The Role of Pulmonary Intravascular Macrophages in the Pathogenesis of African Horse Sickness

J. Comp. Path. 1999 Vol. 121, 25–38

The Role of Pulmonary Intravascular Macrophages in the Pathogenesis of African Horse Sickness L. Carrasco, C. Sa´nchez∗, J. C. Go´mez-Villamandos, M. D. Laviada∗, M. J. Bautista, J. Martı´nez-Torrecuadrada∗, J. M. Sa´nchez-Vizcaı´no∗ and M. A. Sierra Departmento de Anatomı´a y Anatomı´a Patolo´gica Comparadas, Facultad de Veterinaria, Universidad de Co´rdoba, Avenida Medina Azahara s/n, 14005-Co´rdoba and ∗Centro de Investigacio´n en Sanidad Animal, Instituto Nacional de Investigaciones Agrarias, Valdeolmos, Madrid, Spain Summary African horse sickness (AHS) is a disease of equids, characterized by severe pulmonary oedema and caused by an orbivirus. To determine the role of pulmonary intravascular macrophages (PIMs) in the development of pulmonary microvascular changes in this disease, five horses were given an intravenous inoculation of 106 TCID50 of serotype 4 of AHS virus. Viral replication was detected in endothelial cells, PIMs, interstitial macrophages and fibroblasts. Alveolar and interstitial oedema, and changes in pulmonary microvasculature, consisting mainly of the sequestration of neutrophils and the formation of platelet aggregates and fibrinous microthrombi, were related to endothelial changes and to a high degree of PIM activation. This suggested that the PIMs, once activated, contributed to these vascular changes by releasing chemical inflammatory mediators.  1999 W.B. Saunders Company Limited

Introduction In recent years, study of the pulmonary physiology and pathology of pneumonic processes in certain mammalian species has changed dramatically as a result of the description of a resident population of mononuclear cells—pulmonary intravascular macrophages (PIMs). Today, PIMs are recognized as being responsible for blood clearance in pigs (Winkler and Cheville, 1986), goats (Atwal and Saldanha, 1985), sheep (Warner et al., 1987; Miyamoto et al., 1988), cattle (Whiteley et al., 1991a) and deer (Carrasco et al., 1996a). In addition, they influence pulmonary microvascular physiology by the release of inflammatory mediators, which can initiate or enhance acute pulmonary inflammation through the recruitment and activation of neutrophils, the formation of platelet plugs, and activation of clotting cascades. However, only a few studies have focused on the behaviour of PIMs in viral diseases (Winkler and Cheville, 1986; Sierra et al., 1990; Carrasco et al., 1991, 1992; 1996b; Thanawongnuwech et al., 1997). The large number of studies on PIMs in ruminants and pigs contrasts with the small number in horses. PIMs in horses were not demonstrated until the 0021-9975/99/050025+14 $12.00

 1999 W.B. Saunders Company Limited

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early 1990s (Atwal et al., 1992; Longworth et al., 1994). These and other studies evaluated PIM behaviour after the administration of substances such as monostral blue (Atwal et al., 1992; Longworth et al., 1994), halothane (Atwal et al., 1994), liposomes and copper phthalocyanine blue pigment (Longworth et al., 1994), while at the same time showing that PIMs in horses were similar to those present in other species, being characterized mainly by adhesion to pulmonary endothelium and by the presence, on the cell surface, of micropinocytosis vermiformis (Atwal et al., 1992, 1994; Singh et al., 1994). African horse sickness (AHS) is a disease of equids, caused by an orbivirus belonging to the family Reoviridae (Holmes et al., 1995) and characterized by severe pulmonary oedema, mainly in the pulmonary and mixed forms of the disease (Newsholme, 1983; Laegreid et al., 1992; Brown et al., 1994; Wohlsein et al., 1997). Ultrastructurally, natural and experimental AHS infections are characterized by substantial interstitial and alveolar oedema, platelet aggregation, neutrophil sequestration and fibrinous microthrombi in the pulmonary microcirculation (Newsholme, 1983; Laegreid et al., 1992). The separation of endothelial intercellular junctions has also been reported, together with evidence of AHS virus replication in a small proportion of endothelial cells (Laegreid et al., 1992); no explanation has yet been put forward, however, for the low degree of viral replication associated with the changes observed in pulmonary microvasculature (Laegreid et al., 1992). This lack of relation between viral replication and changes in pulmonary microvasculature was reported in in-situ hybridization experiments by Brown et al. (1994). The aim of the present study was to evaluate the role of PIMs in the development of changes in pulmonary microvasculature, and in the subsequent appearance of alveolar oedema in AHS infection. Materials and Methods Virus The strain of AHS virus (serotype 4) used was isolated from the spleen of a Spanish horse in 1989. After three passages on monolayers of Vero cells, the virus was titrated by plaque assay as described previously (Oellermann, 1970) and stored at −80°C. Animals Six mixed breed female horses aged 7 months were used. The horses, which were clinically healthy and seronegative for AHS virus, were housed 2 weeks before the experiment at biocontainment level 3 facilities in the Centro de Investigaciones en Sanidad Animal (INIA, Valdeolmos, Madrid, Spain). They were given free access to water and a balanced feed ration throughout the experiment. Infection Five horses received an intravenous inoculation ( jugular vein) of 106 TCID50 of the virus in 1 ml saline. The animals were painlessly killed with T-61 (American Hoechst Corp., Somerville, NJ, USA), 11–16 days after infection (Table 1) when showing advanced signs of AHS. The sixth animal, used as an uninfected control, was inoculated with 1 ml phosphate-buffered saline (PBS), pH 7·2, and killed at the end of the experiment (day 17). Clinical manifestations of the disease, including rectal temperatures, were monitored from the time of infection until the animals were killed.

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Pulmonary Intravascular Macrophages in AHS

Table 1 Gross post-mortem findings observed in horses experimentally infected with African horse sickness virus (serotype 4) Animal no.

Days postinfection

Macroscopical findings Oedema

Haemorrhages

1

11

Supraorbital, palpebral and intermuscular in neck and thorax. Hydrothorax. Hydropericardium. Pulmonary oedema

Heart, lung, spleen

2

12

Supraorbital, palpebral and intermuscular in neck and thorax. Ascites. Alveolar and interstitial oedema in lung

Heart, lung, spleen

3

12

Supraorbital, palpebral, perineal and intermuscular in neck and thorax

Heart, lung, spleen

4

13

Palpebral and intermuscular in neck and thorax. Hydrothorax. Ascites

Heart, lung, pulmonary artery, skeletal muscle, oesophagus

5

16

Supraorbital, palpebral and intermuscular in neck and thorax. Hydropericardium

Heart, lung, spleen, urinary bladder

The experiment was performed in accordance with the Code of Practice for the Housing and Care of Animals used in Scientific Procedures, approved by the European Economic Community Union in 1986 (86/609/EEC). Histopathological and Ultrastructural Studies Lung samples were fixed in formaldehyde and glutaraldehyde, dehydrated through a graded series of alcohols to xylol (for light microscopy) or propylene oxide (for electron microscopy) and embedded in paraffin wax or Epon 812, respectively. Paraffinwax sections were stained with haematoxylin and eosin. For transmission electron microscopy (TEM), 50-nm sections were stained with uranyl acetate and lead citrate and examined with a Philips CM-10 transmission electron microscope. Morphometrical Study A video-based computer-linked image analyzer was used. To study the PIMs and their phagosomes, two blocks of alveolar tissue were selected from each pulmonary lobe of each animal, and 10 PIMs per block were photographed at ×3900 magnification. Images were captured with a television camera (model VK-C150ED; Hitachi, Japan) with a 50-mm lens, viewed on a monitor screen, and measured by the morphometry software program IMAGO, developed by the Sistemas Inteligentes en Visio´n Artificial (SIVA) research team at the University of Co´rdoba, Spain. The cross-sectional areas of the PIMs and their phagosomes were analyzed with the Statistical Analysis System (SAS) package (SAS Institute Inc., USA). Means and standard deviation were calculated, and differences were tested for significance (P<0·01 and P<0·05) by Student’s t test.

Results Clinical and Post-mortem Findings From 5 days after inoculation, all infected animals were febrile, and from 7 days they showed dyspnoea, tachypnoea and tachycardia, together with

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supraorbital and palpebral oedema and conjunctival haemorrhages. Postmortem examination (Table 1) revealed severe subcutaneous and intramuscular oedema (mainly in the neck), ascites and haemorrhages of the heart, spleen and large intestine. Two animals also showed hydrothorax and hydropericardium. The lungs of infected animals showed widened interlobular septa and two animals had visible alveolar oedema (Table 1). Ultrastructural Findings Septal capillaries of the control (uninfected) animal contained only a small number of erythrocytes, neutrophils, platelets and fusiform PIMs; the latter were characterized by adhesion to endothelial cells and by surface signs of micropinocytosis vermiformis. Animals infected with AHS virus showed thickened alveolar walls (Fig. 1), mainly resulting from invasion of septal capillaries by numerous neutrophils, platelets and mononuclear cells. Some septal capillary endothelial cells displayed areas of high-to-medium electron density, occasionally enclosed by a unit membrane, containing AHS virus-like particles with a circular profile (65–77 nm in diameter), an electron-dense centre (nucleoid), and a halo of lower electron density. Groups of virus-like particles, with or without a nucleoid and bounded by a membrane or free in the cytoplasm, were also visible (Fig. 2a). Paracrystalline (Fig. 2b) and tubular (Fig. 2c) structures were also observed in some endothelial cells. Other changes observed in septal capillary endothelial cells included a dramatic increase in micropinocytosis (Figs 3a and b), phagocyte activation (represented by primary and, occasionally, secondary lysosomes [Fig. 3c]), and the presence of cytoplasmic projections into the vascular lumina. The junctions between endothelial cells showed changes (Fig. 4a) varying from slight separation of the endothelial cells (but maintaining the tonofilament plaques at the junctions) to severe separation; platelets and neutrophils frequently displayed pseudopodial projections towards the basal membrane. The basal membrane of some capillaries was exposed, due to the disappearance of endothelial cells (Fig. 4b), and vascular lumina showed platelet aggregates, sometimes containing degranulated platelets or showing viscous changes, fibrin mesh and cell debris. Most PIMs showed substantial phagocyte activation (Fig. 5), as reflected by an increase in the number and size of lysosomes and phagosomes (Table 2), sometimes containing platelets and neutrophils in various stages of degeneration. This increase in phagocyte activation in infected horses was accompanied by an increase in cell size (Table 2) and by a change from the normal fusiform shape (as seen in the control) to a more rounded shape; as a result, the PIMs almost completely obstructed the capillary lumina (Fig. 6). A small percentage of PIMs showed areas similar to those described in endothelial cells, with circular AHS virus-like structures, 65–77 nm in diameter, possessing a nucleoid and a halo of reduced electron density (Fig. 7). There were also groups of virus-like particles, with and without a nucleoid, either enclosed by a membrane or free in the cytoplasm. Fibrin strands and aggregates of

Pulmonary Intravascular Macrophages in AHS

Fig. 1.

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Enlargement of alveolar wall due to thickening of septal capillaries, which contain PIMs (M), neutrophils, plug of degranulated platelets (P) and fibrin strands (arrows); interstitial oedema (E) is accompanied by fibrin strands (arrowheads) and erythrocytes. TEM. Bar, 5 lm.

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Fig. 2.

L. Carrasco et al.

Endothelial cells of septal capillaries, showing changes associated with AHS virus infection. (a) Viral matrix and a group of virus-like particles (arrow). TEM. Bar, 0·5 lm. (b) Paracrystalline array. Bar, 0·5 lm. (c) Tubular structures. Bar, 0·5 lm.

neutrophils or platelets, or both, were often observed close to PIMs, suggesting activation or virus replication. A further constant feature displayed by alveolar septa was a separation between capillary endothelial cells and alveolar epithelia (Figs 1, 3c and 5), the space being occupied by oedematous fluid and occasional erythrocytes, neutrophils or fibrin strands (Figs 1 and 3c). In addition to these changes, small numbers of interstitial macrophages and fibroblasts were observed in interalveolar septa, with a viral matrix and paracrystalline formations similar to those described in endothelial cells. Alveolar oedema and phagocytic activation of alveolar macrophages were constant features. This activation was occasionally accompanied by fibrin aggregates, the release of neutrophils into alveoli, and degenerative changes in type I pneumocytes, ranging from swelling of cells (Fig. 3a), characterized by disorganization and loss of cytoplasmic organelles, to loss of epithelial cells.

Pulmonary Intravascular Macrophages in AHS

Fig. 3.

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(a) Endothelial cell with a marked increase in the number of vesicles of micropinocytosis adjacent to swelling type I pneumocyte. TEM. Bar, 1 lm. (b) Endothelial cell with a marked increase in the number of vesicles of micropinocytosis adjacent to a PIM (M). Bar, 1 lm. (c) Endothelial cell, with secondary lysosomes (arrowheads), of a septal capillary surrounded by interstitial oedema (E) and fibrin strands (arrow). Bar, 1 lm.

Discussion The viral matrices and virus particles observed were similar to those previously described in AHS and other orbivirus infections (Tsai and Karstad, 1973; Mahrt and Osburn, 1986; Laegreid, 1992), with the presence in some cells of paracrystalline arrays and tubular structures (Mahrt and Osburn, 1986). However, in the present study, evidence of AHS virus replication was detected not only in the endothelial cells of septal capillaries, as previously reported by Laegreid et al. (1992), but also in fibroblasts, interstitial macrophages and PIMs. This may explain previous positive immunohistochemical results in certain mononuclear cells located in septal capillaries (Wohlsein et al., 1997).

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Fig. 4.

L. Carrasco et al.

(a) Plug of platelets, some with pseudopodial projections, in a capillary in which the endothelial cells show separation of intercellular junction (arrowheads). TEM. Bar, 1 lm. (b) Adhesion of a platelet to the basal membrane (arrows) due to the disappearance of the endothelial cell. Bar, 1 lm.

It would also support reports of replication of AHS virus and other orbiviruses in mononuclear phagocytes (Maclachlan et al., 1990; Brown et al., 1994). Alveolar and interstitial oedema are two of the most common lesions described in horses with AHS (Newsholme, 1983; Laegreid et al., 1992; Brown et al., 1994; Wohlsein et al., 1997). However, oedema and changes in pulmonary microvasculature seem at variance with the low percentage of endothelial cells apparently supporting AHS virus replication (Laegreid et al., 1992; Brown et al., 1994; Wohlsein et al., 1997). This suggests that these changes are brought about by various mechanisms, such as (1) increased permeability of cellular membranes of endothelial cells, shown by an increase in micropinocytosis and by the separation of intercellular junctions (Newsholme, 1983; Laegreid et al.,

Pulmonary Intravascular Macrophages in AHS

Fig. 5.

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Alveolar (Æ) and interstitial (E) oedema adjacent to septal capillary containing a PIM with large phagosomes (P). TEM. Bar, 1 lm. Table 2 Mean surface area of pulmonary intravascular macrophages (PIMs) and phagosomes in five horses infected with AHS and one uninfected (control) horse Horse no. 1 2 3 4 5 6 (Control)

Mean∗ ±SD area (lm2) of PIMs

phagosomes

32·77±9·11† 40·50±12·03† 52·79±14·19† 48·24±11·01† 51·95±11·73† 10·11±5·09

6·04±4·22‡ 7·20±3·83‡ 9·03±6·06‡ 11·52±5·64† 10·90±6·08‡ 1·23±0·73

∗ The number of PIMs examinated from each horse was 120. Significances of differences from control: † P<0·01; ‡ P<0·05.

1992), (2) loss of endothelial cells (Laegreid et al., 1992), and (3) haemodynamic alterations originating in the myocardium (Brown et al., 1994). In addition to oedematous fluid, some alveoli contained alveolar macrophages, neutrophils and fibrin strands, and showed disruption of the alveolar epithelial line. The presence of this inflammatory exudate has been described in the cardiac and mixed forms of AHS, and may be due to the initiation of an acute inflammatory process (Newsholme, 1983); disruption of the epithelial alveolar lining is one of the results of neutrophil migration to alveoli, as reported in the primary stages of certain bacterial pneumonias (Whiteley et al., 1991b).

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Fig. 6.

Vascular lumen of a septal capillary occupied by cell debris (∗) and enlarged PIM. TEM. Bar, 2 lm. Inset: Detail of the junctions (arrows) of a PIM with an endothelial cell. Bar, 0·5 lm.

Fig. 7.

Association of platelets on the surface of a PIM with viral matrix (arrowhead) bounded by a membrane. TEM. Bar, 0·5 lm. Inset: Detail of the viral matrix and the virus-like structures (arrows) surrounded by a halo of lower electron density. Bar, 0·5 lm.

Pulmonary Intravascular Macrophages in AHS

35

In the present study, AHS virus replication in septal capillaries may have been responsible for the loss of endothelial cells and consequent exposure of the basal membrane of septal capillaries. The latter may have been responsible for platelet aggregation and the formation of fibrinous microthrombi, as has been described previously (Newsholme, 1983; Laegreid et al., 1992). However, in our study aggregation of platelets was also observed, together the sequestration of neutrophils in pulmonary microvasculature, associated with PIMs. Similar findings have been reported in other species affected by other viruses (Carrasco et al., 1996b), bacteria or toxins (Bertram, 1986; Warner et al., 1987, 1988; Whiteley et al., 1991a, b). AHS virus replication was detected in PIMs, but activation was the predominant feature of these cells, which often showed morphological changes similar to those described in porcine, bovine and ovine PIMs after inoculation with viruses, or bacteria, or treatment with toxins (Bertram, 1986; Winkler and Cheville, 1986; Warner et al., 1987, 1988; Sierra et al., 1990; Whiteley et al., 1991a, b; Carrasco et al., 1992, 1996b; Haschek et al., 1992). These changes consisted of (1) increase in cell size, (2) rounding of cells, and (3) increase in phagocytic activity, shown by occasional phagocytosis of neutrophils and platelets. PIM activation may have been due to one of a number of causes, including exposure of the endothelial basal membrane (West et al., 1993), phagocytosis of cell debris present in vascular lumina (Bertram, 1986), or the action of inflammatory mediators (Carrasco et al., 1996b). The relationship between changes in pulmonary microvasculature and PIM activation suggests that once these cells are activated they release a series of substances capable of inducing an acute inflammatory response, as reported elsewhere (Warner et al., 1987, 1988; Whiteley et al., 1991a, b; Carrasco et al., 1996b). PIMs isolated from pigs produce several metabolites of arachidonic acid (Bertram et al., 1988). These include leukotriene B4, a powerful chemoattractant for neutrophils; its production might account for the accumulation of neutrophils observed in pulmonary microvasculature. Thromboxane A2, a powerful agonist for platelets, might account for the occurrence of platelet aggregates in the vicinity of PIMs; this substance has been shown to alter pulmonary haemodynamics temporarily, thereby increasing vascular resistance, inducing oedema, and hindering alveolar-capillary diffusion (Miyamoto et al., 1988). PIMs may also produce cytokines, such as interleukin (IL)-1 and tumour necrosis factor (TNF)-a (Chitko-McKown et al., 1991). These cytokines, which are chemotactic for neutrophils, act on endothelial cells, inducing a change in endothelial cell shape, widening the intercellular spaces, increasing vascular permeability, and inducing endothelial cells to produce coagulation factors such as platelet activating factor. These cytokines also activate other macrophages, and the release of cytokines by PIMs might account for the activation of alveolar macrophages (Carrasco et al., 1996b) and the subsequent appearance of inflammatory exudate in alveoli. In the present study, the role of PIMs and PIM activation in producing changes in the pulmonary microvasculature in AHS is corroborated by reports of the presence of platelet aggregates in horses (Atwal et al., 1992, 1994; West et al., 1993; Singh et al., 1994; West and Mathieu-Costello, 1994), oedema

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and the release of cells to the interstitium of the alveolar wall (West et al., 1993; West and Mathieu-Costello, 1994), and an increase in pulmonary arterial pressure (Longworth et al., 1994). The results obtained suggest that changes in pulmonary microvasculature and the subsequent oedema are not solely due to the effect of AHS virus on endothelial cells but are also due to PIM activation and the subsequent release of chemical inflammatory mediators. Nevertheless, future studies should also focus on myocardial changes in AHS and their role in the production of pulmonary haemodynamic alterations, which occur mainly in the cardiac and mixed forms of AHS. Acknowledgments The authors thank veterinary students F. Dı´az San Segundo and M. Macia´ for their enthusiastic laboratory work. This work was supported by grants from the Instituto Nacional de Investigaciones Agrarias (SC93-158) and the Plan Andaluz de Investigacio´n (AGR-0137). References Atwal, O. S., McDonell, W., Staempfli, H., Singh, B. and Minhas, K. J. (1994). Evidence that halothane anaesthesia induces intracellular translocation of surface coat and Golgi response in equine pulmonary intravascular macrophages. Journal of Submicroscopic Cytology and Pathology, 26, 369–386. Atwal, O. S. and Saldanha, K. A. (1985). Erythrophagocytosis in alveolar capillaries of goat lung. Ultrastructural properties of blood monocytes. Acta Anatomica, 124, 245–254. Atwal, O. S., Singh, B., Staempfli, H. and Minhas, K. J. (1992). Presence of pulmonary intravascular macrophages in the equine lung: some structuro-functional properties. Anatomical Record, 234, 530–540. Bertram, T. A. (1986). Intravascular macrophages in lungs of pigs infected with Haemophilus pleuropneumoniae. Veterinary Pathology, 23, 681–691 Bertram, T. A., Overby, L. H., Danilowicz, R., Eling, T. E. and Brody, A. R. (1988). Pulmonary intravascular macrophages metabolize arachidonic acid in vitro. American Review of Respiratory Diseases, 138, 936–944. Brown, C. C., Meyer, R. F. and Grubman, M. J. (1994). Presence of African horse sickness virus in equine tissues, as determined by in situ hybridization. Veterinary Pathology, 31, 689–694. Carrasco, L., Chaco´n-M. de Lara, F., Go´mez-Villamandos, J. C., Bautista, M. J., Villeda, C. J., Wilkinson, P. J. and Sierra, M. A. (1996b). The pathogenic role of pulmonary intravascular macrophages in acute African swine fever. Research in Veterinary Science, 61, 193–198. Carrasco, L., Ferna´ndez, A., Go´mez-Villamandos, J. C., Mozos, E., Me´ndez, A. and Jover, A. (1992). Kupffer cells and PIMs in acute experimental African swine fever. Histology and Histopathology, 7, 421–425. Carrasco, L., Go´mez-Villamandos, J. C., Bautista, M. J., Herva´s, J., Pulido, B. and Sierra, M. A. (1996a). Pulmonary intravascular macrophages in deer. Veterinary Research, 27, 71–77. Carrasco, L., Rodrı´guez, F., Martı´n de las Mulas, J., Sierra, M. A., Go´mez-Villamandos, J. C. and Ferna´ndez, A. (1991). Pulmonary intravascular macrophages in rabbits experimentally infected with rabbit haemorrhagic disease. Journal of Comparative Pathology, 105, 345–352. Chitko-McKown, C. G., Chapes, S. K., Brown, R. E., Phillips, R. M., McKown, R. D. and Blecha, F. (1991). Porcine alveolar pulmonary intravascular macrophages:

Pulmonary Intravascular Macrophages in AHS

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comparison of immune functions. Journal of Leukocyte Biology, 50, 364–372. Haschek, W. M., Motelin, G., Ness, D. K., Harlin, K. S., Hall, W. F., Vesonder, R. F., Peterson, R. E. and Beasley, V. R. (1992). Characterization of fumonisin toxicity in orally and intravenously dosed swine. Mycopathologia, 117, 83–96. Holmes, I. H., Boccardo, G., Estes, M. K., Furuichi, M. K., Hoshiro, Y., Joklik, W. K., McCrae, M., Mertens, P. P. C ., Milae, R. G., Samal, K. S. K., Shikata, E., Winton, J. K., Uyeda, Y. and Nuson, D. L. (1995). Family Reoviridae. In: Virus Taxonomy, F. A. Murphy, C. M. Faquet, D. H. L. Bishop, S. A. Ghabrial, A. W. Jarvis, G. P. Martinelli, M. A. Mayo and M. D. Summers, Eds, Springer-Verlag, Wien. Laegreid, W. W., Burrage, T. G., Stone-Marschat, M. and Skowronek, A. (1992). Electron microscopic evidence for endothelial infection by African horsesickness virus. Veterinary Pathology, 29, 554–556. Longworth, K. E., Jarvis, K. A., Tyler, W. S., Steffey, E. P. and Staub, N. C. (1994). Pulmonary intravascular macrophages in horses and ponies. American Journal of Veterinary Research, 55, 382–388. Maclachlan, N. J., Jagels, G., Rossitto, P. V., Moore, P. F. and Heidner, H. W. (1990). The pathogenesis of experimental blue tongue virus infection in calves. Veterinary Pathology, 27, 223–229. Mahrt, C. R. and Osburn, B. I. (1986). Experimental bluetongue infection of sheep; effect of vaccination: pathologic, immunofluorescent and ultrastructural studies. American Journal of Veterinary Research, 47, 1198–1203. Miyamoto, K., Schultz, E., Heath, T., Mitchell, M. D., Albertine, K. H. and Staub, N. C. (1988). Pulmonary intravascular macrophages and hemodynamic effects of liposomes in sheep. Journal of Applied Physiology, 64, 1143–1152. Newsholme, S. J. (1983). A morphological study of the lesions of African horsesickness. Onderstepoort Journal of Veterinary Research, 50, 7–24. Oellermann, R. A. (1970). Plaque formation by African horsesickness virus and characterization of its RNA. Onderstepoort Journal of Veterinary Research, 37, 137–144. Sierra, M. A., Carrasco, L., Go´mez-Villamandos, J. C., Martin de las Mulas, J., Me´ndez, A. and Jover, A. (1990). Pulmonary intravascular macrophages in lungs of pigs inoculated with African swine fever virus of differing virulence. Journal of Comparative Pathology, 102, 323–334. Singh, B., Minhas, K. J. and Atwal, O. S. (1994). Ultracytochemical study of multiple dose effect of monostral blue uptake by equine pulmonary intravascular macrophages (PIMs). Journal of Submicroscopic Cytology and Pathology, 26, 235–243. Thanawongnuwech, R., Thacker, E. L. and Halbur, P. G. (1997). Effect of porcine reproductive and respiratory syndrome virus (PRRSV) (isolate ATCC VR-2385) infection on bactericidal activity of porcine pulmonary intravascular macrophages (PIMs): in vitro comparisons with pulmonary alveolar macrophages (PAMs). Veterinary Immunology and Immunopathology, 59, 323–335. Tsai, K. and Karstad, L. (1973). The pathogenesis of epizootic hemorrhagic disease of deer. American Journal of Pathology, 70, 379–400. Warner, A. E., DeCamp, M. M., Molina, R. M. and Brain, J. D. (1988). Pulmonary removal of circulating endotoxin results in acute lung injury in sheep. Laboratory Investigation, 59, 219–230. Warner, A. E., Molina, R. and Brain, J. D.(1987). Uptake of bloodborne bacteria by pulmonary intravascular macrophages and consequent inflammatory responses in sheep. American Review of Respiratory Diseases, 136, 683–690. West, J. B. and Mathieu-Costello, O. (1994). Stress failure of pulmonary capillaries as a mechanism for exercise-induced pulmonary haemorrhage in the horse. Equine Veterinary Journal, 26, 441–447. West, J. B., Mathieu-Costello, O., Jones, J. H., Birks, E. K., Logemann, R. B., Pascoe, J. R. and Tyler, W. S. (1993). Stress failure of pulmonary capillaries in racehorses with exercise-induced pulmonary hemorrhage. Journal of Applied Physiology, 75, 1097–1109.

38

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Whiteley, L. O., Maheswaran, S. K., Weiss, D. J. and Ames, T. R. (1991a). Morphological and morphometrical analysis of the acute response of the bovine alveolar wall to Pasteurella haemolytica Al-derived endotoxin and leucotoxin. Journal of Comparative Pathology, 104, 23–32. Whiteley, L. O., Maheswaran, S. K., Weiss, D. J. and Ames, T. R. (1991b). Alterations in pulmonary morphology and peripheral coagulation profiles caused by intratracheal inoculation of live and ultraviolet light-killed Pasteurella haemolytica A1 in calves. Veterinary Pathology, 28, 275–285. Winkler, G. C. and Cheville, N. F. (1986). Ultrastructural morphometric investigation of early lesions in the pulmonary alveolar region of pigs during experimental swine influenza infection. American Journal of Pathology, 122, 541–552. Wohlsein, P., Pohlenz, J. F., Davidson, F. L., Salt, J. S. and Hamblin, C. (1997). Immunohistochemical demonstration of African horse sickness viral antigen in formalin-fixed equine tissues. Veterinary Pathology, 34, 568–574.



Received, August 3rd, 1998 Accepted, December 21st, 1998