J. Comp.
Path.
1992
Vol.
106,
333-340
Association of Virulent and Avirulent Strains of Bluetongue Virus Serotype 11 with Premature Births of Late-term Bovine Fetuses A. S. Waldvogel,
G. A. Anderson*, B. I. Osburn
D. L. Phillips*
and
Department of Pathology, School of Veterinary Medicine, University of California, Davis, CA 95616 and *Department of Veterinary Science, University of Nebraska-Lincoln, Lincoln, JVE 68583,U.S.A.
Summary strains of bluetongue virus serotype 11 (BTV 1l), UC-2 avirulent and UC-8 neurovirulent in newborn mice, were inoculated into late-term bovine fetuses to investigate whether infection with these two BTV strains in late Two
gestation would produce congenital infection and pathological changes. Fetuses were inoculated by intramuscular injection through the uterine wall at 243 days gestation and recovered after spontaneous delivery. In calves inoculated with UC-& births occurred 15 to 27 days before expected parturition, resulting in small, weak calves. These calves had a mild encephalitis and were unthrifty at birth. Calves inoculated with UC-2 appeared healthy when born 7 to 11 days prior to expected parturition. No lesions were found in these calves at necropsy. All calves seroconverted by the time of birth. Viraemia was present in the calves inoculated with UC-8 and in one calf inoculated with UC-2. Plasma cortisol concentrations were prematurely elevated, particularly in the calves inoculated with UC-8, indicating that they were stressed by the infection. The elevated cortisol, associated with an active congenital infection caused by bluetongue virus serotype 11 strain UC-8, is capable of causing premature delivery of low birth-weight, weak calves.
Introduction Bluetongue is a non-contagious, insect-transmitted virus disease of domestic and wild ruminants. Bluetongue virus (BTV) infections of susceptible animals are common throughout the southern and western United States (Metcalf, Pearson and Klingsporn, 1981; Osburn, McGowan, Heron, Loomis, Bushnell, Stott and Utterback, 1981). In cattle, fetal death or congenital brain malformations, such as hydranencephaly or porencephaly, can result from infection with BTV (McKercher, Saito and Singh, 1970; Richards, Crenshaw and Bushnell, 197 1; Barnard and Pienaar, 1976; Brown and MacLachlan, 1983; MacLachlan and Osburn, 1983; MacLachlan, Osburn, Ghalib and Stott, 1985). Current strasse
address for A. S. Wkldvogel: Institut 268, CH-8057 Zurich, Switzerland.
0021~-9975/92/040333
+08
$03.00/O
Giir Veterinarpathologie
der
Universitat G
1992
Zurich, Academic
WinterthurerPress
Limited
334
A. S. Waldvogel
et al.
BTV is a member of the genus orbivirus which belongs to the family reoviridae (Knudson and Shope, 1985). The serogroup BTV is further subdivided into 24 serotypes, five of which (serotypes 2, 10, 11, 13 and 17) have been detected in the United States (Gibbs, Greiner, Taylor, Barber, House and Pearson, 1983; Knudson and Shope, 1985). The identification of a virus as BTV is dependent on the demonstration of certain shared group antigens. The BTV serotypes do not define the capacity of the viruses to produce disease (Gorman, 1979). There is increasing evidence that bluetongue virus can be recovered from animals which show no evidence of clinical disease (Osburn et al., 1981; Stott, Anderson, Jochim, Barber and Osburn, 1982). Cattle infected with these viruses seldom show clinical evidence of disease (Osburn, 1985). In addition, there are marked genetic variations between isolates which do not correlate with the serological classification (Rao and Roy, 1983; Squire, Osburn, Chuang and Doi, 1983; Collisson, Barber, Gibbs and Greiner, 1985). A previous report of the results of inoculation of 120-day-old bovine fetuses with two strains of BTV 11, UC-2 and UC-8, indicated that there was a difference in pathogenic potential of these viruses (Waldvogel, Anderson, Phillips and Osburn, 1992). Although there was a marked difference in neurovirulence in newborn BALB/c mice (Waldvogel, Anderson, Higgins and Osburn, 1987), subcutaneous inoculation with both virus strains caused severe necrotizing encephalopathy in 120-day-old bovine fetuses. There were subtle differences in the severity of lesions in the fetuses, suggesting that there may be a modest difference in virulence and pathogenicity between the two virus strains. Little is known about the effect of BTV on late-term bovine fetuses. In sheep, the severity of brain malformations depends on the fetal age at inoculation. Infection of sheep fetuses with BTV at a fetal age of 50 to 55 days results in hydranencephaly, whereas infection of fetuses older than 100 days causes only a mild focal encephalitis (Osburn, Silverstein, Prendergast, Johnson and Parshall, 1971). In cattle, experimental inoculation with BTV of fetal calves of up to 138 days of age resulted in fetal death, hydranencephaly or porencephaly (Barnard and Pienaar, 1976; MacLachlan and Osburn, 1983; MacLachlan et at., 1985). No congenital lesions were reported in calves inoculated with BTV at 5 and 73 months of gestation (Jochim, Luedke and Chow, 1974). However, detailed examinations were not carried out on these calves and it is not clear how the absence of congenital lesions was determined. The objective of this study was to investigate whether inoculation of BTV 11 strains UC-2 and UC-8 in fetal calves at 240 days gestation caused congenital infection and pathological changes and whether there was a difference between the responses to inoculation with UC-2 and UC-8 in these late-term bovine fetuses. Materials
and
Methods
The fetuses of four Hereford cross-bred heifers time-mated with a Simmental bull were inoculated with BTV 11 at 243 days of gestation. Two fetuses were inoculated with lo4 plaque-forming units of UC-2, and two fetuses were inoculated with 10’ plaque-forming units of UC-8. The inoculum was prepared as described previously
Bluetongue
Virus
and Premature
335
Birth
(Waldvogel, Stott, Squire and Osburn, 1986) and the fetal calves were directly inoculated intramuscularly through the uterine wall. Cows were given a tranquillizer followed by local anaesthesia.A laparotomy was performed in the right flank and the pregnant uterus was located. The fetus was located before the direct inoculation was carried out. The calves were recovered after spontaneous delivery; their birth weight was determined, and precolostral blood samples were taken for virus isolation, determination of cortisol concentrations and serology. The calves were killed by electrocution at 1 to 3 days of age. A complete necropsy was carried out and samplesof all major organs were fixed in buffered formalin for microscopy and snap-frozen for immunohistochemistry as previously described (Anderson, Phillips, Waldvogel and Osburn, 1989). Samples of brain, liver and lung were collected for virus re-isolation. Newborn calves from the beef cattle herd of the Department of Veterinary Science of the University Nebraska-Lincoln were usedas negative controls. Cortisol concentrations were determined in the sera by an enzyme immunoassay (Munro and Stabenfeldt, 1985). Virus isolation, serology and immunohistochemistry were performed asdescribed previously (Anderson et al., 1989; Waldvogel et nl., 1992). Results
The calves inoculated with UC-8 (J and L) were born 16 and 28 days after inoculation, after a gestation of 259 and 271 days (Table 1). Their birth weights were 21 and 22 kg, respectively. Both were weak, and calf J, born 16 days after inoculation, did not nurse spontaneously. The two calves inoculated with UC-2, M and N, were born 32 and 35 days after inoculation, after a gestation of 275 and 278 days. These two calves weighed 32 and 34 kg, respectively at birth, and they appeared clinically normal. The cortisol concentrations of the four calves were in the range of 46 to 85 ng per ml (Table 1). Gross lesions were not detected in any calf at necropsy. However, microscopical examination revealed glial nodules in the cerebral cortex, basal ganglia and the thalamus (Fig. 1) of the two calves inoculated with UC-8. Morphological evidence ofimmune stimulation during gestation was detected in the lymph nodes of all four infected calves. Secondary follicles with numerous mitotic figures were found in the cortex (Fig. 2)) and plasma cells were present in the medullary cords.
Duration
J (3) L (8) M ($1 N (9
of gestation,
birth weight gestation
Table 1 and cortisol concentrations with BTV 11 strains UC-2
UC-8 UC-8 UC-2 UC-2
Note: Gestation and birth weight ofAngus order of 286 days and 38 kg, respectively
259f 1 271*1 275fl 278f 1
21 22 32 34
of calves and UC-8
46 58 85 64
and Hereford dams bred by Simmental (Smith el al., 1976).
inoculated
at 243 days
Weak/Small Weak/Small
bulls are normally
in the
336
A. S. Waldvogel
et al.
Fig. 1,
Glial nodule in basal ganglia of calfJ, inoculated with UC-8 and born 16 days after inoculation. HE. Bar= 30 pm.
Fig. 2.
Secondary lymph follicle with numerous mitotic figures in mesenteric inoculated with UC-8, born 28 days after inoculation and necropsied Bar= 50 pm.
Isolation
C&f
of BTV
11 strains
UC-2
Inoculum
(-xl
and
Table 2 UC-8 from newborn days of gestation
Birth
UC-8
L (3) M (6) N (0)
UC-8 UC-2 UC-2
calves
after
afterinoculation (dwi
J (6)
intramuscularly
16 28 32 35
at 243 days gestation lymph node of calf L, at 2 days of age. HE.
in utero
inoculation
at 243
Virus isolation Blood
Brain
Lung end liver
+ + + -
+ + -
+ + -
Bluetongue
Virus
and Premature
Birth
337
BTV was isolated from the brain, blood and pooled liver and lung of calves and L, both inoculated with UC-8, but only from the blood of calf M, inoculated with UC-2 (Table 2). All four calves were serologically positive for BTV and negative for bovine viral diarrhoea virus (BVDV). BTV antigen was detected by the avidin-biotin complex method in the glial nodules of the fetuses inoculated with UC-8 (Anderson et al., 1989).
J
Discussion These experiments indicate that BTV can cause congenital infection in lateterm bovine fetuses. Three of four calves inoculated with bluetongue virus strains UC-2 or UC-8 at 243 days of gestation and born 16 to 32 days after inoculation were viraemic at birth. Calves inoculated with UC-8 had a mild encephalitis. The significance of these lesions for the viability of the calves was not determined. Since the lesions were modest, it is likely that they would be of little or no significance as far as clinical signs are concerned. Jochim and coworkers (1974) reported no malformations in bovine fetuses inoculated at 5 and 7: months of gestation. However, since they did not necropsy calves after birth, it is not known whether their calves had lesions. The gestation period for the two calves inoculated with UC-8 was 27 and 15 days, less than the expected gestation period for these crossbreeds. The birth weights were 45 per cent and 42 per cent below the average for calves from Hereford and Angus dams mated with Simmental bulls (Liggins, 1982) and these calves were weak at birth. In contrast, the calves inoculated with UC-2 appeared healthy at birth. They were born within 8 to 11 days of the expected date of parturition, and their birth weights were only 15 per cent and 11 pel cent below average. This suggests that infection with UC-8 causes more severe infection and brain lesions which result in premature delivery of low birth weight and weak calves. The exact factors contributing to the low birth weights are not understood. The severity of the brain lesions decreases with increasing age of the bovine fetus. This follows the same pattern as the infection in fetal sheep (Osburn e1nl., 197 1). There were either no brain lesions in calves inoculated with UC-2, or only modest lesions in calves inoculated with UC-8. The fact that brain lesions were detected only in the fetuses inoculated with UC-8, but not UC-2, suggests that factors influencing the expression of neurovirulence in newborn BALB/c mice may also be operable in late-term bovine fetuses. If this concept is valid in bovine fetuses, then the ability to prevent UC-2 from reaching the brain must be acquired between 120 and 243 days of gestation, since both strains were neurovirulent in 120-day-old fetuses. MacLachlan and Osburn (1983) report that one of several bovine fetuses inoculated with BTV 10 at a fetal age of approximately 125 days (as determined by rectal palpation) did not develop central nervous system lesions. Although they reported that the fetus was viraemic 50 days after inoculation, no virus was isolated from its brain. Since rectal palpation is not an exact method of age determination, it was suggested that this fetus was older than 125 days at inoculation and therefore did not develop brain lesions. However, infection of fetuses with UC-8 even late in
338
A. S. Waldvogel
et al.
gestation produced glial nodules suggesting that at least two factors, straindependent differences in neurovirulence of BTV and the stage of development of the CNS, influence the outcome of an infection of late-term bovine fetuses. A preparturient surge of fetal cortisol is associated with initiation of parturition in ruminants (Smith, Laster and Gregory, 1976). Normally, cortisol concentrations of approximately 3.6 ng per ml are found in uninfected bovine fetuses of 260 days gestation (Liggins, 1982) and normal newborn calves have cortisol concentrations ranging from 45 to 300 ng per ml (Lin, Oxender and Hafs, 1973). The rise in fetal cortisol in normal parturition begins within 5 to 7 days of parturition. Previous reports indicate that fetal cortisol rises in response to the stressof congenital infections, and this elevation can induce premature delivery (Osburn, Drost and Stabenfeldt, 1972; Stott and Reinhard, 1978). The fact that calves J and L, inoculated with UC-8, had greater morphological evidence of lesions, and that their cortisol was markedly elevated (46 to 85 ng per ml) above expected values for comparably aged normal calves (3.6 ng per ml) strongly suggests that these infected calves were stressed and that this, associated with the UC-8 infection, led to the 15 to 27 day premature delivery of these calves. This study indicates that infection with UC-8 causes congenital infection and modest disease in the form of neural lesions in late-term bovine fetuses. The stressassociated with infection resulted in premature birth of small, weak calves. The UC-8 strain of serotype 11, therefore, is more virulent and pathogenic leading to premature delivery of weak and low birth-weight calves. Acknowledgments
This researchwas supported in part by USDA Special Grant 84-CSRS-2-2504, USDA Animal Health and Disease Grant CSRS-1433, and Western Regional Research Project W 112. The authors thank MS Carrie Schore for manuscript preparation. References
Anderson, G. A., Phillips, D. L., Waldvogel, A. S. and Osburn, B. I. (1989). Detection of bluetongue virus in bovine fetuses using the avidin-biotin complex immunoperoxidase method. Journal of Veterinary Diagnostic Investigation, 1, 45-49. Barnard, B. J. H. and Pienaar, J. G. ( 1976). Bluetongue virus as a cause of hydranencephaly in cattle. Onderstepoort Journal of Veterinary Research, 43, 155-158. Brown, C. C. and MacLachlan, N. J. (1983). Congenital encephalopathy in a calf. Veterinary Pathology, 20, 770-773.
Collisson, E. W., Barber, T. L., Gibbs, E. P. J. and Greiner, E. C. (1985). Two electropherotypes of bluetongue virus serotype 2 from naturally infected calves. Journal of GeneralVirology, 66, 1279-1286. Gibbs, E. P. J., Greiner, E. C., Taylor, W. P., Barber, T. L., House,J. A. and Pearson, J. E. (1983). Isolation of bluetongue virus serotype 2 from cattle in Florida: Serotype of bluetongue virus hitherto unrecognized in the Western hemisphere. American Journal of Veterinary Research, 44, 2226-2228. Gorman, B. M. (1979). Comparison of the genetic material of orbiviruses asa method Symposium on Veterinary of classification. Proceedings of the 2nd International Epidemiology and Economy, Canberra, Australia, W. A. Geering, R. T. Roe, and L. A. Chapman, Eds, Australian Publishing Service, 272-278. Jochim, M. M., Luedke, A. J. and Chow, T. L. (1974). Bluetongue in cattle: Immunogenic and clinical responsesin calves inoculated in utero and after birth. American Journal of Veterinary Research, 35, 517-522.
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Virus
and Premature
Birth
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Knudson, D. L. and Shope, R. E. (1985). 0 verview of the orbiviruses. In Bluetongue and Related Orbiviruses. T. L. Barber and M. M. Jochim, Eds, Alan R. Liss, New York, pp. 255-266. Liggins, G. C. (1982). The fetus and birth. In Reproduction in Mammals, Book 2. Embryonic and Fetal Development, 2nd Edit. C. R. Austin and R. V. Short, Eds, Cambridge University Press, Cambridge, pp. 114-141. Lin, Y. C., Oxender, W. D. and Hafs, H. D. (1973). Fetal and maternal cortisol in the bovine. Journal of Animal Science, 37, 3 19. MacLachlan, N. J., Osburn, B. I., Ghalib, H. W. and Stott, J. L. (1985). Bluetongue virus-induced encephalopathy in fetal cattle. Veterinary Pathology, 22, 415-417. MacLachlan, N. J. and Osburn, B. I. (1983). Bluetongue virus-induced hydranencephaly in cattle. Veterinary Pathology, 20, 563-573. McKercher, D. G., Saito, J. K. and Singh, K. V. (1970). Serologic evidence of an etiologic role for bluetongue virus in hydranencephaly of calves. Journal of the American Veterinary Medical Association, 156, 1044- 1047. Metcalf, H. E., Pearson, J. E. and Klingsporn, A. L. (1981). Bluetongue in cattle: A serologic survey of slaughter cattle in the United States. American Journal of Veterinary Research, 42, 1057-l 161. C. and Stabenfeldt, G. (1985). Development of a cortisol enzyme Munro, immunoassay in plasma. Clinical Chemistry, 31, 956. Osburn, B. I. (1985). Role of the immune system in bluetongue host-viral interactions. In Bluetongue and Related Orbiuiruses. T. L. Barber and M. M. Jochim, Eds, Alan R. Liss, New York, pp. 417-422. Osburn, B. I., Drost, M. and Stabenfeldt, G. H. (1972). Response of fetal adrenal cortex to congenital infections. American Journal of Obstetrics and Gynecology, 114, 622-627. Osburn, B. I., McGowan, B., Heron, B., Loomis, E., Bushnell, R., Stott, J. and Utterback, W. ( 198 1) . Epizootiologic study of bluetongue: Virologic and serologic results. American Journal of Veterinary Research, 42, 885-887. Osburn, B. I., Silverstein, A. M., Prendergast, R. A., Johnson, R. T. and Parshall, C. J. (1971). Experimental viral-induced congenital encephalopathies. I. Pathology of hydranencephaly and porencephaly caused by bluetongue vaccine virus. Laboratory Investigation, 25, 197-205. of bluetongue virus serotype 11 Rao, C. D. and Roy, P. (1983). G enetic variation isolated from host (sheep) and vector (Culicoides variipennis) at the same site. American Journal of Veterinary Research, 44, 9 1 l-9 14. Richards, W. P. C., Crenshaw, G. L. and Bushnell, R. B. (1971). Hydranencephaly of calves associated with natural bluetongue virus infection. Cornell Veterinarian, 61, 336-348. Smith, G. M., Laster, D. B. and Gregory, K. E. ( 1976). Ch aracterization of biologica types of cattle. I. Distocia and preweaning growth. Journal of Animal Science, 43, 27-36. Squire, K. R. E., Osburn, B. I., Chuang, R. Y. and Doi, R. H. (1983). A survey of electropherotype relationships of bluetongue virus isolates from the Western United States. Journal of General Virology, 64, 2103-2115. Stott, G. H. and Reinhard, E. J. (1978). Adrenal function and passive immunity in the dystocial calf. Journal of Dairy Science, 61, 1457-1461. Stott, J. L., Anderson, G. A., Jochim, M. M., Barber, T. L. and Osburn, B. I. (1982). Clinical expression of bluetongue disease in cattle. Proceedings of the Annual Meeting of the US Animal Health Association, 86, 126-131. Waldvogel, A. S., Anderson, C. A., Higgins, R. J. and Osburn, B. I. (1987). Neurovirulence of the UC-2 and UC-8 strains of bluetongue virus serotype 11 in newborn mice. Veterinary Pathology, 24, 404-410. Waldvogel, A. S., Anderson, G. A., Phillips, D. L. and Osburn, B. I. (1992). Infection of bovine fetuses at 120 days gestation with virulent and avirulent strains of bluetongue virus serotype 11. Comparative Immunology, Microbiology and Infectious Diseases, 15, 53-63.
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Waldvogel, A. S., Stott, J. L., Squire, K. Strain-dependent virulence characteristics Journal of General Virology, 67, 765-769.
et al.
R. E. and Osburn, B. I. (1986) of bluetongue virus serotype 11
Received, December 6th, 1991 Accepted, January 18th, 1992
1
Path.
1992
Vol.
106,
333-340
Association of Virulent and Avirulent Strains of Bluetongue Virus Serotype 11 with Premature Births of Late-term Bovine Fetuses A. S. Waldvogel,
G. A. Anderson*, B. I. Osburn
D. L. Phillips*
and
Department of Pathology, School of Veterinary Medicine, University of California, Davis, CA 95616 and *Department of Veterinary Science, University of Nebraska-Lincoln, Lincoln, JVE 68583,U.S.A.
Summary strains of bluetongue virus serotype 11 (BTV 1l), UC-2 avirulent and UC-8 neurovirulent in newborn mice, were inoculated into late-term bovine fetuses to investigate whether infection with these two BTV strains in late Two
gestation would produce congenital infection and pathological changes. Fetuses were inoculated by intramuscular injection through the uterine wall at 243 days gestation and recovered after spontaneous delivery. In calves inoculated with UC-& births occurred 15 to 27 days before expected parturition, resulting in small, weak calves. These calves had a mild encephalitis and were unthrifty at birth. Calves inoculated with UC-2 appeared healthy when born 7 to 11 days prior to expected parturition. No lesions were found in these calves at necropsy. All calves seroconverted by the time of birth. Viraemia was present in the calves inoculated with UC-8 and in one calf inoculated with UC-2. Plasma cortisol concentrations were prematurely elevated, particularly in the calves inoculated with UC-8, indicating that they were stressed by the infection. The elevated cortisol, associated with an active congenital infection caused by bluetongue virus serotype 11 strain UC-8, is capable of causing premature delivery of low birth-weight, weak calves.
Introduction Bluetongue is a non-contagious, insect-transmitted virus disease of domestic and wild ruminants. Bluetongue virus (BTV) infections of susceptible animals are common throughout the southern and western United States (Metcalf, Pearson and Klingsporn, 1981; Osburn, McGowan, Heron, Loomis, Bushnell, Stott and Utterback, 1981). In cattle, fetal death or congenital brain malformations, such as hydranencephaly or porencephaly, can result from infection with BTV (McKercher, Saito and Singh, 1970; Richards, Crenshaw and Bushnell, 197 1; Barnard and Pienaar, 1976; Brown and MacLachlan, 1983; MacLachlan and Osburn, 1983; MacLachlan, Osburn, Ghalib and Stott, 1985). Current strasse
address for A. S. Wkldvogel: Institut 268, CH-8057 Zurich, Switzerland.
0021~-9975/92/040333
+08
$03.00/O
Giir Veterinarpathologie
der
Universitat G
1992
Zurich, Academic
WinterthurerPress
Limited
334
A. S. Waldvogel
et al.
BTV is a member of the genus orbivirus which belongs to the family reoviridae (Knudson and Shope, 1985). The serogroup BTV is further subdivided into 24 serotypes, five of which (serotypes 2, 10, 11, 13 and 17) have been detected in the United States (Gibbs, Greiner, Taylor, Barber, House and Pearson, 1983; Knudson and Shope, 1985). The identification of a virus as BTV is dependent on the demonstration of certain shared group antigens. The BTV serotypes do not define the capacity of the viruses to produce disease (Gorman, 1979). There is increasing evidence that bluetongue virus can be recovered from animals which show no evidence of clinical disease (Osburn et al., 1981; Stott, Anderson, Jochim, Barber and Osburn, 1982). Cattle infected with these viruses seldom show clinical evidence of disease (Osburn, 1985). In addition, there are marked genetic variations between isolates which do not correlate with the serological classification (Rao and Roy, 1983; Squire, Osburn, Chuang and Doi, 1983; Collisson, Barber, Gibbs and Greiner, 1985). A previous report of the results of inoculation of 120-day-old bovine fetuses with two strains of BTV 11, UC-2 and UC-8, indicated that there was a difference in pathogenic potential of these viruses (Waldvogel, Anderson, Phillips and Osburn, 1992). Although there was a marked difference in neurovirulence in newborn BALB/c mice (Waldvogel, Anderson, Higgins and Osburn, 1987), subcutaneous inoculation with both virus strains caused severe necrotizing encephalopathy in 120-day-old bovine fetuses. There were subtle differences in the severity of lesions in the fetuses, suggesting that there may be a modest difference in virulence and pathogenicity between the two virus strains. Little is known about the effect of BTV on late-term bovine fetuses. In sheep, the severity of brain malformations depends on the fetal age at inoculation. Infection of sheep fetuses with BTV at a fetal age of 50 to 55 days results in hydranencephaly, whereas infection of fetuses older than 100 days causes only a mild focal encephalitis (Osburn, Silverstein, Prendergast, Johnson and Parshall, 1971). In cattle, experimental inoculation with BTV of fetal calves of up to 138 days of age resulted in fetal death, hydranencephaly or porencephaly (Barnard and Pienaar, 1976; MacLachlan and Osburn, 1983; MacLachlan et at., 1985). No congenital lesions were reported in calves inoculated with BTV at 5 and 73 months of gestation (Jochim, Luedke and Chow, 1974). However, detailed examinations were not carried out on these calves and it is not clear how the absence of congenital lesions was determined. The objective of this study was to investigate whether inoculation of BTV 11 strains UC-2 and UC-8 in fetal calves at 240 days gestation caused congenital infection and pathological changes and whether there was a difference between the responses to inoculation with UC-2 and UC-8 in these late-term bovine fetuses. Materials
and
Methods
The fetuses of four Hereford cross-bred heifers time-mated with a Simmental bull were inoculated with BTV 11 at 243 days of gestation. Two fetuses were inoculated with lo4 plaque-forming units of UC-2, and two fetuses were inoculated with 10’ plaque-forming units of UC-8. The inoculum was prepared as described previously
Bluetongue
Virus
and Premature
335
Birth
(Waldvogel, Stott, Squire and Osburn, 1986) and the fetal calves were directly inoculated intramuscularly through the uterine wall. Cows were given a tranquillizer followed by local anaesthesia.A laparotomy was performed in the right flank and the pregnant uterus was located. The fetus was located before the direct inoculation was carried out. The calves were recovered after spontaneous delivery; their birth weight was determined, and precolostral blood samples were taken for virus isolation, determination of cortisol concentrations and serology. The calves were killed by electrocution at 1 to 3 days of age. A complete necropsy was carried out and samplesof all major organs were fixed in buffered formalin for microscopy and snap-frozen for immunohistochemistry as previously described (Anderson, Phillips, Waldvogel and Osburn, 1989). Samples of brain, liver and lung were collected for virus re-isolation. Newborn calves from the beef cattle herd of the Department of Veterinary Science of the University Nebraska-Lincoln were usedas negative controls. Cortisol concentrations were determined in the sera by an enzyme immunoassay (Munro and Stabenfeldt, 1985). Virus isolation, serology and immunohistochemistry were performed asdescribed previously (Anderson et al., 1989; Waldvogel et nl., 1992). Results
The calves inoculated with UC-8 (J and L) were born 16 and 28 days after inoculation, after a gestation of 259 and 271 days (Table 1). Their birth weights were 21 and 22 kg, respectively. Both were weak, and calf J, born 16 days after inoculation, did not nurse spontaneously. The two calves inoculated with UC-2, M and N, were born 32 and 35 days after inoculation, after a gestation of 275 and 278 days. These two calves weighed 32 and 34 kg, respectively at birth, and they appeared clinically normal. The cortisol concentrations of the four calves were in the range of 46 to 85 ng per ml (Table 1). Gross lesions were not detected in any calf at necropsy. However, microscopical examination revealed glial nodules in the cerebral cortex, basal ganglia and the thalamus (Fig. 1) of the two calves inoculated with UC-8. Morphological evidence ofimmune stimulation during gestation was detected in the lymph nodes of all four infected calves. Secondary follicles with numerous mitotic figures were found in the cortex (Fig. 2)) and plasma cells were present in the medullary cords.
Duration
J (3) L (8) M ($1 N (9
of gestation,
birth weight gestation
Table 1 and cortisol concentrations with BTV 11 strains UC-2
UC-8 UC-8 UC-2 UC-2
Note: Gestation and birth weight ofAngus order of 286 days and 38 kg, respectively
259f 1 271*1 275fl 278f 1
21 22 32 34
of calves and UC-8
46 58 85 64
and Hereford dams bred by Simmental (Smith el al., 1976).
inoculated
at 243 days
Weak/Small Weak/Small
bulls are normally
in the
336
A. S. Waldvogel
et al.
Fig. 1,
Glial nodule in basal ganglia of calfJ, inoculated with UC-8 and born 16 days after inoculation. HE. Bar= 30 pm.
Fig. 2.
Secondary lymph follicle with numerous mitotic figures in mesenteric inoculated with UC-8, born 28 days after inoculation and necropsied Bar= 50 pm.
Isolation
C&f
of BTV
11 strains
UC-2
Inoculum
(-xl
and
Table 2 UC-8 from newborn days of gestation
Birth
UC-8
L (3) M (6) N (0)
UC-8 UC-2 UC-2
calves
after
afterinoculation (dwi
J (6)
intramuscularly
16 28 32 35
at 243 days gestation lymph node of calf L, at 2 days of age. HE.
in utero
inoculation
at 243
Virus isolation Blood
Brain
Lung end liver
+ + + -
+ + -
+ + -
Bluetongue
Virus
and Premature
Birth
337
BTV was isolated from the brain, blood and pooled liver and lung of calves and L, both inoculated with UC-8, but only from the blood of calf M, inoculated with UC-2 (Table 2). All four calves were serologically positive for BTV and negative for bovine viral diarrhoea virus (BVDV). BTV antigen was detected by the avidin-biotin complex method in the glial nodules of the fetuses inoculated with UC-8 (Anderson et al., 1989).
J
Discussion These experiments indicate that BTV can cause congenital infection in lateterm bovine fetuses. Three of four calves inoculated with bluetongue virus strains UC-2 or UC-8 at 243 days of gestation and born 16 to 32 days after inoculation were viraemic at birth. Calves inoculated with UC-8 had a mild encephalitis. The significance of these lesions for the viability of the calves was not determined. Since the lesions were modest, it is likely that they would be of little or no significance as far as clinical signs are concerned. Jochim and coworkers (1974) reported no malformations in bovine fetuses inoculated at 5 and 7: months of gestation. However, since they did not necropsy calves after birth, it is not known whether their calves had lesions. The gestation period for the two calves inoculated with UC-8 was 27 and 15 days, less than the expected gestation period for these crossbreeds. The birth weights were 45 per cent and 42 per cent below the average for calves from Hereford and Angus dams mated with Simmental bulls (Liggins, 1982) and these calves were weak at birth. In contrast, the calves inoculated with UC-2 appeared healthy at birth. They were born within 8 to 11 days of the expected date of parturition, and their birth weights were only 15 per cent and 11 pel cent below average. This suggests that infection with UC-8 causes more severe infection and brain lesions which result in premature delivery of low birth weight and weak calves. The exact factors contributing to the low birth weights are not understood. The severity of the brain lesions decreases with increasing age of the bovine fetus. This follows the same pattern as the infection in fetal sheep (Osburn e1nl., 197 1). There were either no brain lesions in calves inoculated with UC-2, or only modest lesions in calves inoculated with UC-8. The fact that brain lesions were detected only in the fetuses inoculated with UC-8, but not UC-2, suggests that factors influencing the expression of neurovirulence in newborn BALB/c mice may also be operable in late-term bovine fetuses. If this concept is valid in bovine fetuses, then the ability to prevent UC-2 from reaching the brain must be acquired between 120 and 243 days of gestation, since both strains were neurovirulent in 120-day-old fetuses. MacLachlan and Osburn (1983) report that one of several bovine fetuses inoculated with BTV 10 at a fetal age of approximately 125 days (as determined by rectal palpation) did not develop central nervous system lesions. Although they reported that the fetus was viraemic 50 days after inoculation, no virus was isolated from its brain. Since rectal palpation is not an exact method of age determination, it was suggested that this fetus was older than 125 days at inoculation and therefore did not develop brain lesions. However, infection of fetuses with UC-8 even late in
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gestation produced glial nodules suggesting that at least two factors, straindependent differences in neurovirulence of BTV and the stage of development of the CNS, influence the outcome of an infection of late-term bovine fetuses. A preparturient surge of fetal cortisol is associated with initiation of parturition in ruminants (Smith, Laster and Gregory, 1976). Normally, cortisol concentrations of approximately 3.6 ng per ml are found in uninfected bovine fetuses of 260 days gestation (Liggins, 1982) and normal newborn calves have cortisol concentrations ranging from 45 to 300 ng per ml (Lin, Oxender and Hafs, 1973). The rise in fetal cortisol in normal parturition begins within 5 to 7 days of parturition. Previous reports indicate that fetal cortisol rises in response to the stressof congenital infections, and this elevation can induce premature delivery (Osburn, Drost and Stabenfeldt, 1972; Stott and Reinhard, 1978). The fact that calves J and L, inoculated with UC-8, had greater morphological evidence of lesions, and that their cortisol was markedly elevated (46 to 85 ng per ml) above expected values for comparably aged normal calves (3.6 ng per ml) strongly suggests that these infected calves were stressed and that this, associated with the UC-8 infection, led to the 15 to 27 day premature delivery of these calves. This study indicates that infection with UC-8 causes congenital infection and modest disease in the form of neural lesions in late-term bovine fetuses. The stressassociated with infection resulted in premature birth of small, weak calves. The UC-8 strain of serotype 11, therefore, is more virulent and pathogenic leading to premature delivery of weak and low birth-weight calves. Acknowledgments
This researchwas supported in part by USDA Special Grant 84-CSRS-2-2504, USDA Animal Health and Disease Grant CSRS-1433, and Western Regional Research Project W 112. The authors thank MS Carrie Schore for manuscript preparation. References
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R. E. and Osburn, B. I. (1986) of bluetongue virus serotype 11
Received, December 6th, 1991 Accepted, January 18th, 1992
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