. 1a). Additional findings in histiocytic cells included proliferation and dilation of Golgi complex (Fig. 1a), diffuse proliferation and dilation of rough endoplasmic reticulum (RER) (Fig. 1b), and lysosomal proliferation, which was variably associated with evidence of phagocytosis (Fig. 1c). Whorled myelinoid figures were observed in the cytoplasm of histiocytes, most of them forming large and complex electron-dense ICIs and containing aggregates of VLPs. Whorled myelinoid figures were occasionally seen within damaged mitochondria (Fig. 1d). Increased numbers of plasmalemmal vesicles or proliferation of caveolae was observed in histiocytes of inguinal lymph nodes of pigs No. 1 and 2 (Fig. 1e).

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Fig. 1.

Lymph node, histiocytes; PMWS-affected pigs. Fig. 1a. Pig No. 1. Intracytoplasmic inclusion bodies (arrows) distributed throughout the cytoplasm, next to enlarged and swollen mitochondria (mit); dilated Golgi complex is also present. Bar  =  1 µm. Fig. 1b. Pig No. 3. Proliferation and dilation of rough endoplasmic reticulum (RER). Bar  =  500 nm. Fig. 1c. Pig No. 1. Nuclei with dilated membranes, and the presence of 4 double-membrane-bounded giant phagolysosomes within the cytoplasm (black arrows); some of the phagolysosomes have a granular pattern, which corresponds to aggregates of virus-like particle (white arrows). Bar  =  1 µm. Fig. 1d. Pig. No. 1. Two degenerated mitochondria (mit) with disruption of cristae and a myelinoid figure (arrow). Bar  =  200 nm. Fig. 1e. Pig No. 2. Increased number of plasmalemmal vesicles or proliferation of caveolae (arrows). Bar  =  100 nm. Uranyl acetate and Reynold's lead citrate solution.

ICIs had round, oval, or irregular shapes with variable electron density, and granular or homogeneous appearance (Fig. 2a). They were non-delimited or limited by internal membranes (Fig. 2b) (sometimes with well-defined myelinoid bodies) and were usually dispersed throughout the cytoplasm. ICIs were found in variable number (2–32/cell) and size (0.1–4.5 µm diameter). Small ones (<0.5 µm diameter) were surrounded by a double membrane and almost always contained icosahedral VLPs of approximately 8–17 nm diameter, organized either in paracrystalline arrays (often semicircular); some ICIs had a granular appearance. Large ICIs (>0.5 µm diameter) had a heterogeneous ultrastructural appearance with VLPs organized as a crystalline grille (paracrystalline arrays with a herringbone pattern) (Fig. 2c); some of large ICIs had also a granular appearance, sometimes within whorled myelinoid membranes. ICIs were usually closely related to (sharing the double membrane or in contact with) organelles such as mitochondria, RER, or Golgi complex (Fig. 2d). It was very frequent to find ICIs next to swollen mitochondria that had lysis of matrix, and sometimes disruption of the cisternae and external membrane. Sporadically, small (10–17 nm diameter) icosahedral VLPs were found within damaged mitochondria (Fig. 2e). Some histiocytes also had intranuclear inclusions (INIs), which were less common than ICIs, and were composed of nonmembrane bounded aggregates containing 10- to 13-nm-diameter VLPs (Fig. 2f).

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Fig. 2.

Lymph node, histiocytes; PMWS-affected pigs. Fig. 2a. Pig. No. 2. Large (asterisks) and small (arrows) intracytoplasmic inclusion bodies containing granular content. The granular content corresponds to 15- to 17-nm diameter icosahedral virus-like particles (VLPs). Bar  =  200 nm. Fig. 2b. Pig No. 3. Intracytoplasmic inclusion bodies with 15-nm diameter icosahedral VLPs, with absence of any external membrane. Bar  =  200 nm. Fig. 2c. Pig No. 1. Complex intracytoplasmic inclusion body with multiple features, including VLPs in paracrystalline arrays, herringbone patterns (arrowhead, insert close-up) and amorphous electron-dense aggregates. Bar  =  200 nm. Fig. 2d. Pig No. 1. Large intracytoplasmic inclusion body with double external membrane and small virions inside (10 nm), next to a swollen mitochondria (mit) also containing VLPs. Bar  =  200 nm. Fig. 2e. Pig No. 1. A swollen mitochondria (mit) that contains 17-nm diameter icosahedral VLPs. Bar  =  100 nm. Fig. 2f. Pig No. 2. A large intranuclear inclusion (arrow) surrounded by clear space and composed of aggregates of indistinct VLPs (10–13 nm diameter). Bar  =  500 nm. Uranyl acetate and Reynold's lead citrate solution.

Lymphocyte depletion was moderate to severe in all 4 cases. Lymphocytes, dendritic cells, and endothelial cells from PMWS-affected pigs showed mild alterations than histiocytes, and mitochondrial swelling was the most prominent finding. Lymphocytes also showed detachment of ribosomes from the RER membrane and peripheralization of nuclear chromatin. Cells (mainly lymphocytes) with necrotic and/or apoptotic appearance were occasionally found (in PMWS-affected pigs but not in healthy animals) (Fig 3a), and had nuclear shrinkage or peripheral margination of chromatin, enlargement and swelling of mitochondria, myelinoid figures, vacuolar bodies and dilation of RER. Phagocytosis of lymphocytes with giant lysosomes was sporadically observed in histiocytes of Pig No. 1 (Fig. 3b); such histiocytes occasionally contained VLPs. Loss of dendritic processes was a striking finding of dendritic cells. ICIs and VLPs were not observed in lymphocytes or dendritic cells.

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Fig. 3.

Lymph node; PMWS-affected pigs. Fig. 3a. Pig No. 1. Apoptotic lymphocyte showing nuclear convolution, margination of chromatin and electron-lucent zones. Mitochondrial swelling (arrow) is observed in the cytoplasm. Bar  =  500 nm. Fig. 3b. Pig No. 1. Phagocytosis of a lymphocyte by a histiocyte (arrowhead), with an adjacent intracytoplasmic inclusion containing VLPs (insert close-up). Bar  =  1 µm. Uranyl acetate and Reynold's lead citrate solution.

Discussion

This work represents the first extensive study on ultrastructural findings of lymphoid tissues from pigs with PMWS. ²⁰ Previous studies used electron microscopy to support histopathologic findings in affected pigs, ^3,7,9 showing the existence of inclusion bodies that were sometimes composed of paracrystalline arrays of PCV2-like particles. In the present study, the main significant organellar changes associated with viral infection were observed in histiocytes, affecting mitochondria, RER, Golgi complex, and lysosomes. Similar ultrastructural changes were observed in other cell populations, such as lymphocytes and dendritic cells, although to a lesser frequency. As a whole, the observed changes are indicative of secretory activation and inflammation. ⁵ Most of these ultrastructural changes were associated with the presence of VLPs in the cytoplasm of affected cells (and occasionally in the nucleus), which were similar to those observed with other viruses such as African swine fever, ⁵ classical swine fever, ⁶ and rubella virus. ¹⁵

According to the literature, viral factories (VFs) refer to aggregates of viral particles of different shapes and sizes, which represent viral maturation structures that induce changes in cellular organization. ¹² In the present study, we observed inclusion bodies containing VLPs associated with important alterations in organelles of histiocytes. Therefore, ICIs and INIs can be classified as VFs. In fact, membrane structures of damaged organelles appear to be used to assemble VFs, where immature virions of different sizes (nonenveloped or enveloped by a double membrane) were observed. Therefore, the presence of VFs suggests that viral replication does occur in histiocytes. This result is contradictory with another reports indicating that macrophages do not represent a significant target cell for PCV2 replication in vitro. ⁴ However, other studies have described that at least a certain proportion of macrophages can support virus replication in vitro, perhaps with variability in susceptibility between pigs and also between PCV2 isolates. ¹⁰ Therefore, the results obtained in the present study support the latter results that PCV2 replicates to a certain extent in macrophages in vivo. ¹³

It has been demonstrated that DNA and RNA viruses use RER and Golgi complex for replication. As examples, African swine fever virus (ASFV) capsid assembly and envelopment take place in the RER membrane and cisternae, respectively; ⁵ herpesviruses acquire the secondary envelope and proteins for capsid envelope in the trans-Golgi area; ¹¹ and viral assembly of Bunyamwera is produced in the Golgi region in connection to mitochondria. ¹⁷ In that sense, PCV2 replication is apparently related to proliferation and dilation of RER and Golgi complex, since VLPs were consistently present next to those organelles. PCV2 VFs were also surrounded by proliferated and degenerated mitochondria; this feature has also been described in some DNA viruses such as ASFV, where VFs are observed next to these organelles. ⁵ Of interest, immature PCV2-like particles were also observed within mitochondria, suggesting that the virus might replicate within this organelle. Viral replication within mitochondria is not apparently described in the literature for other viruses; although some RNA viruses can use the mitochondrial membrane for replication, they are not present within the organelle. ¹² In any case, the potential replication of PCV2 within mitochondria deserves further research.

Previous studies have described PCV2 virions by electron microscopy, revealing small nonenveloped VLPs of 12 to 23 nm diameter in cytoplasm, and 10 to 12 nm diameter in nucleus. ^3,7,9 The present study further confirms published data, in which icosahedral/granular 12–18 nm diameter VLPs were mainly found in double-membrane–bounded VFs containing electron-dense material, and icosahedral 15–18 nm diameter VLPs were found in non-membrane-bounded, irregular VFs. Large electron-dense VFs with paracrystalline arrays and whorled myelinoid membranes occupied wide cytoplasmic areas, as it has been described for PCV1 in PK-15 cells. ²² These viral macrostructures probably contain accumulations of large quantities of protein, as it is usually described for viruses in general. ¹² In contrast, non-membrane-bounded intranuclear VFs were occasionally seen in histiocytes, as it has been also described for other circoviruses including PCV1. ^21,22 The potential relationship between presence of intranuclear VFs and nuclear changes could not be verified in the present study.

Although apoptosis is considered insignificant as a potential mechanism for lymphocyte depletion in natural occurring PMWS, ¹⁴ apoptosis of lymphocytes was observed in PMWS-affected pigs but not in healthy ones in the present study. This apparent incongruence may be explained by the different methodologies used to measure apoptosis. ¹⁴ It has also been suggested that lymphocytes might support PCV2 replication, although to a low degree. ¹³ However, VLPs were not observed in lymphocytes in the present study.

In summary, this is the first detailed study of the ultrastructural findings in lymphoid tissues from PMWS-affected pigs. This work should be further complemented with the development of specific techniques that allow viral detection and localization in cellular organelles of tissues from PMWS-affected animals, and also with studies on morphogenesis of the virus in vitro. The accomplishment of these objectives would allow a better understanding of the pathogenesis of PMWS and PCV2 infection.