Vet Pathol 29:554-556 (1992)
Electron Microscopic Evidence for Endothelial Infection by African Horsesickness Virus W. W.
LAEGREID,
T. G.
BURRAGE, M. STONE-MARSCHAT, AND
A.
SKOWRONEK
Key words: African horsesickness; endothelium; horses.
African horsesickness (AHS) is an arboviral disease of horses, donkeys and mules. The causative agent, classified in the Reoviridae, genus Orbivirus, is transmitted primarily by biting midges of the genus Culicoides. African horsesickness virus (AHSV) infection results in severe pulmonary edema and pleural and pericardial effusion in naive horses.>' The mechanism(s) by which these alterations in fluid balance occur has been the subject of limited research. The results of one recent study of the morphologic basis for the development of pulmonary edema in AHS revealed no ultrastructural evidence for endothelial damage or infection by AHS virus .. The present report describes the ultrastructural evidence for infection of pulmonary microvascular endothelial cells of horses experimentally infected with AHSV. Two groups of four horses each were used. Animals in group NO.1 were infected by intravenous injection with 102 CCID so of AHSV type 4 (a recent field isolate, passed three times in Vera cells, provided by Dr. J. M. Sanchez-Vizcaino, Instituto Nacional de Investigaciones Agrarias (INIA), Madrid, Spain). Animals in group No.2 were sham inoculated with virus-free medium. Both groups were evaluated clinically daily. When an animal was in extremis, based on presence of high fever and respiratory rate >40 breaths per minute along with depression or recumbency, the animal was euthanatized by intravenous injection of T-61 (American Hoechst Corp., Somerville, NJ) and a complete necropsy was performed. Tissues for routine histologic examination were fixed in 10% neutral buffered formalin and embedded in paraffin. Samples oflung were obtained from multiple sites, minced, and fixed by immersion for 2 hours in 2% formaldehyde (freshly prepared from paraformaldehyde) and 1% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). Tissues were post-fixed in 2% osmium tetroxide in 0.1 M cacodylate buffer and stained en bloc with uranyl acetate. Thin sections from at least five blocks of lung from each horse were mounted on slot (1- x 2-mm) grids for transmission electron microscopic examination. Animals infected with AHSV developed typical high fevers (40 C [104 F]) by post-infection day 4 and showed signs of depression. On post-infection day 6, all infected animals had mild to moderate increases in respiratory rate and effort. One animal died very rapidly following the onset of clinical signs (post-infection day 6) and the others were euthanatized (postinfection day 7). None of the control animals had signs or
lesions indicative ofAHSV infection. The lungs ofall AHSVinfected animals failed to collapse on opening the thoracic cavity. The lungs were mottled red with pronounced widening of the interlobular septa. Cut surfaces were moist and glistening with accumulations of pale yellow gelatinous material in the interlobular spaces. Histologically, many alveolar spaces, irregularly distributed in large patches, were filled with homogenous, pale eosinophilic edema fluid. Alveolar capillaries were either distended with erythrocytes or nearly empty of cells. As previously described, many arterioles and small arteries in the lung had moderate to large numbers of mononuclear cells within the adventitia and media; there was no cellular infiltration of the intima of these vessels.'> Some arterioles contained increased numbers of monocytes and lymphocytes with a few neutrophils, occasionally nearly filling their lumina. Larger vessels, small veins, and conducting airways were normal. Ultrastructural changes in the lung were most prominent in the alveolar capillaries. Numerous capillaries contained discrete aggregations of platelets, monocytes, and occasional neutrophils (microthrombi) that were quite closely apposed to the endothelial cells in some sections. Small to moderate amounts of irregular electron-dense fibrillar material (fibrin) were associated with most of the microthrombi. The capillary lumina adjacent to these microthrombi were often greatly enlarged and packed with erythrocytes. No such changes were present in comparable sections from control animals. There were changes indicative of capillary endothelial damage diffusely distributed throughout sections oflung from all blocks examined from each of the AHSV-infected horses; endothelial damage was not present in sections from control horses. The most common finding in endothelial cells was loss of electron density of the cytoplasm accompanied by increased numbers of cavealae on both the luminal and basal margins of the cells. Pronounced cell swelling with some loss of integrity ofplasma membranes was present in the most severely affected cells. Tight junctions between endothelial cells were intact in many sections. However, widely separated junctions (up to I «rn apart) were occasionally present, as were greater numbers ofjunctions that appeared to have lost integrity but did not have widened junctional spaces. Infrequently, there were distinct accumulations of coarse, granular electron-dense material in the cytoplasm of capillary endothelial cells (Fig. I). This material was morphologically similar to the viral
Fig. 1. Lung; horse. The alveolar capillary contains an electron-dense mass of granular viral matrix and mature viral particles (arrow). Bar = 0.5 Mm. Inset: Enlargement of area indicated by arrow in Fig. I. Bar = 100 nm. Fig. 2. Lung; horse. Note alveolar capillary endothelial cell with perinuclear viral matrix and viral particles. M = viral matrix, G = Golgi complex. Bar = 0.5 Mm. 554 Downloaded from vet.sagepub.com at KAI NAN UNIV on March 6, 2015
Vet Pathol 29:6, 1992
Brief Communications and Case Reports
Downloaded from vet.sagepub.com at KAI NAN UNIV on March 6, 2015
555
Brief Communications and Case Reports
556
matrix associated with AHSV and other orbivirus replication.i-> Within this matrix in some cells were mature virus
particles (Fig. I, inset). These particles were 78 nm in diameter with an electron-dense core surrounded by a less dense outer capsid, morphology typical of intracellular AHSV (Fig. I, inset). I The viral matrix and particles tended to be located in the perinuclear and Golgi regions ofthe endothelial cells (Fig. 2). Tubular structures associated with orbivirus replication were not recognized in pulmonary microvascular endothelial cells infected with AHSV.u In this experiment, the changes seen in the pulmonary microvasculature of horses infected with AHSV are quite similar to those seen in other significant orbiviral diseases of animals, such as bluetongue and epizootic hemorrhagic disease of deer.2.3.5·!o A previous study failed to detect any AHS-related changes in pulmonary vascular endothelium, lymph nodes, or spleen from infected horses." Possible reasons for the failure of other investigators to identify endotheliallesions in AHS include differences in the virulence of virus isolates and irregular distribution of the lesions within the lung. However, the accumulations of viral matrix are quite small and only a small percentage of capillary endothelial cells contained viral matrix in quantities sufficient for ultrastructural identification. Even fewer cells contained identifiable viral particles.' The changes described in the lungs ofAHSV -infected horses may account for the severe pulmonary edema associated with acute AHS; however, the presence of direct virus infection of some endothelial cells, loss of junctional integrity in other endothelial cells without evidence ofdirect infection, and the presence of microthrombi suggest that multiple mechanisms may be involved in the development of pulmonary microvascular leakage. Whichever mechanism predominates in a given animal may determine the rapidity of
Vet Pathol 29:6, 1992
onset of clinical signs, severity of disease, and perhaps which form of AHS an infected animal develops. The results presented in this report are the first description of endothelial damage and infection by AHSV. Mature viral particles in endothelial cells represent the first localization of AHSV in a particular cell type in the horse and may at least partially explain the development of pulmonary edema in AHS-infected horses. These findings provide significant insight into the pathogenesis of AHS and establish a firm morphologic foundation for future studies in this area.
References
2
3 4
5
6
Lecatsas G, Erasmus BJ: Electron microscopic study of the formation of African horse-sickness virus. Arch Gesamte Virusforsch 22:442-450, 1967 Mahrt CR, Osburn BI: Experimental bluetongue infection of sheep; effect of vaccination: pathologic, immunofluorescent and ultrastructural studies. Am J Vet Res 47: 1198-1203,1986 Maurer FD, McCully RM: African horse-sickness-with emphasis on pathology. Am J Vet Res 24:235-266, 1963 Newsholme SJ: A morphological study of the lesions of African horsesickness. Onderstepoort J Vet Res 50:7-24, 1983 Stair EL: The pathogenesis ofbluetongue in sheep: a study by immunofluorescence and histopathology. PhD Diss., Texas A&M University, College Station, TX, 1968 Tsai K, Karstad L: The pathogenesis of epizootic hemorrhagic disease of deer. Am J Pathol 70:379-400, 1973
Request reprints from Dr. W. W. Laegreid, Molecular Pathology, Plum Island Animal Disease Center, USDA, ARS, PO Box 848, Greenport, NY 11944-0848 (USA).
Vet Pathol 29:556-559 (1992)
Chronic Proliferative Rhinitis Associated with Salmonella arizonae in Sheep 1. T.
MEEHAN,
R. C.
K. A.
BROGDEN,
CUTLIP, AND
H. D.
C.
COURTNEY,
LEHMKUHL
Key words: Proliferative rhinitis; Salmonellae arizonae; sheep. Salmonella arizonae has been isolated frequently from sheep in North America and the United Kingdom.1.4.6.9.11.13 In two reports, S. arizonae isolations were associated with abortion or enteritis.v' In other reports, S. arizonae was considered host adapted to sheep because it was either isolated from intestine in the absence of lesions or was a secondary finding to other pathogens.t-" The following is a report of two cases of proliferative rhinitis associated with S. arizonae infection in sheep. Sheep No. I was a clinically normaI2-year-old Columbian
ewe at the National Animal Disease Center (Ames, IA) that was experimentally infected with bovine respiratory syncytial virus. At 4 days post-inoculation, the sheep was euthanatized by administering Sleepaway (Fort Dodge Laboratories) intravenously for postmortem examination. The cranial portion of the left ventral nasal concha had a locally extensive area (I em) of nodules (2-3 mm) on the mucosa. Some nodules had pale central areas (Fig. I). For histologic evaluation, turbinates were fixed in 4% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.6) and pro-
Downloaded from vet.sagepub.com at KAI NAN UNIV on March 6, 2015
Electron Microscopic Evidence for Endothelial Infection by African Horsesickness Virus W. W.
LAEGREID,
T. G.
BURRAGE, M. STONE-MARSCHAT, AND
A.
SKOWRONEK
Key words: African horsesickness; endothelium; horses.
African horsesickness (AHS) is an arboviral disease of horses, donkeys and mules. The causative agent, classified in the Reoviridae, genus Orbivirus, is transmitted primarily by biting midges of the genus Culicoides. African horsesickness virus (AHSV) infection results in severe pulmonary edema and pleural and pericardial effusion in naive horses.>' The mechanism(s) by which these alterations in fluid balance occur has been the subject of limited research. The results of one recent study of the morphologic basis for the development of pulmonary edema in AHS revealed no ultrastructural evidence for endothelial damage or infection by AHS virus .. The present report describes the ultrastructural evidence for infection of pulmonary microvascular endothelial cells of horses experimentally infected with AHSV. Two groups of four horses each were used. Animals in group NO.1 were infected by intravenous injection with 102 CCID so of AHSV type 4 (a recent field isolate, passed three times in Vera cells, provided by Dr. J. M. Sanchez-Vizcaino, Instituto Nacional de Investigaciones Agrarias (INIA), Madrid, Spain). Animals in group No.2 were sham inoculated with virus-free medium. Both groups were evaluated clinically daily. When an animal was in extremis, based on presence of high fever and respiratory rate >40 breaths per minute along with depression or recumbency, the animal was euthanatized by intravenous injection of T-61 (American Hoechst Corp., Somerville, NJ) and a complete necropsy was performed. Tissues for routine histologic examination were fixed in 10% neutral buffered formalin and embedded in paraffin. Samples oflung were obtained from multiple sites, minced, and fixed by immersion for 2 hours in 2% formaldehyde (freshly prepared from paraformaldehyde) and 1% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). Tissues were post-fixed in 2% osmium tetroxide in 0.1 M cacodylate buffer and stained en bloc with uranyl acetate. Thin sections from at least five blocks of lung from each horse were mounted on slot (1- x 2-mm) grids for transmission electron microscopic examination. Animals infected with AHSV developed typical high fevers (40 C [104 F]) by post-infection day 4 and showed signs of depression. On post-infection day 6, all infected animals had mild to moderate increases in respiratory rate and effort. One animal died very rapidly following the onset of clinical signs (post-infection day 6) and the others were euthanatized (postinfection day 7). None of the control animals had signs or
lesions indicative ofAHSV infection. The lungs ofall AHSVinfected animals failed to collapse on opening the thoracic cavity. The lungs were mottled red with pronounced widening of the interlobular septa. Cut surfaces were moist and glistening with accumulations of pale yellow gelatinous material in the interlobular spaces. Histologically, many alveolar spaces, irregularly distributed in large patches, were filled with homogenous, pale eosinophilic edema fluid. Alveolar capillaries were either distended with erythrocytes or nearly empty of cells. As previously described, many arterioles and small arteries in the lung had moderate to large numbers of mononuclear cells within the adventitia and media; there was no cellular infiltration of the intima of these vessels.'> Some arterioles contained increased numbers of monocytes and lymphocytes with a few neutrophils, occasionally nearly filling their lumina. Larger vessels, small veins, and conducting airways were normal. Ultrastructural changes in the lung were most prominent in the alveolar capillaries. Numerous capillaries contained discrete aggregations of platelets, monocytes, and occasional neutrophils (microthrombi) that were quite closely apposed to the endothelial cells in some sections. Small to moderate amounts of irregular electron-dense fibrillar material (fibrin) were associated with most of the microthrombi. The capillary lumina adjacent to these microthrombi were often greatly enlarged and packed with erythrocytes. No such changes were present in comparable sections from control animals. There were changes indicative of capillary endothelial damage diffusely distributed throughout sections oflung from all blocks examined from each of the AHSV-infected horses; endothelial damage was not present in sections from control horses. The most common finding in endothelial cells was loss of electron density of the cytoplasm accompanied by increased numbers of cavealae on both the luminal and basal margins of the cells. Pronounced cell swelling with some loss of integrity ofplasma membranes was present in the most severely affected cells. Tight junctions between endothelial cells were intact in many sections. However, widely separated junctions (up to I «rn apart) were occasionally present, as were greater numbers ofjunctions that appeared to have lost integrity but did not have widened junctional spaces. Infrequently, there were distinct accumulations of coarse, granular electron-dense material in the cytoplasm of capillary endothelial cells (Fig. I). This material was morphologically similar to the viral
Fig. 1. Lung; horse. The alveolar capillary contains an electron-dense mass of granular viral matrix and mature viral particles (arrow). Bar = 0.5 Mm. Inset: Enlargement of area indicated by arrow in Fig. I. Bar = 100 nm. Fig. 2. Lung; horse. Note alveolar capillary endothelial cell with perinuclear viral matrix and viral particles. M = viral matrix, G = Golgi complex. Bar = 0.5 Mm. 554 Downloaded from vet.sagepub.com at KAI NAN UNIV on March 6, 2015
Vet Pathol 29:6, 1992
Brief Communications and Case Reports
Downloaded from vet.sagepub.com at KAI NAN UNIV on March 6, 2015
555
Brief Communications and Case Reports
556
matrix associated with AHSV and other orbivirus replication.i-> Within this matrix in some cells were mature virus
particles (Fig. I, inset). These particles were 78 nm in diameter with an electron-dense core surrounded by a less dense outer capsid, morphology typical of intracellular AHSV (Fig. I, inset). I The viral matrix and particles tended to be located in the perinuclear and Golgi regions ofthe endothelial cells (Fig. 2). Tubular structures associated with orbivirus replication were not recognized in pulmonary microvascular endothelial cells infected with AHSV.u In this experiment, the changes seen in the pulmonary microvasculature of horses infected with AHSV are quite similar to those seen in other significant orbiviral diseases of animals, such as bluetongue and epizootic hemorrhagic disease of deer.2.3.5·!o A previous study failed to detect any AHS-related changes in pulmonary vascular endothelium, lymph nodes, or spleen from infected horses." Possible reasons for the failure of other investigators to identify endotheliallesions in AHS include differences in the virulence of virus isolates and irregular distribution of the lesions within the lung. However, the accumulations of viral matrix are quite small and only a small percentage of capillary endothelial cells contained viral matrix in quantities sufficient for ultrastructural identification. Even fewer cells contained identifiable viral particles.' The changes described in the lungs ofAHSV -infected horses may account for the severe pulmonary edema associated with acute AHS; however, the presence of direct virus infection of some endothelial cells, loss of junctional integrity in other endothelial cells without evidence ofdirect infection, and the presence of microthrombi suggest that multiple mechanisms may be involved in the development of pulmonary microvascular leakage. Whichever mechanism predominates in a given animal may determine the rapidity of
Vet Pathol 29:6, 1992
onset of clinical signs, severity of disease, and perhaps which form of AHS an infected animal develops. The results presented in this report are the first description of endothelial damage and infection by AHSV. Mature viral particles in endothelial cells represent the first localization of AHSV in a particular cell type in the horse and may at least partially explain the development of pulmonary edema in AHS-infected horses. These findings provide significant insight into the pathogenesis of AHS and establish a firm morphologic foundation for future studies in this area.
References
2
3 4
5
6
Lecatsas G, Erasmus BJ: Electron microscopic study of the formation of African horse-sickness virus. Arch Gesamte Virusforsch 22:442-450, 1967 Mahrt CR, Osburn BI: Experimental bluetongue infection of sheep; effect of vaccination: pathologic, immunofluorescent and ultrastructural studies. Am J Vet Res 47: 1198-1203,1986 Maurer FD, McCully RM: African horse-sickness-with emphasis on pathology. Am J Vet Res 24:235-266, 1963 Newsholme SJ: A morphological study of the lesions of African horsesickness. Onderstepoort J Vet Res 50:7-24, 1983 Stair EL: The pathogenesis ofbluetongue in sheep: a study by immunofluorescence and histopathology. PhD Diss., Texas A&M University, College Station, TX, 1968 Tsai K, Karstad L: The pathogenesis of epizootic hemorrhagic disease of deer. Am J Pathol 70:379-400, 1973
Request reprints from Dr. W. W. Laegreid, Molecular Pathology, Plum Island Animal Disease Center, USDA, ARS, PO Box 848, Greenport, NY 11944-0848 (USA).
Vet Pathol 29:556-559 (1992)
Chronic Proliferative Rhinitis Associated with Salmonella arizonae in Sheep 1. T.
MEEHAN,
R. C.
K. A.
BROGDEN,
CUTLIP, AND
H. D.
C.
COURTNEY,
LEHMKUHL
Key words: Proliferative rhinitis; Salmonellae arizonae; sheep. Salmonella arizonae has been isolated frequently from sheep in North America and the United Kingdom.1.4.6.9.11.13 In two reports, S. arizonae isolations were associated with abortion or enteritis.v' In other reports, S. arizonae was considered host adapted to sheep because it was either isolated from intestine in the absence of lesions or was a secondary finding to other pathogens.t-" The following is a report of two cases of proliferative rhinitis associated with S. arizonae infection in sheep. Sheep No. I was a clinically normaI2-year-old Columbian
ewe at the National Animal Disease Center (Ames, IA) that was experimentally infected with bovine respiratory syncytial virus. At 4 days post-inoculation, the sheep was euthanatized by administering Sleepaway (Fort Dodge Laboratories) intravenously for postmortem examination. The cranial portion of the left ventral nasal concha had a locally extensive area (I em) of nodules (2-3 mm) on the mucosa. Some nodules had pale central areas (Fig. I). For histologic evaluation, turbinates were fixed in 4% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.6) and pro-
Downloaded from vet.sagepub.com at KAI NAN UNIV on March 6, 2015
