PATHOLOGY OF CONGENITAL BOVINE EPIZOOTIC ARTHROGRYPOSIS AND HYDRANENCEPHALY AND ITS RELATIONSHIP TO AKABANE VIRUS W, J. HARTLEY*, D.Sc., M.V.Sc., F.R.C.V.S., F.R.C.Path., W. G. De SARAM~, M.V.Sc., Ph.D., A. J. DELLA-PORTAS, B.Sc., PhD., W. A. SNOWDON$, B.V.Sc. and N. C. SHEPHERD$, B.V.Sc. SUMMARY: The 1974 epizootic of congenital bovine arthrogryposis/ hydranencephaly syndrome in southeastern New South Wales afforded the opportunity to study the pathology, serology and virology of field cases from the outbreak. For ease of description, the lesions observed were grouped into 5 stages which possibly approximate to the gestational age of the foetus at the time of initial insult. Group 7: calves born incoordinate and microscopic examination revealed a universal mild to moderate non-suppurative acute encephalomyelitis. Group 2: calves born incoordinate or showed mild arthrogryposis and microscopically a mild to moderately severe acute Wallerian type degeneration of all zones of the spinal cord except the dorsal funiculi was observed. Group 3: calves born with arthrogryposis and microscopically there was a severe diffuse loss of myelinated fibres in the lateral and ventral funiculi of affected areas of the spinal cord. There was also a moderate to severe atrophy of the skeletal musculature. Group 4: calves born with hydranencephaly, sometimes associated with arthrogryposis, with almost complete replacement of both cerebral hemispheres by a fluid filled cavity. Group 5: calves born with micrencephaly and hydranencephaly and sometimes arthrogryposis and loss of anterior and mid brain stem and cerebellar cavitation. There was often an overlap of the lesions between groups and the groups have been placed i n the order i n which they were observed in the field during this epizootic. Although no virus was isolated from affected calves, there was a complete correlation with the presence of Akabane virus neutralising antibodies in the pre-colostrum serum of calves affected with arthrogryposis and hydranencephaly (Groups 3 and 4). Epidemiological evidence and some serological evidence, would suggest that lesions in Groups 1, 2 and 5 may be associated with Akabane virus. It is concluded that epizootic bovine congenital arthrogryposis/hydranencephaly syndrome associated with infection of the foetus with Akabane virus should be called Akabane disease. IntodUCtiOU
The syndrome of congenital bovine arthrogryposis and hydranencephaly (AG/HE) has been recognised in New South Wales for at least 30 years (Blood 1956; Whittem 1957; Hartley and Wanner 1974). A similar congenital deformity has been observed in cattle in Israel (Markusfeld and Mayer 1971; Nobel et a2 1971) and in Japan (Nosaka et al 1973; Omori 1973; Kanno 1973). Japanese workers (Miura et al 1974; Kurogi et a2 1975) recently demonstrated that the AG/ HE syndrome was associated with serum neutralising antibodies to Akabane virus in the serums of afEected calves before they had sucked. Akabane virus was first isolated from mosquitoes in Japan (Oya et a2 1961) and later from the biting midge Crrlicoides brevitarsis in Australia Department of Veterinary Medicine, University of Sydney, Camden New South Wales 2570 Research Station,' Glenfield, New South Wales. 2617 t CSIRO Division of Animal Health. Animal Health Research ' +boratory, Private Bag No. 1, P.O., Parkville, Victoria, 3052 0 Dstrict Veterinary Office, P.O. Box 389, Goulburn, New South W a l ~ s ,2580, presently located at National Parks and Wildlife Semce, P.O. Box N189, Grosvenot Street Post Office, Sydney, New South Wales, 2000
t Veterinh
Australian Veterinary Journal. Vol. 53, July, 1977
(Doherty et al 1972) and is a member of the Simbu serological sub-group of arboviruses (Doherty et a1 1972). The association between the presence of serum neutralising antibodies to Akabane virus and the AG/HE syndrome has been confirmed in preliminary observations made in New South Wales (Hartley et al 1975). The occurrence of an epizootic of bovine AG/HE in south-eastem New South Wales during 1974 provided the opportunity to further investigate this syndrome. This paper describes the results of the pathological, serological and virological examinations carried out on deformed calves. Other papers in this series describe the field aspects of the epizootic (N.C. Shepherd, C. D. Gee, T. Jessep, G. Timmins, S. N. Carrol and R. A. Bonner, personal communication) and a serological survey of the distribution of serum neutralising antibodies to Akabane virus in Australian cattle (Della-Porta et al 1976). Materials and Methods The Study From ApriI 1974, field veterinarians in south-eastern New South Wales reported the birth of many calves which were either incoordinated or were unable to 3 19
stand. This newly recognised condition was followed by cases of congenital arthrogryposis in calves from later calving cows in the same geographical areas and often on the same property. Later still, many blind calves were born and towards the end of the outbreak, in October, several calves were born with a variety of other abnormalities. Samples were taken for pathological examination throughout all stages of the outbreak and for serological and virological examination towards the end of the outbreak.
in broth and inoculated into further mice. If there were no signs of ill health in mice 8 to 10 days after inoculation a suspension was made from a pool of brains from all mice in the litter and inoculated into additional mice. All samples were passaged 2 to 4 times in suckling mice before being discarded as being negative for virus.
Results
Pathology of the Central Nervous System For ease of description the lesions observed have been classed into 5 groups or stages, probPathology During the period April-October 1974 specimens were ably approximating the gestational age of the examined from 130 calves that were born in the Eden, foetus at the time of the initial insult. However, Braidwood, Goulburn, Bowral, Camden, Mudgee, Young there was often some overlap of lesions between and Cooma districts of New South Wales. Eighty calves were submitted alive or dead and brain and spinal cord these various classes, particularly in group 5 . fixed in formalin were submitted from the remaining Group I - Affected calves in this group were 50. All were believed to have been affected with various seen at the start of the epizootic, mainly in April, stages of the same disease process. Following a clinical examination of live animals, May and June. They were either incoordinate or these, and the dead ones, were autopsied. The brain and unable to stand at birth. There were no gross spinal cord were removed and a gross examination was lesions, but on microscopical examination of the made. In some cases the brains were weighed. From CNS there was a universal mild to moderate most of these cases the central nervous system (CNS) non-suppurative acute encephalomyelitis, which was fixed by immersion in 10% neutral formalin. Samples of peripheral nerves and a complete range of was most evident in the grey matter of the mid other organs and tissues were also taken from some and posterior brain stem (Figure 1). calves. Following fixation, the brain was sliced transGroup 2 - Calves in this group showed either versely and gross lesions recorded. Blocks were taken from representative levels of the brain and cord. These incoordination, flaccid paralysis or mild arthroand blocks from other organs were processed for histo- gryposis. Microscopically there was a mild to logical examination by conventional methods. Sections moderately severe active Wallerian type degenerawere stained with haematoxylin and eosin (H and E) tion of all zones of the spinal cord except the and selected sections from the CNS were also stained with Lux01 fast blue - periodic acid Schiff - haema- dorsal funiculi (Figure 2). This was associated with mild to extensive degeneration and/or loss toxylin (LFB, PAS, H) and Gomori’s method for iron. of ventral horn neurones, Wallerian degeneration Serology Towards the end of the epizootic, serums were col- of ventral spinal nerves and usually a mild nonlected from cattle on 30 properties, mainly in the suppurative encephalomyelitis (Figure 3 ) . Goulburn, Braidwood and Mudgee districts, where hy- Group 3 - Arthrogryposis was observed in dranencephaly was the predominant congenital defect. calves born between June and September. ClinicSerums were taken from 84 cows, some of which had given birth to affected calves. Serums were also col- ally affected calves had fixed flexion or sometimes lected from 84 calves, 32 of which were apparently extension of one or more limbs and occasionally normal and had sucked and 52 of which were affected cervical scoliosis. Brain and cords appeared (31 had sucked and. 21 had not). In addition, fluid, grossly normal, although on the cut surface the presumably cerebrospinal fluid (CSF), was taken from the hydranencephalic cavity of 20 affected calves. ventral and lateral white matter of the cord Serums and CSF were examined for neutralising anti- appeared somewhat translucent. Microscopically bodies to Akabane virus (strain B 8935; Doherty et al there was a severe diffuse loss of myelinated 1972) using a microtitre or a tube neutralising test with fibres in the lateral and ventral funiculi of affected Vero cells (Delia-Porta et aZ 1976) and using 12-25 TCIDso of virus or 100 TCID, of virus in each test, areas of the spinal cord, together with loss of respectively. Serums were taken to be positive if they ventral horn neurones and marked loss of nerve neutralised the virus infectivity at a dilution of 1 in 2 fibres in ventral spinal nerves (Figure 4). The or greater. lesions were associated with a moderate to severe ViroZogy atrophy of the skeletal musculature supplied by During September and October samples were taken the ventral spinal nerves and/or adipose tissue from 18 calves with hydranencephaly. These samples replacement. included cerebral remnants, cerebellum, cord, heart, Group 4 - Hydranencephaly was seen towards spleen and CSF. Tissue suspensions were inoculated into suckling mice the end of the epizootic, July till October and was 1 to 3 days of age. Ten per cent suspensions of tissue sometimes associated with lesions of arthrosamples were made in heart infusion broth, p H 7.4 and gryposis. Calves with hydranencephaly alone were inoculated by the intracerebral route. The mice were examined daily and any mice showing signs of sickness able to walk quite well although blind. They were killed, their brains removed, suspensions prepared often had a slightly domed cranium and were 3 20
Australian Veterinary Journal, Vol. 53, July, 1977
somewhat dejected. In those calves with a domed cranium, the affected cranial bones were thinner than normal. Two aborted calves were seen with hydranencephaly and arthrogryposis. In the majority of affected calves there was an almost complete replacement of both cerebral hemispheres by a fluid filled cavity. Brain weights ranged from 35-85 g (normal 180-250 8). Spinal cords appeared normal in size. Virtually all that remained of the cerebral hemispheres were the leptomeninges (Figure 5). However the anterior brain stem structures could be readily identified although reduced in size. In a few calves born late in the epizootic, the hydranencephaly was less severe and consisted of extensive cavitation or porencephaly of the white matter of the dorsal and ventrolateral cerebral hemispheres with dilatation of the lateral ventricles (Figure 6 ) . Occasionally the hemispheric lesions were asymmetrical and occasionally there was focal symmetrical cavitation of the mid brain stem. Microscopically there were no significant lesions in the brain parenchyma adjacent to hydranencephalic cavitation. Myelination appeared to be normal and there was no evidence of peripheral gliosis. In several brains with small focal cavitations there were pigmented, iron-containing macrophages, both in the lumen and in the peripheral brain parenchyma and sometimes mild to moderate perivascular cuffing. Group 5 - The 12 calves in this category were born mainly in September/October towards the end of the epizootic. They came from 12 different properties scattered throughout the affected area, all but 2 of which had experienced other losses from the AG/HE syndrome. These 12 are grouped because they showed a variety of additional congenital abnormalities of the CNS. Four of the 12 had severe and 2 mild arthrogryposis. Most of the others could stand but showed incoordination. Nine had thickened cranial bones and an obvious reduction in the size of the cranial cavity. The 9 had severe to extreme hydranencephalv, which in 5 calves also involved the anterior and mid brain stem (Figure 7). Brain weights of these ranged from 12-85 g . In 2 others there was a mild degree of micrencephaly with focal cavitation of cerebral hemispheric white matter. One of the latter and 5 other calves had a moderate to marked reduction in the size of the cerebellum (Figures 7 and 8 ) . Microscopically there was cavitation and collapse of the cerebellar white matter. Most folia had normal looking external and internal granule cell layers, but in several calves there was considerable disruption of folial architecture. Australiari Veterinary Jotrrrinl. Vol. 53, July, 1977
The spinal cord from one severe case of arthrogryposis was much smaller than normal and there was a complete absence of all elements except the dorsal columns and small parts of dorsal horns. Pathology of Other Tissues and Organs There were no consistent associated lesions outside the CNS and peripheral nerves except in cases of arthrogryposis, where there was mild to moderate neurogenic skeletal muscle atrophy and/or adipose tissue replacement. However, at least 15 calves submitted alive with hydranencephaly and which were autopsied from 1 to 7 days after birth, had one or more of the following: shallow erosions about the external nares and muzzle and between distal digits, fibrinous polyarticular synovitis, fibrinous leptomeningitis, fibrinous ependymitis, fibrinous navel infection, ophthalmia, cataract and presternal steatosis. No infective agents were seen in histological sections of any of these inflammatory lesions. Further, routine microbiological examination failed to isolate any bacteria, mycoplasma or arboviruses. Also no organisms could be seen in joint and lepto-meningeal exudates examined with the electron microscope. Serology All 18 cows giving birth to confirmed cases of hydranencephaly had positive serum neutralising antibody titres for Akabane virus. In addition, 20 of the 23 cows which had affected calves, according to owner assessment, also had positive antibody titres as did 28 of 36 cows on the same properties at the same time, which had apparently normal calves. Serums from 31 calves which had hydranencephaly and that had sucked colostrum, contained neutralising antibodies against Akabane virus, as did 10 calves with hydranencephaly or arthrogryposis that had not sucked. Twenty-three of 32 apparently normal calves that sucked also possessed neutralising antibodies. Serums were available from 11 calves in group 5 and from 7 of their dams. Three dam serums were positive for neutralising antibodies, 1 questionably positive and 3 negative. Four calf serums were positive, 2 questionably positive and 5 were negative. One of the 4 positive calves had sucked, the other 3 were reported not to have sucked. There were no apparent differences in congenital abnormalities between those calves with positive and negative antibody titres. All 3 calves with positive titres had small brain cavities, with severe hydraneiicephaly and 2 had absent anterior and 321
?? m
ii
322
2 .-m LL
Australian Veterinary Journal, Vol. 5 3 , July, 1917
-m>; S Q 0 d) C
c L
R t e P
in c
0
m
c._ %
c
m m a 0 ._ c ._
-e L
m 0
0
5
c
d
E
f
w"
mid brain stem and a small cerebellum. One had mild arthrogryposis. All 20 CSF samples from calves with hydranencephaly did not possess neutralising antibodies against Akabane virus. Virology All specimens examined for virus were negative. Discussion
The 1974 epizootic of bovine congenital AG/ HE syndrome seen in south-eastern New South Wales (Shepherd et al, personal communication) was probably more severe and more extensive than the epizootic j. previously observed (Blood 1956; Whittem 1957; Hartley and Wanner 1974). In addition to the common abnormalities, arthrogryposis and hydranencephaly, there were many abortions and stillbirths (Shepherd et al, personal communication) and also calves with encephalomyelitis, secondary demyelination of the spinal cord and motor spinal nerves, micrencephaly and cerebellar cavitation. In years prior to the 1974 outbreak, sporadic cases of congenital bovine encephalomyelitis and active secondary demyelination of motor tracts and nerves had been seen in specimens examined at both the Veterinary Research Station of the New South Wales Department of Agriculture (Glenfield) and at the Department of Veterinary Medicine of the University of Sydney (Camden). However, as these cases were seen in the autumn months, they were not at the time thought to be associated with the AG/HE syndrome. The condition in Murray Grey calves reported by Hartley and Wanner (1974) was in all probability also part of this same syndrome. The epizootics reported from Israel (Markusfeld and Mayer 1971; Nobel et a1 1971) and in Japan (Nosaka et a1 1973; Konno et a1 1975) consisted of abortions, stillbirths, arthrogryposis and hydranencephaly with mild inflammatory lesions in some cases. In the Israel outbreak arthrogryposis and hydranencephaly were also seen in lambs and goat kids (Nobel et a1 1971). Both Inaba (cited in Inaba et a1 1975) in Japan and Parsonson et a1 (1975) in Australia have demonstrated transmission of Akabane virus across the placenta in pregnant goats, cows and ewes and a proportion of these animals gave birth to congenitally deformed foetuses. The former has produced arthrogryposis and hydranencephaly in a kid and in a calf. The latter have produced micrencephaly, arthrogryposis, hydranencephaly and porencephaly in a number of lambs. There have been no reports of concomitant congenital CNS disease in lambs in the areas of 324
New South Wales where cattle were affected with the AG/HE syndrome, but this may be a matter of different husbandry (e.g. mating at different times of the year). However, small outbreaks of congenital micrencephaly, arthrogryposis and hydranencephaly have been seen in lambs in the Camden area of New South Wales in the spring of both 1971 and 1973 (Hartley and Haughey 1974 and W. J. Hartley and K. G. Haughey, unpublished data). These were years in which there were only sporadic cases of the AG/HE syndrome in cattle in that area. In 1973 there was a high prevalence of serum neutralising antibodies to Akabane virus in the dams of affected lambs and also in ewes with normal lambs (W. J. Hartley, K. G. Haughey and A. J. Della-Porta, unpublished data). From a morphological study of many cases of the bovine AG/HE syndrome it is possible to follow the sequential development of the pathological process. The type of lesion encountered would appear to depend on the stage of development of the foetal CNS at the time of the initial insult. Groups 1 to 3 represent infection probably occurring within the last 1 to 3 months of gestation. These were associated with a non-suppurative encephalomyelitis, followed by degeneration of, and loss of, ventral horn neurones, Wallerian degeneration and loss of motor tracts in the spinal cord and peripheral motor nerves and consequential neurological muscle atrophy. Group 4 probably represents infection occurring in mid gestation. Most of the calves in Group 5 probably represent infection at an earlier date. From material so far available, it is not possible to be certain of the pathogenesis of the brain cavitation and hydranencephaly. The lesions could start in a similar manner to that reported (Young and Cordy 1964; Osburn et al 1971) in experimental congenital blue tongue virus infection in sheep. That is, oedema and necrosis of the cerebral white matter followed by lysis and replacement by a fluid filled cavity bounded by leptomeninges. In cases of hydranencephaly in the AG/HE syndrome the probability is that there has been destruction of preformed brain parenchyma. In Group 4, the cranial cavity appeared either normal in size or possibly, in those with a domed cranium, slightly enlarged. For most calves in Group 5, the cranial cavity was considerably reduced in size and most had severe to extreme hydranencephaly. The exact relationship between the type of lesion and foetal gestational age at infection must await detailed experimental observations. It will Atrstralian Veferinury Journal, Vol. 53, July, 1977
also be necessary to ascertain if placental lesions occur in cases of abortion. In calves affected with arthrogryposis, hydranencephaly, or both (Groups 3 and 4), there was a complete correlation with the presence of Akabane virus neutralising antibodies in the serum of these calves collected before they had ingested colostrum. The same relationship has also been demonstrated in Japan (Miura et al 1974; Kurogi et a1 1975) and in Australia (Hartley et a1 1975) for 2 sporadic cases of calves with hydranencephaly and micrencephaly born at Wallabadah in north-eastern New South Wales in 1973. The epidemiological evidence and the sequential pathological findings strongly suggest that all or most of the calves in Group 5 belong to the epizootic AG/HE syndrome. However, the serological data do not fully support this, as 50 per cent of affected calves did not possess serum neutralising antibodies to Akabane virus. The absence of detectable neutralising antibodies in the foetus does not necessarily indicate that the foetus was not infected, as observed (Parsonson et a1 1975) with a lamb that had porencephaly produced by Akabane virus infection, especially if the virus was eliminated before the foetus became immunocompetent. However, the absence of neutralising antibodies also in the serum of the dam, in the light of present knowledge, would mitigate against the Akabane virus being the cause of all of these deformities. It is thus possible that another agent may be implicated in this epizootic. The unavailability of serums and tissues for virological examination from calves early in the outbreak, Groups 1 and 2, precludes any linkage between these cases and infection by the Akabane virus. The failure to isolate Akabane virus from the affected calves sampled towards the end of the outbreak suggests that either the virus was no longer present or that its presence was masked by antibodies to Akabane virus. A similar situation exists with mucosal disease virus infection of the bovine foetus, in which the virus could not be isolated (Braun et a1 1973) when neutralising antibodies to the virus were present. The presence of acute fibrinous lesions in the CNS, joints and elsewhere of a number of calves, some of which were less than 24 hours old at autopsy, suggests that the inflammatory process may have commenced before birth. Although no microorganisms could be demonstrated in these cases, it is conceivable, but unlikely, that the lesions may have resulted from exacerbation of a latent Akabane virus infection. Australian Veterinary loirrnul, Vol. 53, July, 1977
In conclusion, the epidemiological evidence from the 1974 epizootic of the AG/HE syndrome, backed up by the pathological findings and augmented by the demonstration of Akabane neutralising antibodies in most affected calves sampled before ingestion of colostrum, strongly suggest that most, if not all, lesions seen in this outbreak are part of the same disease process and are in all probability due to the direct effect of foetal infection by the Akabane virus. The authors concur with Inaba et a1 (1975) that the term AG/HE syndrome associated with Akabane virus now be renamed Akabane Disease. Acknowledgments
We wish to acknowledge the technical assistance of Mrs P. Lowen, D. Duivenvoorden and Mr K. McWilliam. References Blood. D. C. (1956)-Aust. vet. I . 3 2 125. Braun; R. K., Osburn, B. I. and Kendrick, J. W. (1973) -Am. I. vet. Res. 34: 1127. Della-Porta. A. J.. Murrav. M. D. and Cvbinski. D. H. (1976)-Aust. vet. I. 52:'496. Doherty, R. L., Carley, J. G., Standfast, H. A., Dyce, A. L. and Snowdon, W. A. (1972)-Aust. vet. I. 48: 81. Hartley, W. J . and Haughey, K. G. (1974)-Aust. vet. J . 50: 55. Hartley, W. J. and Wanner, R. A. (1974)--Aust. vet. J. 50: 185. Hartley, W. J., Wanner, R. A., Della-Porta, A. J. and Snowdon, W. A. (1975)--Aust. vet. J . 51: 103. Inaba, Y., Kurogi, H. and Ornori, T. (1975)-Aust. vet. J. 51: 584. Konno, S. ( 1 9 7 3 k - J . l a p . vet. med. Ass. 26: 515. Konno, S., Moriwaki, M., Nakagawa, M., Uchimura, M., Kamimiyata, M. and Tojinbara, K. (1975)Nat. Inst. Arrim. Hlth. Quart. (Japan) 15: 52. Kuroni. H.. Inaba. Y.. Goto. Y . . Miura. Y.. Takohashi. H.,-Sato,' K., Omori, T. and Matumoto, M. (1975)Arch. Virol. 47: 71. Markusfeld, 0. and Mayer, E. (1971)-Refuah v e f . 28: 51. Miura, Y.,Hayashi, S., Ishihara, T., Inaba, Y . , Omori, T. and Matumoto, M. (1974)-Arck. ges. Virsforsch. 46: 377. Nobel, T. A., Klopfer, U. and Neurnann, F. (1971)Refuak vet. 28: 144. Nosaka, D., Tateyama, S., Ashizawa, H., Nakamura, N., Yago, H., Shimizu, T. and Murakami, T. (1973)Bull. Miyazaki Univ. Agricul. Faculty 20: 3 11. Omori, T. (1973)-J. l a p . vet. rned. Ass. 26: 510. 0-burn, B. I., Silverstein, A. M., Prendergast, R. A,, Johnson, R. T. and Parshall, C. J. (1971)--Lah Invest. 25: 197. Oya, A., Okuno, T., Ogata, T., Kobayashi, I. and Matsuyama, T. (1961)-Jop 1 rned Sci ,Biol 14: 101.
Parsonson, I. M., Della-Porta, A. J., Snowdon, W. A. and Murray, M. D. (1975)-Aust. \vet. 1. 51: 5 8 5 . Whittem, J. H. (1957)-J. Path. Bact. 73: 375. Young, S. and Cordy, D. R. (1964)-J. Neuroputh. exp. Neurol. 23: 635. (Received for publication 15 June 1976) 325
The syndrome of congenital bovine arthrogryposis and hydranencephaly (AG/HE) has been recognised in New South Wales for at least 30 years (Blood 1956; Whittem 1957; Hartley and Wanner 1974). A similar congenital deformity has been observed in cattle in Israel (Markusfeld and Mayer 1971; Nobel et a2 1971) and in Japan (Nosaka et al 1973; Omori 1973; Kanno 1973). Japanese workers (Miura et al 1974; Kurogi et a2 1975) recently demonstrated that the AG/ HE syndrome was associated with serum neutralising antibodies to Akabane virus in the serums of afEected calves before they had sucked. Akabane virus was first isolated from mosquitoes in Japan (Oya et a2 1961) and later from the biting midge Crrlicoides brevitarsis in Australia Department of Veterinary Medicine, University of Sydney, Camden New South Wales 2570 Research Station,' Glenfield, New South Wales. 2617 t CSIRO Division of Animal Health. Animal Health Research ' +boratory, Private Bag No. 1, P.O., Parkville, Victoria, 3052 0 Dstrict Veterinary Office, P.O. Box 389, Goulburn, New South W a l ~ s ,2580, presently located at National Parks and Wildlife Semce, P.O. Box N189, Grosvenot Street Post Office, Sydney, New South Wales, 2000
t Veterinh
Australian Veterinary Journal. Vol. 53, July, 1977
(Doherty et al 1972) and is a member of the Simbu serological sub-group of arboviruses (Doherty et a1 1972). The association between the presence of serum neutralising antibodies to Akabane virus and the AG/HE syndrome has been confirmed in preliminary observations made in New South Wales (Hartley et al 1975). The occurrence of an epizootic of bovine AG/HE in south-eastem New South Wales during 1974 provided the opportunity to further investigate this syndrome. This paper describes the results of the pathological, serological and virological examinations carried out on deformed calves. Other papers in this series describe the field aspects of the epizootic (N.C. Shepherd, C. D. Gee, T. Jessep, G. Timmins, S. N. Carrol and R. A. Bonner, personal communication) and a serological survey of the distribution of serum neutralising antibodies to Akabane virus in Australian cattle (Della-Porta et al 1976). Materials and Methods The Study From ApriI 1974, field veterinarians in south-eastern New South Wales reported the birth of many calves which were either incoordinated or were unable to 3 19
stand. This newly recognised condition was followed by cases of congenital arthrogryposis in calves from later calving cows in the same geographical areas and often on the same property. Later still, many blind calves were born and towards the end of the outbreak, in October, several calves were born with a variety of other abnormalities. Samples were taken for pathological examination throughout all stages of the outbreak and for serological and virological examination towards the end of the outbreak.
in broth and inoculated into further mice. If there were no signs of ill health in mice 8 to 10 days after inoculation a suspension was made from a pool of brains from all mice in the litter and inoculated into additional mice. All samples were passaged 2 to 4 times in suckling mice before being discarded as being negative for virus.
Results
Pathology of the Central Nervous System For ease of description the lesions observed have been classed into 5 groups or stages, probPathology During the period April-October 1974 specimens were ably approximating the gestational age of the examined from 130 calves that were born in the Eden, foetus at the time of the initial insult. However, Braidwood, Goulburn, Bowral, Camden, Mudgee, Young there was often some overlap of lesions between and Cooma districts of New South Wales. Eighty calves were submitted alive or dead and brain and spinal cord these various classes, particularly in group 5 . fixed in formalin were submitted from the remaining Group I - Affected calves in this group were 50. All were believed to have been affected with various seen at the start of the epizootic, mainly in April, stages of the same disease process. Following a clinical examination of live animals, May and June. They were either incoordinate or these, and the dead ones, were autopsied. The brain and unable to stand at birth. There were no gross spinal cord were removed and a gross examination was lesions, but on microscopical examination of the made. In some cases the brains were weighed. From CNS there was a universal mild to moderate most of these cases the central nervous system (CNS) non-suppurative acute encephalomyelitis, which was fixed by immersion in 10% neutral formalin. Samples of peripheral nerves and a complete range of was most evident in the grey matter of the mid other organs and tissues were also taken from some and posterior brain stem (Figure 1). calves. Following fixation, the brain was sliced transGroup 2 - Calves in this group showed either versely and gross lesions recorded. Blocks were taken from representative levels of the brain and cord. These incoordination, flaccid paralysis or mild arthroand blocks from other organs were processed for histo- gryposis. Microscopically there was a mild to logical examination by conventional methods. Sections moderately severe active Wallerian type degenerawere stained with haematoxylin and eosin (H and E) tion of all zones of the spinal cord except the and selected sections from the CNS were also stained with Lux01 fast blue - periodic acid Schiff - haema- dorsal funiculi (Figure 2). This was associated with mild to extensive degeneration and/or loss toxylin (LFB, PAS, H) and Gomori’s method for iron. of ventral horn neurones, Wallerian degeneration Serology Towards the end of the epizootic, serums were col- of ventral spinal nerves and usually a mild nonlected from cattle on 30 properties, mainly in the suppurative encephalomyelitis (Figure 3 ) . Goulburn, Braidwood and Mudgee districts, where hy- Group 3 - Arthrogryposis was observed in dranencephaly was the predominant congenital defect. calves born between June and September. ClinicSerums were taken from 84 cows, some of which had given birth to affected calves. Serums were also col- ally affected calves had fixed flexion or sometimes lected from 84 calves, 32 of which were apparently extension of one or more limbs and occasionally normal and had sucked and 52 of which were affected cervical scoliosis. Brain and cords appeared (31 had sucked and. 21 had not). In addition, fluid, grossly normal, although on the cut surface the presumably cerebrospinal fluid (CSF), was taken from the hydranencephalic cavity of 20 affected calves. ventral and lateral white matter of the cord Serums and CSF were examined for neutralising anti- appeared somewhat translucent. Microscopically bodies to Akabane virus (strain B 8935; Doherty et al there was a severe diffuse loss of myelinated 1972) using a microtitre or a tube neutralising test with fibres in the lateral and ventral funiculi of affected Vero cells (Delia-Porta et aZ 1976) and using 12-25 TCIDso of virus or 100 TCID, of virus in each test, areas of the spinal cord, together with loss of respectively. Serums were taken to be positive if they ventral horn neurones and marked loss of nerve neutralised the virus infectivity at a dilution of 1 in 2 fibres in ventral spinal nerves (Figure 4). The or greater. lesions were associated with a moderate to severe ViroZogy atrophy of the skeletal musculature supplied by During September and October samples were taken the ventral spinal nerves and/or adipose tissue from 18 calves with hydranencephaly. These samples replacement. included cerebral remnants, cerebellum, cord, heart, Group 4 - Hydranencephaly was seen towards spleen and CSF. Tissue suspensions were inoculated into suckling mice the end of the epizootic, July till October and was 1 to 3 days of age. Ten per cent suspensions of tissue sometimes associated with lesions of arthrosamples were made in heart infusion broth, p H 7.4 and gryposis. Calves with hydranencephaly alone were inoculated by the intracerebral route. The mice were examined daily and any mice showing signs of sickness able to walk quite well although blind. They were killed, their brains removed, suspensions prepared often had a slightly domed cranium and were 3 20
Australian Veterinary Journal, Vol. 53, July, 1977
somewhat dejected. In those calves with a domed cranium, the affected cranial bones were thinner than normal. Two aborted calves were seen with hydranencephaly and arthrogryposis. In the majority of affected calves there was an almost complete replacement of both cerebral hemispheres by a fluid filled cavity. Brain weights ranged from 35-85 g (normal 180-250 8). Spinal cords appeared normal in size. Virtually all that remained of the cerebral hemispheres were the leptomeninges (Figure 5). However the anterior brain stem structures could be readily identified although reduced in size. In a few calves born late in the epizootic, the hydranencephaly was less severe and consisted of extensive cavitation or porencephaly of the white matter of the dorsal and ventrolateral cerebral hemispheres with dilatation of the lateral ventricles (Figure 6 ) . Occasionally the hemispheric lesions were asymmetrical and occasionally there was focal symmetrical cavitation of the mid brain stem. Microscopically there were no significant lesions in the brain parenchyma adjacent to hydranencephalic cavitation. Myelination appeared to be normal and there was no evidence of peripheral gliosis. In several brains with small focal cavitations there were pigmented, iron-containing macrophages, both in the lumen and in the peripheral brain parenchyma and sometimes mild to moderate perivascular cuffing. Group 5 - The 12 calves in this category were born mainly in September/October towards the end of the epizootic. They came from 12 different properties scattered throughout the affected area, all but 2 of which had experienced other losses from the AG/HE syndrome. These 12 are grouped because they showed a variety of additional congenital abnormalities of the CNS. Four of the 12 had severe and 2 mild arthrogryposis. Most of the others could stand but showed incoordination. Nine had thickened cranial bones and an obvious reduction in the size of the cranial cavity. The 9 had severe to extreme hydranencephalv, which in 5 calves also involved the anterior and mid brain stem (Figure 7). Brain weights of these ranged from 12-85 g . In 2 others there was a mild degree of micrencephaly with focal cavitation of cerebral hemispheric white matter. One of the latter and 5 other calves had a moderate to marked reduction in the size of the cerebellum (Figures 7 and 8 ) . Microscopically there was cavitation and collapse of the cerebellar white matter. Most folia had normal looking external and internal granule cell layers, but in several calves there was considerable disruption of folial architecture. Australiari Veterinary Jotrrrinl. Vol. 53, July, 1977
The spinal cord from one severe case of arthrogryposis was much smaller than normal and there was a complete absence of all elements except the dorsal columns and small parts of dorsal horns. Pathology of Other Tissues and Organs There were no consistent associated lesions outside the CNS and peripheral nerves except in cases of arthrogryposis, where there was mild to moderate neurogenic skeletal muscle atrophy and/or adipose tissue replacement. However, at least 15 calves submitted alive with hydranencephaly and which were autopsied from 1 to 7 days after birth, had one or more of the following: shallow erosions about the external nares and muzzle and between distal digits, fibrinous polyarticular synovitis, fibrinous leptomeningitis, fibrinous ependymitis, fibrinous navel infection, ophthalmia, cataract and presternal steatosis. No infective agents were seen in histological sections of any of these inflammatory lesions. Further, routine microbiological examination failed to isolate any bacteria, mycoplasma or arboviruses. Also no organisms could be seen in joint and lepto-meningeal exudates examined with the electron microscope. Serology All 18 cows giving birth to confirmed cases of hydranencephaly had positive serum neutralising antibody titres for Akabane virus. In addition, 20 of the 23 cows which had affected calves, according to owner assessment, also had positive antibody titres as did 28 of 36 cows on the same properties at the same time, which had apparently normal calves. Serums from 31 calves which had hydranencephaly and that had sucked colostrum, contained neutralising antibodies against Akabane virus, as did 10 calves with hydranencephaly or arthrogryposis that had not sucked. Twenty-three of 32 apparently normal calves that sucked also possessed neutralising antibodies. Serums were available from 11 calves in group 5 and from 7 of their dams. Three dam serums were positive for neutralising antibodies, 1 questionably positive and 3 negative. Four calf serums were positive, 2 questionably positive and 5 were negative. One of the 4 positive calves had sucked, the other 3 were reported not to have sucked. There were no apparent differences in congenital abnormalities between those calves with positive and negative antibody titres. All 3 calves with positive titres had small brain cavities, with severe hydraneiicephaly and 2 had absent anterior and 321
?? m
ii
322
2 .-m LL
Australian Veterinary Journal, Vol. 5 3 , July, 1917
-m>; S Q 0 d) C
c L
R t e P
in c
0
m
c._ %
c
m m a 0 ._ c ._
-e L
m 0
0
5
c
d
E
f
w"
mid brain stem and a small cerebellum. One had mild arthrogryposis. All 20 CSF samples from calves with hydranencephaly did not possess neutralising antibodies against Akabane virus. Virology All specimens examined for virus were negative. Discussion
The 1974 epizootic of bovine congenital AG/ HE syndrome seen in south-eastern New South Wales (Shepherd et al, personal communication) was probably more severe and more extensive than the epizootic j. previously observed (Blood 1956; Whittem 1957; Hartley and Wanner 1974). In addition to the common abnormalities, arthrogryposis and hydranencephaly, there were many abortions and stillbirths (Shepherd et al, personal communication) and also calves with encephalomyelitis, secondary demyelination of the spinal cord and motor spinal nerves, micrencephaly and cerebellar cavitation. In years prior to the 1974 outbreak, sporadic cases of congenital bovine encephalomyelitis and active secondary demyelination of motor tracts and nerves had been seen in specimens examined at both the Veterinary Research Station of the New South Wales Department of Agriculture (Glenfield) and at the Department of Veterinary Medicine of the University of Sydney (Camden). However, as these cases were seen in the autumn months, they were not at the time thought to be associated with the AG/HE syndrome. The condition in Murray Grey calves reported by Hartley and Wanner (1974) was in all probability also part of this same syndrome. The epizootics reported from Israel (Markusfeld and Mayer 1971; Nobel et a1 1971) and in Japan (Nosaka et a1 1973; Konno et a1 1975) consisted of abortions, stillbirths, arthrogryposis and hydranencephaly with mild inflammatory lesions in some cases. In the Israel outbreak arthrogryposis and hydranencephaly were also seen in lambs and goat kids (Nobel et a1 1971). Both Inaba (cited in Inaba et a1 1975) in Japan and Parsonson et a1 (1975) in Australia have demonstrated transmission of Akabane virus across the placenta in pregnant goats, cows and ewes and a proportion of these animals gave birth to congenitally deformed foetuses. The former has produced arthrogryposis and hydranencephaly in a kid and in a calf. The latter have produced micrencephaly, arthrogryposis, hydranencephaly and porencephaly in a number of lambs. There have been no reports of concomitant congenital CNS disease in lambs in the areas of 324
New South Wales where cattle were affected with the AG/HE syndrome, but this may be a matter of different husbandry (e.g. mating at different times of the year). However, small outbreaks of congenital micrencephaly, arthrogryposis and hydranencephaly have been seen in lambs in the Camden area of New South Wales in the spring of both 1971 and 1973 (Hartley and Haughey 1974 and W. J. Hartley and K. G. Haughey, unpublished data). These were years in which there were only sporadic cases of the AG/HE syndrome in cattle in that area. In 1973 there was a high prevalence of serum neutralising antibodies to Akabane virus in the dams of affected lambs and also in ewes with normal lambs (W. J. Hartley, K. G. Haughey and A. J. Della-Porta, unpublished data). From a morphological study of many cases of the bovine AG/HE syndrome it is possible to follow the sequential development of the pathological process. The type of lesion encountered would appear to depend on the stage of development of the foetal CNS at the time of the initial insult. Groups 1 to 3 represent infection probably occurring within the last 1 to 3 months of gestation. These were associated with a non-suppurative encephalomyelitis, followed by degeneration of, and loss of, ventral horn neurones, Wallerian degeneration and loss of motor tracts in the spinal cord and peripheral motor nerves and consequential neurological muscle atrophy. Group 4 probably represents infection occurring in mid gestation. Most of the calves in Group 5 probably represent infection at an earlier date. From material so far available, it is not possible to be certain of the pathogenesis of the brain cavitation and hydranencephaly. The lesions could start in a similar manner to that reported (Young and Cordy 1964; Osburn et al 1971) in experimental congenital blue tongue virus infection in sheep. That is, oedema and necrosis of the cerebral white matter followed by lysis and replacement by a fluid filled cavity bounded by leptomeninges. In cases of hydranencephaly in the AG/HE syndrome the probability is that there has been destruction of preformed brain parenchyma. In Group 4, the cranial cavity appeared either normal in size or possibly, in those with a domed cranium, slightly enlarged. For most calves in Group 5, the cranial cavity was considerably reduced in size and most had severe to extreme hydranencephaly. The exact relationship between the type of lesion and foetal gestational age at infection must await detailed experimental observations. It will Atrstralian Veferinury Journal, Vol. 53, July, 1977
also be necessary to ascertain if placental lesions occur in cases of abortion. In calves affected with arthrogryposis, hydranencephaly, or both (Groups 3 and 4), there was a complete correlation with the presence of Akabane virus neutralising antibodies in the serum of these calves collected before they had ingested colostrum. The same relationship has also been demonstrated in Japan (Miura et al 1974; Kurogi et a1 1975) and in Australia (Hartley et a1 1975) for 2 sporadic cases of calves with hydranencephaly and micrencephaly born at Wallabadah in north-eastern New South Wales in 1973. The epidemiological evidence and the sequential pathological findings strongly suggest that all or most of the calves in Group 5 belong to the epizootic AG/HE syndrome. However, the serological data do not fully support this, as 50 per cent of affected calves did not possess serum neutralising antibodies to Akabane virus. The absence of detectable neutralising antibodies in the foetus does not necessarily indicate that the foetus was not infected, as observed (Parsonson et a1 1975) with a lamb that had porencephaly produced by Akabane virus infection, especially if the virus was eliminated before the foetus became immunocompetent. However, the absence of neutralising antibodies also in the serum of the dam, in the light of present knowledge, would mitigate against the Akabane virus being the cause of all of these deformities. It is thus possible that another agent may be implicated in this epizootic. The unavailability of serums and tissues for virological examination from calves early in the outbreak, Groups 1 and 2, precludes any linkage between these cases and infection by the Akabane virus. The failure to isolate Akabane virus from the affected calves sampled towards the end of the outbreak suggests that either the virus was no longer present or that its presence was masked by antibodies to Akabane virus. A similar situation exists with mucosal disease virus infection of the bovine foetus, in which the virus could not be isolated (Braun et a1 1973) when neutralising antibodies to the virus were present. The presence of acute fibrinous lesions in the CNS, joints and elsewhere of a number of calves, some of which were less than 24 hours old at autopsy, suggests that the inflammatory process may have commenced before birth. Although no microorganisms could be demonstrated in these cases, it is conceivable, but unlikely, that the lesions may have resulted from exacerbation of a latent Akabane virus infection. Australian Veterinary loirrnul, Vol. 53, July, 1977
In conclusion, the epidemiological evidence from the 1974 epizootic of the AG/HE syndrome, backed up by the pathological findings and augmented by the demonstration of Akabane neutralising antibodies in most affected calves sampled before ingestion of colostrum, strongly suggest that most, if not all, lesions seen in this outbreak are part of the same disease process and are in all probability due to the direct effect of foetal infection by the Akabane virus. The authors concur with Inaba et a1 (1975) that the term AG/HE syndrome associated with Akabane virus now be renamed Akabane Disease. Acknowledgments
We wish to acknowledge the technical assistance of Mrs P. Lowen, D. Duivenvoorden and Mr K. McWilliam. References Blood. D. C. (1956)-Aust. vet. I . 3 2 125. Braun; R. K., Osburn, B. I. and Kendrick, J. W. (1973) -Am. I. vet. Res. 34: 1127. Della-Porta. A. J.. Murrav. M. D. and Cvbinski. D. H. (1976)-Aust. vet. I. 52:'496. Doherty, R. L., Carley, J. G., Standfast, H. A., Dyce, A. L. and Snowdon, W. A. (1972)-Aust. vet. I. 48: 81. Hartley, W. J . and Haughey, K. G. (1974)-Aust. vet. J . 50: 55. Hartley, W. J. and Wanner, R. A. (1974)--Aust. vet. J. 50: 185. Hartley, W. J., Wanner, R. A., Della-Porta, A. J. and Snowdon, W. A. (1975)--Aust. vet. J . 51: 103. Inaba, Y., Kurogi, H. and Ornori, T. (1975)-Aust. vet. J. 51: 584. Konno, S. ( 1 9 7 3 k - J . l a p . vet. med. Ass. 26: 515. Konno, S., Moriwaki, M., Nakagawa, M., Uchimura, M., Kamimiyata, M. and Tojinbara, K. (1975)Nat. Inst. Arrim. Hlth. Quart. (Japan) 15: 52. Kuroni. H.. Inaba. Y.. Goto. Y . . Miura. Y.. Takohashi. H.,-Sato,' K., Omori, T. and Matumoto, M. (1975)Arch. Virol. 47: 71. Markusfeld, 0. and Mayer, E. (1971)-Refuah v e f . 28: 51. Miura, Y.,Hayashi, S., Ishihara, T., Inaba, Y . , Omori, T. and Matumoto, M. (1974)-Arck. ges. Virsforsch. 46: 377. Nobel, T. A., Klopfer, U. and Neurnann, F. (1971)Refuak vet. 28: 144. Nosaka, D., Tateyama, S., Ashizawa, H., Nakamura, N., Yago, H., Shimizu, T. and Murakami, T. (1973)Bull. Miyazaki Univ. Agricul. Faculty 20: 3 11. Omori, T. (1973)-J. l a p . vet. rned. Ass. 26: 510. 0-burn, B. I., Silverstein, A. M., Prendergast, R. A,, Johnson, R. T. and Parshall, C. J. (1971)--Lah Invest. 25: 197. Oya, A., Okuno, T., Ogata, T., Kobayashi, I. and Matsuyama, T. (1961)-Jop 1 rned Sci ,Biol 14: 101.
Parsonson, I. M., Della-Porta, A. J., Snowdon, W. A. and Murray, M. D. (1975)-Aust. \vet. 1. 51: 5 8 5 . Whittem, J. H. (1957)-J. Path. Bact. 73: 375. Young, S. and Cordy, D. R. (1964)-J. Neuroputh. exp. Neurol. 23: 635. (Received for publication 15 June 1976) 325
