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Development of microscopic lesions in splenic cords ofpigs infected with African swine fever virus

L Carrasco, Mj Bautista, Jc Gómez-Villamandos, J Martin de Las Mulas, FChacón-M de Lara, Pj Wilkinson, Ma Sierra

To cite this version:L Carrasco, Mj Bautista, Jc Gómez-Villamandos, J Martin de Las Mulas, F Chacón-M de Lara, etal.. Development of microscopic lesions in splenic cords of pigs infected with African swine fever virus.Veterinary Research, BioMed Central, 1997, 28 (1), pp.93-99. <hal-00902462>

Short note

Development of microscopic lesions in splenic cordsof pigs infected with African swine fever virus

L Carrasco MJ Bautista JC Gómez-Villamandos J Martin

de las Mulas F Chacón-M de Lara PJ Wilkinson MA Sierra

! Departnmento de !tnafo!ta y Anatomía Pato16gica Comparadas, Facultad de Veteri!ar!a,(/nit’f!;daJJg Córdoba, A vda Medina Azahara 7, 74005 Córdoba, Spaí!;

2 Institute.f(!r Animal Health, PirbrightLaboratory, Ash Road, Pirbright, Woking CU24 ONF, UK

(Received 29 February 1996; accepted 9 July 1996)

Summary &horbar; Acute forms of African swine fever are characterized by hemorrhagic lesions in the lym-phoid organs. This paper reports the evolution of lesions in the splenic cords of pigs inoculatedwith African swine fever (ASF) virus (strain Malawi’83). Ultrastructural examination of the spleniccords of the infected pigs revealed numerous macrophages attached to the muscle cells harboring virusreplication center and cytopathic effects at 3 dpi (days post-infection). From 5 dpi, the splenic cordscontained a large number of erythrocytes associated with abundant fibrin deposits, mainly arrangedaround the muscle cells, from which macrophages had disappeared. It is likely that the ASF virus repli-cation, and consequent cytopathic effects, observed in the fixed macrophages of splenic cords, maybe responsible for the fibrin deposition.

pig / virus / African swine fever / macrophage / spleen

Résumé &horbar; Évolution des lésions microscopiques dans les cordons spléniques de porcs infectéspar le virus de la peste porcine africaine. Les formes aiguës de la peste porcine africaine se carac-térisent par des lésions hémorragiques dans les organes lymphoïdes. Nous avons étudié l’évolutiondes lésions observées au niveau des cordons spléniques de porcs auxquels on avait inoculé le virusde la peste porcine africaine (souche Malawi’83). L’étude ultrastructurale des cordons spléniques desporcs infectés a revéle la présence de nombreux macrophages adhérant aux cellules musculairesavec réplication du virus et un effet cytopathique à 3 jours post-inoculation (jpi). À partir de 5 jpi les

* Correspondence and reprintsTel: (34) 57 21 86 82; fax: (34) 57 21 86 66; e-mail: anleaotl@lucano.uco.es

cordons spléniques contenaient une grande quantité d’érythrocytes associés à d’abondants dépôts defibrine, disposés essentiellement autour des cellules musculaires, où les macrophages avaient disparu.Nous en avons conclu que la réplication virale du virus de la peste porcine africaine, et l’effet cyto-pathique consécutif, dans les macrophages fixés des cordons spléniques, pouvaient être respon-sables des dépôts de fibrine.

porc / virus / peste porcine africaine / macrophages / rate

INTRODUCTION

African swine fever (ASF) is caused by a largeenveloped icosahedral DNA virus consideredto belong to a separate family (Brown, 1986).Virus replication takes place in the cytoplasm,chiefly in cells belonging to the mononuclearphagocyte system (MPS) (Mebus, 1988), and isconsidered to play a key role in the pathogene-sis of the disease (Mebus, 1988). ).

Acute forms of the disease are characterized

by death within 6 or 7 dpi (days post-infection), ),and by the presence of hemorrhagic lesions,mainly in the lymphoid organs (Moulton andCoggins, 1968; Nunes Petisca and MartinsGonyalves, 1976). Macroscopic lesions of thespleen have been variously described assplenomegaly (Moulton and Coggins, 1968;Konno et al, 1972), hyperemic splenomegaly(Konno et al, 1972), or hemorrhagicsplenomegaly (Nunes Petisca and MartinsGon!alves, 1976). Microscopic analysis revealsthe retention of a large number of erythrocytes,together with abundant cellular debris (Moul-ton and Coggins, 1968; Colgrove et al, 1969;Konno et al, 1972; Mebus and Dardiri, 1979;Mebus, 1988) which for some authors resultsfrom the destruction of reticulin fibers (Moultonand Coggins, 1968; Konno et al, 1972; NunesPetisca and Martins Goncaives, 1976). ).

Immunohistochemical studies performed toidentify the main cells involved in the patho-genesis of spleen lesions have revealed the pres-ence of the viral antigen in macrophages locatedprimarily in the red pulp, while the lymphoidstructures are reported to be practically nega-tive (Colgrove et al, 1969; Mebus, 1988;Minguez et al, 1988; Galo and Nunes Petisca,

1990; Ferndndez et al, 1992a, b; Gonzalez-Juar-rero et al, 1992; Pérez et al, 1994).

Although splenic cords are traditionallydescribed as being composed of a network ofreticular fibers totally engulfed by star-shapedreticular cells, smooth muscle fibers have alsobeen reported in the pig spleen (Ramis et al,1991; Ueda et al, 1991; Carrasco et al, 1995).The structure of porcine splenic cords hasrecently been described (Carrasco et al, 1995)as a network of smooth muscle cells surrounded

by a new population of fixed macrophages shar-ing certain characteristics with pulmonaryintravascular macrophages and Kupffer cells.The reticular cells in swine splenic cords arereported to be poorly developed, and are foundonly in the adventitia of venous sinuses, scat-tered along the splenic cords and, more rarely,adhering to muscle cells (Carrasco et al, 1995). ).

This paper describes the development ofsplenic lesions in acute ASF.

MATERIALS AND METHODS

Ten Large White x Landrace pigs of both sexes,weighing roughly 30 kg at the start of the experi-ment, were used for this study. Two pigs wereused as controls, and the remainder were inoc-ulated im with 105 HADso of ASF virus strain

Malawi’83 (Haresnape, 1984), classified ashighly virulent and hemadsorbent. The animalsexhibited no behavioral changes due to theexperimental conditions and developed symp-toms characteristic of ASF (Colgrove et al,1969; Wilkinson, 1989). The inoculated ani-mals were slaughtered in pairs at 1, 3, 5 and 7dpi. The experiment was performed at the Insti-

tute of Animal Health, Pirbright (UK), in accor-dance with the Practice Code for the Hous-

ing and Care ofAnimals u.sed in Scientific Pro-cedures.

The tissues were fixed by vascular perfusionusing 2.5% glutaraldehyde in 0. M phosphatebuffer (pH 7.4), at a pressure of 120 mmHg.Prior to perfusion, the animals were tranquil-ized with azaperone (Stresnil, Janssen AnimalHealth, Belgium) and anesthetized with sodiumthiopental (Thiovet, C-Vet, UK). The sampleswere embedded in paraffin and Epon 812 (Fluka,Buchs, Switzerland). For the ultrastructural anal-

ysis, 4 pm thick sections were cut and stainedwith hematoxylin and eosin. Thin sections (1 Ipm) were cut and stained with toluidine blue (ina 1 % aqueous solution). For transmission elec-tron microscopy, 50 nm samples were stainedwith uranyl acetate and lead citrate and viewedthrough a Philips CM-10 transmission electronmicroscope.

RESULTS

The examination of the splenic cords in the con-trol and experimental animals sacrificed 1 dpirevealed the virtual absence of circulating bloodcells due to perfusion. There remained an inter-trabecular network of myocytes surrounded bymacrophages. The few reticular cells observedwere generally located on the adventitial aspectof the venous sinuses, scattered along the spleniccords and, more rarely, adhering to muscle cells.The only difference observed between controlsand experimental animals slaughtered 1 dpi wasthat the macrophages in the splenic cords of thelatter group presented an increase in both sizeand phagosome number.

The animals slaughtered 3 dpi exhibitedhyperemic splenomegaly, despite the perfusion.The splenic cords contained large numbers oferythrocytes, some of them with viral particlesattached to the membrane, and an increase in

macrophage numbers. Most of these

macrophages, which were observed bothattached to the muscle fibers and free in the

splenic cords, showed peripheral marginationof their nuclear chromatin and a clear, rounded

organelle-free area of cytoplasm containing com-plete and incomplete viral particles (fig 1 Themacrophages containing virus replication centers,and in particular those attached to muscle cells,had fewer filopodia and projections envelopingmuscle fibers, with occasional loss of linear den-sities in the membrane; they presented a roundedprofile with progressive detachment from themuscle cell (fig 1 B). In some areas of the spleniccords, myocytes were surrounded by cellulardebris (fig 2) interspersed with occasional viralparticles (fig 2A), fibrin deposits and degranu-lated platelets which presented pseudopodia inclose contact with the collagen fibers which weresurrounding the myocytes (fig 2B).

At 5 dpi, the animals exhibited intense hyper-emic splenomegaly. The red pulp was com-

pletely filled with erythrocytes, fibrin depositsand cellular debris. The smooth muscle fibers

were surrounded by abundant fibrin depositsand cellular debris (fig 3), among which degran-ulated platelets (fig 3A) and virions (fig 3B)were frequently observed. Macrophages attachedto muscle cells were scarce, and contained virus

replication centers.

At 7 dpi, the hyperemic splenomegalyremained intense. The red pulp was completelyfilled with large masses of cellular debris inter-spersed with erythrocytes, a few neutrophils,monocytes and fibrin bundles and enmeshed

erythrocytes and cellular debris, among whichviral particles were observed (fig 4).

DISCUSSION

The most significant finding in the splenic cordsof pigs infected with a highly virulent strain wasthe virus replication in the macrophages attached

to the network of muscle cells. These

macrophages disappeared and were replaced bya large number of erythrocytes associated withabundant fibrin deposits.

The viral replication and cytopathic effectsobserved in the macrophages attached to themuscle cells shared the characteristics previ-ously reported for other macrophage popula-tions in ASF (Sierra et al, 1990; Carrasco et al,1992; G6mez-Villamandos et al, 1995a, b). The

cytopathic effects included peripheral margina-

tion of the chromatin, cytoplasmic vacuoliza-tion and, in pulmonary intravascular

macrophages following the inoculation of a vir-ulent ASF isolate, the swelling of the cells result-ing in a loss of intercellular junctions (Sierra etal, 1990; Carrasco et al, 1992). In the spleen,the change in the shape and the loss of intercel-lular junctions in the macrophages attached tomuscle cells resulted in the exposure of the col-

lagen of the basal lamina of the muscle cells toblood plasma. This fact is responsible for theactivation of the platelets and the fibrin depo-sition observed in these areas (Chen and Weiss,1972; Carrasco et al, 1995). This activation of

platelets and fibrin deposition around the musclecells was more obvious when the macrophageswere in necrosis due to virus replication.

The attached macrophages undergoing necro-sis due to ASF virus replication probably cor-respond to the reticular cells described elsewhereas degenerating (Moulton and Coggins, 1968;Nunes Petisca and Martins Gonqalves, 1976;Mebus and Dardiri, 1979). The deposition offibrin around the muscle cells from which

macrophages had disappeared, and subsequently

within splenic cords, may give rise to the accu-mulation of erythrocytes and the structural dis-organization reported by many authors (Moultonand Coggins, 1968; Konno et at, 1972; Mebusand Dardiri, 1979; Mebus, 1988). Moreover, the

appearance of fibrin in the splenic cords from3 dpi onwards coincides with the appearance ofthe gross lesions most characteristic of the spleenin acute ASF, and described as hyperemic orhemorrhagic splenomegaly (Moulton and Cog-gins, 1968; Konno et at, 1972; Nunes Petiscaand Martins Gonqalves, 1976; Mebus and Dard-iri, 1979; Mebus, 1988).

Following the inoculation of the same ASFvirus strain (Malawi’83), fibrin deposition hasbeen reported in the liver from the earliest stageof the disease (3 dpi) (Gomez-Villamandos et al,1995a). A characteristic feature of the liver isthe presence of a macrophage population withinthe hepatic sinusoids. Similar findings arereported here for the spleen. Although the splenicmacrophages are not located within blood ves-sels, the splenic cords are interposed in thebloodstream, and thus the intravascular deposi-tion of fibrin may be related to virus replicationin the intravascular macrophages (G6mez-Vil-lamandos et al, 1995a). The possible relation-ship between replication in MPS cells and thedeposition of fibrin (Gomez-Villamandos et at,1995a, b) is also supported by the resultsobtained for the kidney following inoculationof the same ASF virus strain. There the fibrin

is not detected until later stages of the infection

(5 dpi), coinciding with the observation ofmonocytes supporting virus replication in renalcapillaries (G6mez-Villamandos et at, 1995b). Inthe liver and kidney, the deposition of fibrin isrelated to an indirect mechanism. The virus repli-cation in the macrophages is responsible for theactivation of the endothelial cells (Gomez-Vil-lamandos et al, 1995a, b) and this endothelialactivation produces a disseminated intravascularcoagulation process. In the spleen, the depositionof fibrin could be a direct consequence of virus

replication, subsequent necrosis and loss ofmacrophages attached to muscle cells of spleniccords. This loss of macrophages exposes the

basal lamina, which in turn exposes the musclecells to the plasma blood. Platelet activation andfibrin generation around the muscle cells occuras a result. This direct mechanism in the spleenis supported by the absence of endothelial acti-vation in the spleen. The necrosis of spleenmacrophages may be related to an increase infibrin monomers in the blood. As a result the

main function of the spleen, blood clearance, isimpaired.

In this paper we described the splenic lesionsrelated to the fixed macrophages of splenic cords.A pathogenic mechanism was proposed; thecytopathic effects and necrosis of these cellsmight have been responsible for the depositionof fibrin in the splenic cords, the subsequentretention of a large number of erythrocytes inthe spleen and the appearance of considerableamounts of cellular debris. These mechanisms

may be present in other species in which thesemacrophages are present.

ACKNOWLEDGMENTS

We wish to thank A Jover for his support in this exper-iment; SM Williams and GH Hutchings for technicalassistencc in the laboratory; T Kinsella and L Fitz-patrick for their assistance in the animal unit; and to theveterinary student B Pulido for her enthusiastic labo-ratory work. This work was support by grants fromCAICYT and Junta de Andalucia (Spain) and the Min-istry of Agriculture, Fisheries and Food (UK).

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