Vet. Pathol. 12: 394-404 (1975)
Pathogenetic Studies of Infection of the Bovine Fetus with Bovine Viral Diarrhea Virus 11. Ocular Lesions
T.T. BROWN,S . I. BISTNER, A. DELAHUNTA, F. W. SCOTTand K. MCENTEE Departments of Large Animal Medicine, Obstetrics and Surgery, Anatomy and Microbiology, New York State Veterinary College at Cornell University, Ithaca, N.Y.
Absrrucr. Twenty-three susceptible pregnant heifers were inoculated with bovine viral diarrhea virus at 150k 1 days of gestation. Seven additional heifers were inoculated between 65 and 115 days of gestation. Acute ocular lesions were seen in fetuses taken 17-21 days after inoculation of their dams at 150 days. By the fourth week, the acute lesions were beginning to resolve, and in newborn animals focal to total retinal atrophy was seen. The acute lesions were characterized by a mild to moderate retinitis that resulted in various degrees of destruction of the different layers, mononuclear cuffing of inner retinal vessels, proliferation of pigment epithelium, and choroiditis. Residually there was an absence of cellular elements in the atrophied areas of the retina, frequently a loss of layering and various numbers of pigment-containing cells. Moderately severe acute inflammation was seen in the retina of the fetus taken at 22 days after inoculation of its dam at 95 days. Ocular lesions did not occur in the other fetuses taken from heifers inoculated at earlier stages of gestation.
Congenital infection of human fetuses with rubella virus and cytomegalovirus may result in ocular lesions in addition to lesions in other tissues [14, 15, 19, 291. Experimental viral infection of the fetuses or neonates of some laboratory animals results in the consistent production of ocular lesions [l, 2,6, 13,21,26,27]. Inoculation of lamb fetuses with blue tongue vaccine virus and newborn rats with lymphocytic choriomeningitis virus and simian virus 40 results in retinopathy [13, 21, 26, 271. The ocular lesions observed after the inoculation of these viruses into the appropriate laboratory animal are similar in several respects. They attack the immature retina, primarily damaging either the developing neuroblastic layer or one or both of the recently formed nuclear layers. Viral infection or lesions, or both, are also observed in the pigment epithelium. Endothelial damage or perivascular
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mononuclear cuffing, or both [13,21,26], are usually seen in the inner retina. Residually there is retinal atrophy with or without rosette formation. Infection of susceptible pregnant cows with bovine viral diarrhea virus may result in ocular lesions in their offspring [2, 4-6, 24, 281. This paper describes the evolution of ocular lesions in bovine fetuses after inoculation of this virus into susceptible pregnant heifers.
Materials and Methods Twenty-three pregnant heifers with no detectable serum neutralizing antibody to bovine viral diarrhea virus were inoculated intravenously with 250 OOO TCID,, (median tissue culture infective dose) of a cytopathogenic strain (Holmes) of virus at 150+ I days of gestation. Details for preparation of the virus inoculum are given by BROWNet al. [5]. Seven additional heifers were inoculated between 65 and 115 days of gestation. Three control heifers with serum neutralizing titers to the virus were inoculated at 150+ 1 days of gestation. The fetuses of the susceptible heifers were surgically taken at sequential times after inoculation (table I). Their eyes were examined with a direct ophthalmoscope (Vistadial Giantscope, American Optical Corp., Buffalo, N.Y.); the fetuses were then exsanguinated by intracardiac puncture. Two susceptible and one control heifers were allowed to calve normally, and the calves were clinically examined before being electrocuted. Shortly after death, one eye was fixed in Zenker’s solution for 6-12 h. It was placed in 50% alcohol for 24 h and then stored in 70% alcohol. Paraffin-embedded sections were cut at 6 pm and stained with hematoxylin and eosin (HE).
Results Gross lesions were observed only in one animal. The F45 calf taken at 140 days was almost totally blind. Ophthalmoscopic examination showed increased pigmentation bilaterally and adherence of a persistent pupillary membrane to the anterior capsule of the lens of the right eye. The occurrence of microscopic ocular lesions in the fetuses or calves is listed in table I. Lesions were restricted to the retina and choroid. Acute inflammatory lesions occurred in fetuses taken between 17 and 21 days from dams inoculated at 150 f 1 days of gestation and in a 117-day fetus whose dam was inoculated at 95 days of gestation. Resolving or resolved lesions were seen in fetuses taken after 21 days from heifers inoculated at 150 days. The acute lesions in two fetuses were mild. In fetus F78 taken at 17 days, small focal hemorrhages were occasionally seen in the inner nuclear layer of the nontapetal retina. A few small, subtle areas of necrosis were seen in
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Table I. Ocular lesions in fetuses from heifers inoculated with bovine viral diarrhea virus during pregnancy
Fetus No. F44 F47 F87 F54 F1902
Gestational age inoculation cesarotomy 150 150
149 150 150
154 157 161 163 164
Days after inoculation
14
150 150
F94 F48 F8974 F55 F56
150 150 150 150 150
168 169 170 171 171
18 19 20 21 21
F49 F48a F57 F1901 F53
150 150
174 177 191 206 219
24 27 42 56 70
232 27 1 290
83 120 140
149 150 150
F5O F56a F45
149 150
149 149 151
150
15
16 17 17
F70* F41 F02 F95 F38
115
95 95 85 80
137 112 117 106 113
22 17 22 21 33
F66 F80
75 65
96 86
21 21
Control F6 150 F5I 150 F52 151
168 178 284
18 28 133
- = None;
inflammation necrosis
4 7 12 13 14
163 165 166 167 167
FOI F63 F62 F46 F78
Ocular lesions'
+ = least severe; +f+i- = most severe.
* At cesarotomy, fetus was dead.
atrophy
Ocular Lesions in Bovine Diarrhea Virus
397 I
Fig. 1. Fetus F8974, 170 days. Focal retinitis with extensive necrosis in the nuclear layers and mononuclear perivascular cuffing of inner retinal vessels. Also, choroiditis and reactive pigment epithelial cells. HE.
the outer nuclear layer of the nontapetal retina of the F56 fetus taken at 21 days. They appeared as small, clear areas in which cellular debris could be seen. In one or two places, the necrosis extended to the inner nuclear layer. The acute lesions in fetuses F8974, taken at 20 days, and F02, taken 22 days after inoculation of its dam at 95 days of gestation, were considerably more severe. In both fetuses, there were various sized foci of retinitis that occurred mainly in the nontapetal retina. There was severe necrosis in the inner and outer nuclear layers of F8974 (fig. 1). Pyknotic nuclei and fragmented cellular debris were prominent, and normally separated layers blended together. In such areas, the rod and cone layer was destroyed and the adjacent pigment epithelium was hypertrophied and hyperplastic. Cellular debris was intermixed with the reactive cells of the pigment epithelium. Associated with these areas of retinitis was a mononuclear inflammatory infiltrate in the inner portion of the choroid. Many of the inner retinal vessels were cuffed by mononuclear cells, most of which were undifferentiated but some of which
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Fig. 2. Fetus F02, 117 days. Serial sections of a small focus of retinal inflammation show the apparent spread of inflammation to the choroid through a small break in the external limiting membrane. a Inflammation of the neuroblastic layer (arrow), pigment epithelium and choroid with an intact external limiting membrane, and separation of retina from pigment epithelium. b Same inflammatory focus 30 pm lateral to a. Focal disruption of external limiting membrane with extension of inflammation from retina t o pigment epithelium and choroid. c Same inflammatory focus 12 prn lateral to b a n d opposite a. Changes similar to those in a. HE.
were identifiable as lymphocytes or plasma cells. Necrosis of ganglion cells could occasionally be seen. Similar lesions were seen in the F02 fetus, in which the undifferentiated neuroblastic layer was the principal site of damage. Necrosis was not as severe as in F8974 but of sufficient severity to make the Miiller cells prominent in some areas. Perivascular cuffing did not occur in the inner retina, but there was choroidal inflammation similar to that seen in F8974. Adjacent to areas of choroidal inflammation, the pigment epithelium was reactive, and there was separation of the retina from the pigment epithelium. In areas where necrosis in the neuroblastic layer was extensive, patchy breaks were present in the external limiting membrane, and pseudorosettes were occasionally seen. In areas where inflammation was less severe, that in the retina and choroid appeared to have been the result of separate processes; however, serial sectioning of such areas showed, at greater
Ocular Lesions in Bovine Diarrhea Virus
399
Fig. 3. Fetus F53, 219 days. Atrophied, actively inflamed retina. Inflammatory cells tend to aggregate around vessels. Numerous lymphocytes with plasma cells among them. HE.
depths, small breaks in the external limiting membrane. At such points, there was a continuation of inflammation from the neuroblastic layer to the pigment epithelium and adjacent choroid, suggesting that inflammation spread outward from the neuroblastic layer through the small breaks in the external limiting membrane to the choroid (fig. 2). The retinal lesions in the F55 fetus taken at 21 days were resolving. A few small focal areas of atrophy were seen in the nontapetal and tapetal retina, in which there was a focal depletion of cells in the outer and, to a lesser extent, the inner nuclear layers. Cellular debris was not evident, and there were no signs of active inflammation in the retina or choroid. However, the presence of cells containing pigment in the depleted areas attested to a prior inflammatory episode. In areas where there was a depletion of cells in the nuclear layers, the rod and cone layer was destroyed, and there was hypertrophy of the pigment epithelium. Focal to total retinal atrophy was seen in the fetuses taken at 42 days or afterwards. With the exception of the F53 fetus taken at 70 days, active inflammation was not observed in the retina or choroid of these fetuses. In some places, cells containing pigment were present in the atrophied retinal tissues. In the F53 fetus, the entire retina was damaged, and mononuclear inflammatory cells were still numerous (fig. 3). Many of the inflammatory cells were either lymphocytes or plasma cells and were aggregated in the
400
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Fig. 4. Term fetus F56a, 271 days. Focal retinal atrophy. Outer layers of the retina have been destroyed, whereas the inner layers are intact. HE. Fig. 5. Fetus F50, 232 days. Retinal fold at a point of detachment of the retina from the proliferated pigment epithelium. HE.
markedly altered inner layers most abundantly around vessels. The nuclear layers had been severely damaged, and only a single layer of cells, three to four cells wide, remained in the outermost retina. Retinal atrophy was similar in the eyes of the animals taken at later stages of gestation or after birth. The nuclear layers were most noticeably damaged. In places, there was total destruction of the nuclear layers, the intervening outer plexiform layer, and the layer of rods and cones, whereas the inner retinal layers appeared more normal (fig. 4). Total retinal atrophy was seen in other eyes with a loss of layering and glial scars. Few retinal folds were present in the eye of the F56a fetus taken at 120 days (fig. 5). These folds were created by a detachment and infolding of the external limiting membrane and outer nuclear layer. The pigment epithelium adjacent to these areas was hypertrophied and hyperplastic. Ocular lesions were not seen in the control fetuses nor in fetuses, other than F02, from heifers inoculated at earlier stages of gestation.
Ocular Lesions in Bovine Diarrhea Virus
40 I
Discussion
The acute lesions seen in the bovine fetuses were similar to those seen in lambs infected in utero with bluetongue virus and in rat neonates infected with lymphocytic choriomeningitis virus or simian virus 40, in that the principal damage occurred in the neuroblastic layer or in the nuclear layers that derive from it [13,21,26,27]. Mononuclear perivascular cuffs of inner retinal vessels were common to all these viral-induced retinopathies. The cuffs were usually part of the acute lesion and were not extensive. However, in the bovine fetus taken at 70 days (F53), the retinal inflammation persisted until later in gestation than it did in the other fetuses and was more intense and the inflammatory cells better differentiated. These findings suggest a more prolonged antigenic stimulation in the eye of this fetus than in other fetuses. Retinal inflammation of a sufficient degree to cause destruction of cells in the nuclear layers is frequently accompanied by destruction of the adjacent pigment epithelium and basilar lamina, allowing inflammation to extend to the choroid [lo]. Apparently, this is what occurred in the eyes of the bovine fetuses. When choroidal inflammation was seen, it occurred adjacent to areas of retinal inflammation, and the intervening pigment epithelium was usually reactive. An increasing number of viruses are known or thought to be responsible for damage to retinal pigment epithelium. Maternal infections with rubella, variola, varicella, mumps, chickenpox, or influenza have been incriminated as the cause of clinically diagnosed pigmentary retinopathies in human patients [9, 19,20,23]. Hypertrophy and atrophy of the retinal pigment epithelium have been reported as histopathological changes in the rubellaocular syndrome [29], and retinitis with widespread involvement of the pigment epithelium has been seen in patients with rubella [18]. Swelling and proliferation of pigment epithelial cells with and without evidence of other retinal damage have been recognized for years as intraocular lesions in dogs with distemper [16, 221, and typical canine distemper inclusions have more recently been described in some reactive pigment epithelial cells [ l l , 121. In neonatal rats inoculated with lymphocytic choriomeningitis virus and simian virus 40, viral antigen was detected in the pigment epithelium by immunofluorescence without accompanying lesions. In lambs inoculated with bluetongue virus, reactive changes were seen in the pigment epithelium, but no information was given as to the presence or absence of viral antigen [13,21, 26,271. Experimental postnatal infection with canine herpes virus in Beagle dogs 1-4 days of age has produced retinal inflammation and retinal dysplasia. Pigment epithelial changes were folding, hypertrophy, and reduplica-
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tion. Other areas show pigment epithelial necrosis. Virus could be demonstrated within the pigment epithelial tissue by fluorescent antibody techniques [ 11. In this study, hypertrophy and hyperplasia of the pigment epithelium were frequently seen in the acute lesions. However, it was not possible t o determine if the reactive changes occurred solely in response t o retinal inflammation [lo] or if the virus were more intimately involved in the development of the lesions. SILVERSTEIN et al. [25-271 saw retinal folds in lamb fetuses after inoculation of bluetongue virus. The dysplasia occurred without detachment of the retina from the pigment epithelium. Experimental infection of I - t o 4-dayold Beagle dogs with canine herpes virus produced full-thickness retinal folds with adhesions between the folds and formation of two- and three-layered tubes. These retinal changes were associated with abnormalities of the pigment epithelium that produced loss of intimate attachment between pigment epithelium and normal retina [l]. In this study, retinal folds were seen only in the eye of the F56a fetus. In contrast t o what was seen in lamb fetuses, the retina in this fetus was separated from the reactive pigment epithelium, suggesting that the loss of the organizing influence of the pigment epithelium on the retina was responsible for the development of this dysplastic lesion [26]. The residual ocular lesions in calves whose dams were experimentally infected with the virus during gestation were more severe than the lesions in these fetuses and included lenticular lesions that we did not see [2]. The most plausible explanation for the different findings in the two studies is the different strains of viruses used. In the study reported [2], a noncytopathogenic strain of virus was used, whereas we used a cytopathogenic strain. In support of the impression that different strains of virus may have different degrees of virulence for the bovine fetus are other studies [3, 7, 81 in which bovine fetuses were inoculated at different stages of gestation with a cytopathogenic strain of virus different from the one used in the present study. Ocular lesions were not seen [3, 7, 81. The significance of the absence of eye lesions in all but one of the fetuses from the heifers inoculated between 65 and 115 days of gestation is not known. Fetus F70 was dead at the time of cesarotomy, which probably explains the absence of ocular lesions. A cknowledgetnents Supported by US Public Health Service grants R R 05462, A1 06137, H D 05757 and NS 02658.
Ocular Lesions in Bovine Diarrhea Virus
403
References 1 ALBERT, D.: Personal commun. 2 BISTNER, S. I. ; RUBIN,L. F., and SAUNDERS, L. Z. : The ocular lesions of bovine viral diarrhea-mucosal disease. Path. vet. 7: 275-286 (1970). R. K.; OSBURN, B. I., and KENDRICK, J. W.: Immunologic response of bovine 3 BRAUN, fetus to bovine diarrhea virus. Am. J. vet. Res. 34: 1127-1132 (1973). 4 BROWN,T.T.: Pathogenetic studies of bovir,e viral diarrhea infection in the bovine fetus. I. Gross and histopathological lesions. 11. Serological studies; PhD thesis Cornell University, Ithaca (1973). A.; BISTNER, S.I.; SCOTT,F. W., and MCENTEE,K. : 5 BROWN,T.T.; DELAHUNTA, Pathogenetic studies of infection of the bovine fetus with bovine viral diarrhea virus. I. Cerebellar atrophy. Vet. Pathol. (in press). 6 BROWN,T.T.; DELAHUNTA, A.; SCOTT,F.W.; KAHRS,R.F.; MCENTEE,K., and GILLESPIE,J.H.: Virus induced congenital anomalies of the bovine fetus. 11. Histopathology of cerebellar degeneration (hypoplasia) induced by the virus of bovine viral diarrhea-mucosal disease. Cornell Vet. 63: 561 (1973). A.P.E.: The pathological and immunological response of the bovine fetus 7 CASARO, to bovine viral diarrhea virus; PhD thesis University of California, Davis (1969). A. P.E.; KENDRICK, J. W., and KENNEDY, P.C.: Response of the bovine fetus 8 CASARO, to bovine viral diarrhea-mucosal disease virus. Am. J. vet. Res. 32: 1543-1562 (1971). G . ; Zu RHEIN,G. M.; PRATT,M.V., and KISKEN, W.: Cytomegalic inclu9 DEVENECIA, sion retinitis in an adult. Archs Ophthal., N.Y. 86: 44-57 (1971). 10 DUKE-ELDER, S. and DOBREE, J.H.: Diseases of the retina; in DUKE-ELDER System of ophthalmology, vol. 10, p. 199 (Mosby, St. Louis 1967). 11 FISCHER, C. A.: Retinal and retinochoroidal lesions in early neuropathic canine distemper. J. Am. vet. med. Ass. 158: 740-752 (1971). 12 FISCHER,C. A.: Correspondence. Affinity for retinal pigment epithelium by certain viruses. Am. J. Ophthal. 75: 164-166 (1973). 13 FRIEDMAN, A.H.; BELLHORN, R. W., and HENKIND, P. : Simian virus 40-induced retinopathy in the rat. Investve Ophthal. 12: 591-595 (1973). T.B.; EHRLICH, F.; BLANC,W.A., and BECKER, M.H.: New observations 14 GUYTON, in generalized cytomegalic-inclusion disease of newborn : report of a case with chorioretinitis. New Engl. J. Med. 257: 803-807 (1957). J.B.: Cytomegaloviruses. Virol. Monogr. 3: 1-23 (1968). I5 HANSHAW, L. Z., and COATES, H. V. : The intraocular lesions of canine 16 JUBB,K. V. ; SAUNDERS, distemper. J. comp. Path. Ther. 67: 21-29 (1957). A.: Epidemiological observations on 17 KAHRS,R.F.; SCOTT,F. W., and DELAHUNTA, bovine viral diarrhea-mucosal disease virus-induced congenital cerebellar hypoplasia and ocular defects in calves. Teratology 3: 181-184 (1970). F. W., and CHISHTI, M. I.: Fluorescein studies in diseases affect18 KRILL,A.E.; NEWELL, ing the retinal pigment epithelium. Am. J. Ophthal. 66: 4 7 M 8 4 (1968). 19 LONN,L.I. : Neonatal cytomegalic inclusion disease chorioretinitis. Archs Ophthal., N.Y. 88: 434-438 (1972). M.E. : Pigmentary retinopathy following maternal measles 20 METZ,H.S. and HARKEY, (morbilli) infection. Am. J. Ophthal. 66: 1107-1 110 (1968).
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21 MONJAN,A. A.; SILVERSTEIN, A.M., and COLE,G.A.: Lymphocytic choriomeningitis virus induced retinopathy in newborn rats. Investve Ophthal. 11: 85W356 (1972). 22 PARRY,H.B.: Degenerations of the dog retina. IV. Retinopathies associated with dog distemper-complex infections. Br. J. Ophthal. 38: 295-309 (1954). 23 SCHEIE,H.G. and MORSE,P.H.: Rubeola retinopathy. Archs Ophthal., N.Y. 88: 341344 (1972). 24 SCOTT, F.W.; KAHRS,R.F.; DELAHUNTA, A.; BROWN,T.T.; MCENTEE,K., and GILLESPIE, J. H.: Virus-induced congenital anomalies of the bovine fetus. I. Cerebellar degeneration (hypoplasia), ocular lesions and fetal mummification following experimental infection with bovine viral diarrhea-mucosal disease virus. Cornell Vet. 63: 536 (1 973). A.M.; OSBURN,B. I., and PRENDERCAST, R. A. : The pathogenesis of 25 SILVERSTEIN, retinal dysplasia. Am. J. Ophthal. 72: 13-21 (1971). A.M.; PARSHALL, C. J.; OSBURN,B.I., and PRENDERCAST, R.A.: An 26 SILVERSTEIN, experimental virus-induced retinal dysplasia in the fetal lamb. Am. J. Ophthal. 72: 22-34 (1971). 27 SILVERSTEIN, A.M. : The immunologic medulation of infectious disease pathogenesis. Investve Ophthal. 13: 560-571 (1974). S. J.; MCENTEE,K., and GILLESPIE, J.H.: A study of experi28 WARD,G . M.; ROBERTS, mentally induced bovine viral diarrhea-mucosal disease in pregnant cows and their progeny. Cornell Vet. 59: 525-539 (1969). D.B., and SCHEIE,H.G.: Rubella ocular syndrome. Trans. 29 YANOFF,M.; SCHAFFER, Am. Acad. Ophthal. Oto-lar. 72: 896-902 (1968). L. E. : Histopathologic basis for ocular manifestations of congenital 30 ZIMMERMAN, rubella syndrome. Am. J. Ophthal. 65: 837-862 (1968).
Dr. TALMACE T. BROWN,jr., Department of Pathology, College of Veterinary Medicine, Oklahoma State University, StiNnater, O K 74074 (USA)
Pathogenetic Studies of Infection of the Bovine Fetus with Bovine Viral Diarrhea Virus 11. Ocular Lesions
T.T. BROWN,S . I. BISTNER, A. DELAHUNTA, F. W. SCOTTand K. MCENTEE Departments of Large Animal Medicine, Obstetrics and Surgery, Anatomy and Microbiology, New York State Veterinary College at Cornell University, Ithaca, N.Y.
Absrrucr. Twenty-three susceptible pregnant heifers were inoculated with bovine viral diarrhea virus at 150k 1 days of gestation. Seven additional heifers were inoculated between 65 and 115 days of gestation. Acute ocular lesions were seen in fetuses taken 17-21 days after inoculation of their dams at 150 days. By the fourth week, the acute lesions were beginning to resolve, and in newborn animals focal to total retinal atrophy was seen. The acute lesions were characterized by a mild to moderate retinitis that resulted in various degrees of destruction of the different layers, mononuclear cuffing of inner retinal vessels, proliferation of pigment epithelium, and choroiditis. Residually there was an absence of cellular elements in the atrophied areas of the retina, frequently a loss of layering and various numbers of pigment-containing cells. Moderately severe acute inflammation was seen in the retina of the fetus taken at 22 days after inoculation of its dam at 95 days. Ocular lesions did not occur in the other fetuses taken from heifers inoculated at earlier stages of gestation.
Congenital infection of human fetuses with rubella virus and cytomegalovirus may result in ocular lesions in addition to lesions in other tissues [14, 15, 19, 291. Experimental viral infection of the fetuses or neonates of some laboratory animals results in the consistent production of ocular lesions [l, 2,6, 13,21,26,27]. Inoculation of lamb fetuses with blue tongue vaccine virus and newborn rats with lymphocytic choriomeningitis virus and simian virus 40 results in retinopathy [13, 21, 26, 271. The ocular lesions observed after the inoculation of these viruses into the appropriate laboratory animal are similar in several respects. They attack the immature retina, primarily damaging either the developing neuroblastic layer or one or both of the recently formed nuclear layers. Viral infection or lesions, or both, are also observed in the pigment epithelium. Endothelial damage or perivascular
BROWNet at.
395
mononuclear cuffing, or both [13,21,26], are usually seen in the inner retina. Residually there is retinal atrophy with or without rosette formation. Infection of susceptible pregnant cows with bovine viral diarrhea virus may result in ocular lesions in their offspring [2, 4-6, 24, 281. This paper describes the evolution of ocular lesions in bovine fetuses after inoculation of this virus into susceptible pregnant heifers.
Materials and Methods Twenty-three pregnant heifers with no detectable serum neutralizing antibody to bovine viral diarrhea virus were inoculated intravenously with 250 OOO TCID,, (median tissue culture infective dose) of a cytopathogenic strain (Holmes) of virus at 150+ I days of gestation. Details for preparation of the virus inoculum are given by BROWNet al. [5]. Seven additional heifers were inoculated between 65 and 115 days of gestation. Three control heifers with serum neutralizing titers to the virus were inoculated at 150+ 1 days of gestation. The fetuses of the susceptible heifers were surgically taken at sequential times after inoculation (table I). Their eyes were examined with a direct ophthalmoscope (Vistadial Giantscope, American Optical Corp., Buffalo, N.Y.); the fetuses were then exsanguinated by intracardiac puncture. Two susceptible and one control heifers were allowed to calve normally, and the calves were clinically examined before being electrocuted. Shortly after death, one eye was fixed in Zenker’s solution for 6-12 h. It was placed in 50% alcohol for 24 h and then stored in 70% alcohol. Paraffin-embedded sections were cut at 6 pm and stained with hematoxylin and eosin (HE).
Results Gross lesions were observed only in one animal. The F45 calf taken at 140 days was almost totally blind. Ophthalmoscopic examination showed increased pigmentation bilaterally and adherence of a persistent pupillary membrane to the anterior capsule of the lens of the right eye. The occurrence of microscopic ocular lesions in the fetuses or calves is listed in table I. Lesions were restricted to the retina and choroid. Acute inflammatory lesions occurred in fetuses taken between 17 and 21 days from dams inoculated at 150 f 1 days of gestation and in a 117-day fetus whose dam was inoculated at 95 days of gestation. Resolving or resolved lesions were seen in fetuses taken after 21 days from heifers inoculated at 150 days. The acute lesions in two fetuses were mild. In fetus F78 taken at 17 days, small focal hemorrhages were occasionally seen in the inner nuclear layer of the nontapetal retina. A few small, subtle areas of necrosis were seen in
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Table I. Ocular lesions in fetuses from heifers inoculated with bovine viral diarrhea virus during pregnancy
Fetus No. F44 F47 F87 F54 F1902
Gestational age inoculation cesarotomy 150 150
149 150 150
154 157 161 163 164
Days after inoculation
14
150 150
F94 F48 F8974 F55 F56
150 150 150 150 150
168 169 170 171 171
18 19 20 21 21
F49 F48a F57 F1901 F53
150 150
174 177 191 206 219
24 27 42 56 70
232 27 1 290
83 120 140
149 150 150
F5O F56a F45
149 150
149 149 151
150
15
16 17 17
F70* F41 F02 F95 F38
115
95 95 85 80
137 112 117 106 113
22 17 22 21 33
F66 F80
75 65
96 86
21 21
Control F6 150 F5I 150 F52 151
168 178 284
18 28 133
- = None;
inflammation necrosis
4 7 12 13 14
163 165 166 167 167
FOI F63 F62 F46 F78
Ocular lesions'
+ = least severe; +f+i- = most severe.
* At cesarotomy, fetus was dead.
atrophy
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397 I
Fig. 1. Fetus F8974, 170 days. Focal retinitis with extensive necrosis in the nuclear layers and mononuclear perivascular cuffing of inner retinal vessels. Also, choroiditis and reactive pigment epithelial cells. HE.
the outer nuclear layer of the nontapetal retina of the F56 fetus taken at 21 days. They appeared as small, clear areas in which cellular debris could be seen. In one or two places, the necrosis extended to the inner nuclear layer. The acute lesions in fetuses F8974, taken at 20 days, and F02, taken 22 days after inoculation of its dam at 95 days of gestation, were considerably more severe. In both fetuses, there were various sized foci of retinitis that occurred mainly in the nontapetal retina. There was severe necrosis in the inner and outer nuclear layers of F8974 (fig. 1). Pyknotic nuclei and fragmented cellular debris were prominent, and normally separated layers blended together. In such areas, the rod and cone layer was destroyed and the adjacent pigment epithelium was hypertrophied and hyperplastic. Cellular debris was intermixed with the reactive cells of the pigment epithelium. Associated with these areas of retinitis was a mononuclear inflammatory infiltrate in the inner portion of the choroid. Many of the inner retinal vessels were cuffed by mononuclear cells, most of which were undifferentiated but some of which
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BROWNet al.
Fig. 2. Fetus F02, 117 days. Serial sections of a small focus of retinal inflammation show the apparent spread of inflammation to the choroid through a small break in the external limiting membrane. a Inflammation of the neuroblastic layer (arrow), pigment epithelium and choroid with an intact external limiting membrane, and separation of retina from pigment epithelium. b Same inflammatory focus 30 pm lateral to a. Focal disruption of external limiting membrane with extension of inflammation from retina t o pigment epithelium and choroid. c Same inflammatory focus 12 prn lateral to b a n d opposite a. Changes similar to those in a. HE.
were identifiable as lymphocytes or plasma cells. Necrosis of ganglion cells could occasionally be seen. Similar lesions were seen in the F02 fetus, in which the undifferentiated neuroblastic layer was the principal site of damage. Necrosis was not as severe as in F8974 but of sufficient severity to make the Miiller cells prominent in some areas. Perivascular cuffing did not occur in the inner retina, but there was choroidal inflammation similar to that seen in F8974. Adjacent to areas of choroidal inflammation, the pigment epithelium was reactive, and there was separation of the retina from the pigment epithelium. In areas where necrosis in the neuroblastic layer was extensive, patchy breaks were present in the external limiting membrane, and pseudorosettes were occasionally seen. In areas where inflammation was less severe, that in the retina and choroid appeared to have been the result of separate processes; however, serial sectioning of such areas showed, at greater
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399
Fig. 3. Fetus F53, 219 days. Atrophied, actively inflamed retina. Inflammatory cells tend to aggregate around vessels. Numerous lymphocytes with plasma cells among them. HE.
depths, small breaks in the external limiting membrane. At such points, there was a continuation of inflammation from the neuroblastic layer to the pigment epithelium and adjacent choroid, suggesting that inflammation spread outward from the neuroblastic layer through the small breaks in the external limiting membrane to the choroid (fig. 2). The retinal lesions in the F55 fetus taken at 21 days were resolving. A few small focal areas of atrophy were seen in the nontapetal and tapetal retina, in which there was a focal depletion of cells in the outer and, to a lesser extent, the inner nuclear layers. Cellular debris was not evident, and there were no signs of active inflammation in the retina or choroid. However, the presence of cells containing pigment in the depleted areas attested to a prior inflammatory episode. In areas where there was a depletion of cells in the nuclear layers, the rod and cone layer was destroyed, and there was hypertrophy of the pigment epithelium. Focal to total retinal atrophy was seen in the fetuses taken at 42 days or afterwards. With the exception of the F53 fetus taken at 70 days, active inflammation was not observed in the retina or choroid of these fetuses. In some places, cells containing pigment were present in the atrophied retinal tissues. In the F53 fetus, the entire retina was damaged, and mononuclear inflammatory cells were still numerous (fig. 3). Many of the inflammatory cells were either lymphocytes or plasma cells and were aggregated in the
400
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Fig. 4. Term fetus F56a, 271 days. Focal retinal atrophy. Outer layers of the retina have been destroyed, whereas the inner layers are intact. HE. Fig. 5. Fetus F50, 232 days. Retinal fold at a point of detachment of the retina from the proliferated pigment epithelium. HE.
markedly altered inner layers most abundantly around vessels. The nuclear layers had been severely damaged, and only a single layer of cells, three to four cells wide, remained in the outermost retina. Retinal atrophy was similar in the eyes of the animals taken at later stages of gestation or after birth. The nuclear layers were most noticeably damaged. In places, there was total destruction of the nuclear layers, the intervening outer plexiform layer, and the layer of rods and cones, whereas the inner retinal layers appeared more normal (fig. 4). Total retinal atrophy was seen in other eyes with a loss of layering and glial scars. Few retinal folds were present in the eye of the F56a fetus taken at 120 days (fig. 5). These folds were created by a detachment and infolding of the external limiting membrane and outer nuclear layer. The pigment epithelium adjacent to these areas was hypertrophied and hyperplastic. Ocular lesions were not seen in the control fetuses nor in fetuses, other than F02, from heifers inoculated at earlier stages of gestation.
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Discussion
The acute lesions seen in the bovine fetuses were similar to those seen in lambs infected in utero with bluetongue virus and in rat neonates infected with lymphocytic choriomeningitis virus or simian virus 40, in that the principal damage occurred in the neuroblastic layer or in the nuclear layers that derive from it [13,21,26,27]. Mononuclear perivascular cuffs of inner retinal vessels were common to all these viral-induced retinopathies. The cuffs were usually part of the acute lesion and were not extensive. However, in the bovine fetus taken at 70 days (F53), the retinal inflammation persisted until later in gestation than it did in the other fetuses and was more intense and the inflammatory cells better differentiated. These findings suggest a more prolonged antigenic stimulation in the eye of this fetus than in other fetuses. Retinal inflammation of a sufficient degree to cause destruction of cells in the nuclear layers is frequently accompanied by destruction of the adjacent pigment epithelium and basilar lamina, allowing inflammation to extend to the choroid [lo]. Apparently, this is what occurred in the eyes of the bovine fetuses. When choroidal inflammation was seen, it occurred adjacent to areas of retinal inflammation, and the intervening pigment epithelium was usually reactive. An increasing number of viruses are known or thought to be responsible for damage to retinal pigment epithelium. Maternal infections with rubella, variola, varicella, mumps, chickenpox, or influenza have been incriminated as the cause of clinically diagnosed pigmentary retinopathies in human patients [9, 19,20,23]. Hypertrophy and atrophy of the retinal pigment epithelium have been reported as histopathological changes in the rubellaocular syndrome [29], and retinitis with widespread involvement of the pigment epithelium has been seen in patients with rubella [18]. Swelling and proliferation of pigment epithelial cells with and without evidence of other retinal damage have been recognized for years as intraocular lesions in dogs with distemper [16, 221, and typical canine distemper inclusions have more recently been described in some reactive pigment epithelial cells [ l l , 121. In neonatal rats inoculated with lymphocytic choriomeningitis virus and simian virus 40, viral antigen was detected in the pigment epithelium by immunofluorescence without accompanying lesions. In lambs inoculated with bluetongue virus, reactive changes were seen in the pigment epithelium, but no information was given as to the presence or absence of viral antigen [13,21, 26,271. Experimental postnatal infection with canine herpes virus in Beagle dogs 1-4 days of age has produced retinal inflammation and retinal dysplasia. Pigment epithelial changes were folding, hypertrophy, and reduplica-
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tion. Other areas show pigment epithelial necrosis. Virus could be demonstrated within the pigment epithelial tissue by fluorescent antibody techniques [ 11. In this study, hypertrophy and hyperplasia of the pigment epithelium were frequently seen in the acute lesions. However, it was not possible t o determine if the reactive changes occurred solely in response t o retinal inflammation [lo] or if the virus were more intimately involved in the development of the lesions. SILVERSTEIN et al. [25-271 saw retinal folds in lamb fetuses after inoculation of bluetongue virus. The dysplasia occurred without detachment of the retina from the pigment epithelium. Experimental infection of I - t o 4-dayold Beagle dogs with canine herpes virus produced full-thickness retinal folds with adhesions between the folds and formation of two- and three-layered tubes. These retinal changes were associated with abnormalities of the pigment epithelium that produced loss of intimate attachment between pigment epithelium and normal retina [l]. In this study, retinal folds were seen only in the eye of the F56a fetus. In contrast t o what was seen in lamb fetuses, the retina in this fetus was separated from the reactive pigment epithelium, suggesting that the loss of the organizing influence of the pigment epithelium on the retina was responsible for the development of this dysplastic lesion [26]. The residual ocular lesions in calves whose dams were experimentally infected with the virus during gestation were more severe than the lesions in these fetuses and included lenticular lesions that we did not see [2]. The most plausible explanation for the different findings in the two studies is the different strains of viruses used. In the study reported [2], a noncytopathogenic strain of virus was used, whereas we used a cytopathogenic strain. In support of the impression that different strains of virus may have different degrees of virulence for the bovine fetus are other studies [3, 7, 81 in which bovine fetuses were inoculated at different stages of gestation with a cytopathogenic strain of virus different from the one used in the present study. Ocular lesions were not seen [3, 7, 81. The significance of the absence of eye lesions in all but one of the fetuses from the heifers inoculated between 65 and 115 days of gestation is not known. Fetus F70 was dead at the time of cesarotomy, which probably explains the absence of ocular lesions. A cknowledgetnents Supported by US Public Health Service grants R R 05462, A1 06137, H D 05757 and NS 02658.
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References 1 ALBERT, D.: Personal commun. 2 BISTNER, S. I. ; RUBIN,L. F., and SAUNDERS, L. Z. : The ocular lesions of bovine viral diarrhea-mucosal disease. Path. vet. 7: 275-286 (1970). R. K.; OSBURN, B. I., and KENDRICK, J. W.: Immunologic response of bovine 3 BRAUN, fetus to bovine diarrhea virus. Am. J. vet. Res. 34: 1127-1132 (1973). 4 BROWN,T.T.: Pathogenetic studies of bovir,e viral diarrhea infection in the bovine fetus. I. Gross and histopathological lesions. 11. Serological studies; PhD thesis Cornell University, Ithaca (1973). A.; BISTNER, S.I.; SCOTT,F. W., and MCENTEE,K. : 5 BROWN,T.T.; DELAHUNTA, Pathogenetic studies of infection of the bovine fetus with bovine viral diarrhea virus. I. Cerebellar atrophy. Vet. Pathol. (in press). 6 BROWN,T.T.; DELAHUNTA, A.; SCOTT,F.W.; KAHRS,R.F.; MCENTEE,K., and GILLESPIE,J.H.: Virus induced congenital anomalies of the bovine fetus. 11. Histopathology of cerebellar degeneration (hypoplasia) induced by the virus of bovine viral diarrhea-mucosal disease. Cornell Vet. 63: 561 (1973). A.P.E.: The pathological and immunological response of the bovine fetus 7 CASARO, to bovine viral diarrhea virus; PhD thesis University of California, Davis (1969). A. P.E.; KENDRICK, J. W., and KENNEDY, P.C.: Response of the bovine fetus 8 CASARO, to bovine viral diarrhea-mucosal disease virus. Am. J. vet. Res. 32: 1543-1562 (1971). G . ; Zu RHEIN,G. M.; PRATT,M.V., and KISKEN, W.: Cytomegalic inclu9 DEVENECIA, sion retinitis in an adult. Archs Ophthal., N.Y. 86: 44-57 (1971). 10 DUKE-ELDER, S. and DOBREE, J.H.: Diseases of the retina; in DUKE-ELDER System of ophthalmology, vol. 10, p. 199 (Mosby, St. Louis 1967). 11 FISCHER, C. A.: Retinal and retinochoroidal lesions in early neuropathic canine distemper. J. Am. vet. med. Ass. 158: 740-752 (1971). 12 FISCHER,C. A.: Correspondence. Affinity for retinal pigment epithelium by certain viruses. Am. J. Ophthal. 75: 164-166 (1973). 13 FRIEDMAN, A.H.; BELLHORN, R. W., and HENKIND, P. : Simian virus 40-induced retinopathy in the rat. Investve Ophthal. 12: 591-595 (1973). T.B.; EHRLICH, F.; BLANC,W.A., and BECKER, M.H.: New observations 14 GUYTON, in generalized cytomegalic-inclusion disease of newborn : report of a case with chorioretinitis. New Engl. J. Med. 257: 803-807 (1957). J.B.: Cytomegaloviruses. Virol. Monogr. 3: 1-23 (1968). I5 HANSHAW, L. Z., and COATES, H. V. : The intraocular lesions of canine 16 JUBB,K. V. ; SAUNDERS, distemper. J. comp. Path. Ther. 67: 21-29 (1957). A.: Epidemiological observations on 17 KAHRS,R.F.; SCOTT,F. W., and DELAHUNTA, bovine viral diarrhea-mucosal disease virus-induced congenital cerebellar hypoplasia and ocular defects in calves. Teratology 3: 181-184 (1970). F. W., and CHISHTI, M. I.: Fluorescein studies in diseases affect18 KRILL,A.E.; NEWELL, ing the retinal pigment epithelium. Am. J. Ophthal. 66: 4 7 M 8 4 (1968). 19 LONN,L.I. : Neonatal cytomegalic inclusion disease chorioretinitis. Archs Ophthal., N.Y. 88: 434-438 (1972). M.E. : Pigmentary retinopathy following maternal measles 20 METZ,H.S. and HARKEY, (morbilli) infection. Am. J. Ophthal. 66: 1107-1 110 (1968).
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21 MONJAN,A. A.; SILVERSTEIN, A.M., and COLE,G.A.: Lymphocytic choriomeningitis virus induced retinopathy in newborn rats. Investve Ophthal. 11: 85W356 (1972). 22 PARRY,H.B.: Degenerations of the dog retina. IV. Retinopathies associated with dog distemper-complex infections. Br. J. Ophthal. 38: 295-309 (1954). 23 SCHEIE,H.G. and MORSE,P.H.: Rubeola retinopathy. Archs Ophthal., N.Y. 88: 341344 (1972). 24 SCOTT, F.W.; KAHRS,R.F.; DELAHUNTA, A.; BROWN,T.T.; MCENTEE,K., and GILLESPIE, J. H.: Virus-induced congenital anomalies of the bovine fetus. I. Cerebellar degeneration (hypoplasia), ocular lesions and fetal mummification following experimental infection with bovine viral diarrhea-mucosal disease virus. Cornell Vet. 63: 536 (1 973). A.M.; OSBURN,B. I., and PRENDERCAST, R. A. : The pathogenesis of 25 SILVERSTEIN, retinal dysplasia. Am. J. Ophthal. 72: 13-21 (1971). A.M.; PARSHALL, C. J.; OSBURN,B.I., and PRENDERCAST, R.A.: An 26 SILVERSTEIN, experimental virus-induced retinal dysplasia in the fetal lamb. Am. J. Ophthal. 72: 22-34 (1971). 27 SILVERSTEIN, A.M. : The immunologic medulation of infectious disease pathogenesis. Investve Ophthal. 13: 560-571 (1974). S. J.; MCENTEE,K., and GILLESPIE, J.H.: A study of experi28 WARD,G . M.; ROBERTS, mentally induced bovine viral diarrhea-mucosal disease in pregnant cows and their progeny. Cornell Vet. 59: 525-539 (1969). D.B., and SCHEIE,H.G.: Rubella ocular syndrome. Trans. 29 YANOFF,M.; SCHAFFER, Am. Acad. Ophthal. Oto-lar. 72: 896-902 (1968). L. E. : Histopathologic basis for ocular manifestations of congenital 30 ZIMMERMAN, rubella syndrome. Am. J. Ophthal. 65: 837-862 (1968).
Dr. TALMACE T. BROWN,jr., Department of Pathology, College of Veterinary Medicine, Oklahoma State University, StiNnater, O K 74074 (USA)
