Malignant catarrhal fever: experimental transmission of the ‘sheep-associated’ form of the disease from cattle and deer to cattle, deer, rabbits and hamsters

Research in Veterinary Science /986, 4/, 76-8/

Malignant catarrhal fever: experimental transmission of the 'sheepassociated' form of the disease from cattle and deer to cattle, deer, rabbits and hamsters H. W. REID, D. BUXTON, I. POW, J. FINLAYSON, Moredun Research Institute, 408 Gilmerton Road,

Edinburgh EHI7 7JH

Attempts to transmit malignant catarrhal fever (MCF) from 16 bovine cases of the 'sheep-associated' form of the disease are described. On two occasions disease was transmitted to bovine calves but transmission to red deer (Cervus elaphus) was not achieved. In addition, MCF was transmitted from one experimentally affected calf to a rabbit and on another occasion directly to rabbits with material from a field case which failed to transmit to a bovine calf or red deer. Subsequently each of these isolates was readily passaged through rabbits and one was also passaged to Syrian hamsters. Tissue from MCF-affected red deer consistently produced disease on inoculation into rabbits and deer but failed to cause disease in bovine calves. Contact infection between red deer occurred once and roe deer iCapreotus capreo!us) were also shown to be susceptible to infection by inoculation. Passage of MCF in rabbits with an isolate from red deer failed to produce evidence of further adaptation even after 125 serial passages. Despite the failure to transmit disease from cattle to deer or from deer to cattle it is considered probable that there is only one sheep-associated agent which causes MCF in both species. The reasons for the anomalies in transmission of this form of the disease are discussed. MALIGNANT catarrhal fever (MCF) caused by the herpesvirus of wildebeest can readily be transmitted to cattle and rabbits by inoculation of intact cells from infected animals (Plowright 1968). Where this virus is not responsible for the disease the available evidence suggests that a similar virus of sheep is the cause. This putative virus, referred to as the 'sheepassociated' agent of MCF, has not yet been identified and even experimental transmission of disease from affected cattle to healthy cattle has been achieved only irregularly (Pierson et a11974, Selman et a11978) and transmission from cattle to laboratory animals has never been established. In contrast, transmission of sheep-associated MCF from affected deer to both rabbits and deer has been effected with relative facility (Buxton and Reid 1980, Westbury and

Denholm 1982, Oliver et al 1983, 1985). The reason for the difference in transmissibility is not clear but as both cattle and deer appear to become affected following contact with sheep (Selman et al 1974, Buxton and Reid 1980) it seems likely that the causal agent is the same in both species. The present report describes further studies on the experimental transmission of the sheep-associated agent from deer to deer, cattle and rabbits and reports attempts to transmit the disease from cattle to deer, cattle and laboratory animals. Materials and methods

Experimental animals The cattle used were conventionally reared male Ayrshire or Jersey calves aged under 12 months, which were housed in loose boxes. Farm-reared red deer (Cervus elaphus) aged under 18 months were used. Wherever feasible one inoculated deer was housed with an untreated animal to examine the possibility of lateral transmission. The deer were kept in loose boxes within an enclosure from which all other stock were excluded. Bedding and foodstores were maintained exclusively for the deer and the animal attendant had contact with no other domestic animals. Two hand-reared roe deer (Capreolus capreolus) were also used in these studies. The laboratory animals used were adult Lop and New Zealand White rabbits bred at the Moredun Research Institute. Syrian hamsters bred at the institute were inoculated either within 48 hours of birth or as weanlings aged four to eight weeks.

Clinical and pathological assessment All inoculated animals were examined daily for obvious signs of ill-health and, in addition, rectal temperatures of rabbits were measured daily. Animals were deemed to have reacted on the day when clinical signs were first observed or, in the case of rabbits, when a rectal temperature higher than 76

Malignant catarrhal fever transmission 40°C was recorded. The interval between inoculation and reaction was designated the 'reaction time'. Animals that reacted either died or were destroyed and all were examined after death, a diagnosis of MCF being confirmed by histological examination of selected tissues including at least kidney, lung, liver, mesenteric lymph node, spleen and appendix (Buxton and Reid 1980, Buxton et al 1984). Laboratory animals were considered not to have reacted if clinical signs were not detected during an observation period of 100 days while cattle and deer were observed for six months before being assessed as negative.

77

inoculum while rabbits received l/lOth of this dose. Peripheral blood leucocyte suspensions were also prepared by collecting approximately 2 litres of blood into 2 x 10" iu heparin sodium (Evans Medical). The blood was sedimented at 1200 g for 30 minutes, the plasma removed and the 'buffy coat' resuspended in 100 ml Iscove's medium containing 2 per cent FBS. Either the whole 100 ml was inoculated into a bovine calf or it was divided equally between the calf and a deer. Inocula derived from rabbits and hamsters consisted of suspensions prepared from mesenteric lymph nodes and, or, spleen except with the first 46 passages of 01 I in rabbits where a variety of tissues was used. Subsequent passes of Oil were generally Transmission made by inoculating two rabbits so that one received From cattle. Transmission attempts were made lOB and the other 106 mesenteric lymph node cells. from a total of 16 cases of MCF referred through the Inocula prepared as above and which had been stored veterinary investigation service of the United, in medium supplemented with 20 per cent FBS and 10 Kingdom and only those cases that were positively per cent dimethylsulphoxide at - 80°C in a mechdiagnosed as MCF have been included in the analysis. anical freezer or in liquid nitrogen were also used. Diagnosis was based on the history, clinical signs and the detection of the characteristic gross and histoInjection. For the first 46 passages of Oil in rabbits pathological changes (Berkman et al 1960, Selman et between 5 and 20 ml of approximately 10 per cent al 1974, 1978, Liggitt et al 1978, Liggitt and tissue suspensions were injected intraperitoneally DeMartini 1980). On each occasion that transmission while later passages were made by intravenous injecwas attempted one bovine calf and two rabbits were tion of suspensions of lymph node cells. Hamsters inoculated and on nine occasions a red deer was also were always inoculated intraperitoneally, while indiinjected. vidual cattle and deer were inoculated both by the intravenous and intraperitoneal routes, the latter after From deer. The 'isolate' of MCF obtained from a sedation with xylazine (Rompun; Bayer). red deer and transmitted to rabbits (Buxton and Reid 1980) which was used both for serial transfer through rabbits and to challenge some deer and cattle was Results designated OIl. An additional isolate, 012, was Transmissionfrom cattle derived from a red deer that was spontaneously affected at the institute. It was one of two deer Transmission of bovine MCF to cattle was obtained before the initiation of this work and had attempted from 16 cases but on only two occasions been housed at the institute for nine months in a loose was the outcome successful and in only one of the two box adjacent to other boxes containing sheep. cases was an additional sub passage achieved (Fig I). None of the nine deer inoculated with bovine cells reacted but on two occasions disease was transmitted Inocula from calves to rabbits. In the first instance (C/I) Preparation. Inocula were prepared from selected disease was transmitted only to the calf and no further tissues of affected animals by chopping finely with transmissions were achieved. In the second instance scissors and dispersing cells into Iscove's medium MCF was transmitted from a field case (C/3) to a calf (Difco) supplemented with 2 per cent fetal bovine from which one further passage was made. However serum (FBS) by means of a stomacher 400 (Seward disease was not transmitted thereafter. While the Laboratory). Cell suspensions were either pipetted original material and lymph node cell suspensions directly from the tissue homogenate or passed from the second serially affected calf failed to through a muslin filter to remove gross fragments, transmit disease to rabbits, one of two rabbits sedimented by centrifugation at 500 g and resus- inoculated with 8 x lOS cells from the first experipended in Iscove's medium with 2 per cent FBS. Deer mental calf reacted on day 17. The other successful and cattle cells were prepared from pools of mesen- transmission from a field case (CI2) was to two teric, mediastinal and carcase lymph nodes and final rabbits which reacted on days 13 and 14 respectively suspensions for inoculation contained between 4 x 107 after inoculation with 109 lymph node cells. A third and I· 5 X 108 cells ml '. Recipient cattle and deer rabbit inoculated with 5 x lOS cells that had been were inoculated with between 50 and 150 ml of stored at - 80°C for 91 days from the same case

78

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(lJll)lI

~

--/<1'\ /\ V~ v ,:w-~

H. W. Reid, D. Buxton, I. Pow, J. Finlayson

I \\ / \ 1\\ Vcf)o~V V U V (5/13)

~V V (17/NR)

(49)

4j) (24/45)

(12/15)

FIG 1: Attempted transmission of MCFfrom three naturally occurring bovine cases of MCFto bovine calves, red deer, rabbits and hamsters. Shaded animals represent those that reacted and the figures in brackets indicate the day of reaction. NR No reaction

reacted, while a fourth rabbit given 108 of these stored cells did not. Cattle and deer inoculated with 1010 of the fresh cells did not react. Both C/2 and C/3 were readily passaged to other rabbits by injecting intravenously 108 to 1()6 lymph node cells from reacting rabbits. Each isolate was passaged in series three times and all rabbits developed typical MCF five to 23 days after inoculation with modes of 13 and 12 days for C/2 and C/3 respectively. However, a red deer and a calf inoculated with 2' 8 X 109 cells from a pool of lymph node cells from four rabbits reacting with C/2 did not react while two of 23 one-day-old hamsters each inoculated with 1 x 107 cells from a rabbit with MCF induced by C/2 reacted on days 24 and 45 respectively after inoculation, All of 14 one-day-old hamsters each inoculated intraperitoneally with 4· 5 x 1()4 lymph node cells derived from the hamster that reacted on day 45 developed typical signs of MCF, 12 to 47 days following inoculation. A further four passages of C/2 were

made by intraperitoneal inoculation of three-weekold hamsters, all of which reacted 12 to 42 days (mean 25 days) following inoculation.

Transmissionfrom deer All six red deer injected with lymph node cells obtained from affected deer reacted after an interval of 16 to 42 days and in addition a single roe deer given infected red deer cells also developed typical MCF on day 16 after inoculation. These transmissions were achieved with cells that had been derived from animals that were infected with either D/l or D/2 and were successful when as few as lOS cells were inoculated. Both fresh and stored cells transmitted disease. None of the six bovine calves injected with between 2 x lOS and 1 x 1010 cells from affected red deer reacted. The two rabbits inoculated with tissues from the spontaneous case of MCF at the institute reacted on days 11 and 12 respectively and their tissues, desig-

Malignant catarrhal fever transmission

79

TABLE 1: Day of onsat of clinical disease in 154 rabbits given aithar 106 or 108 mesenteric lymph node cells from affected rabbits representing the 60th to 125th serial passage of the 0/1 isolate of MCF Number of cells inoculated

10B

Total

10&

Day of reaction"

0 0 0

2

3

4

5

6

7

8

9

10

11

12

13

14

>15

AT

3t 0 3

4 0 4

3 0 3

1 0 1

0 0 0

0 1 1

0 1 1

3 1 4

6 0 6

16 11 27

17 11 29

9 21 30

1 13 14

6 12 18

7 4 11

NR Total

1 2 3

n n

154

AT Atypical reaction

NR No reaction " Day when rectal temperature first >40·0 o C t Number of rabbits reacting on a given day

nated the DI2 isolate, were serially passaged through other rabbits. Tissues from all nine experimental red deer were inoculated into rabbits and caused disease. From one deer suspensions of lymph node cells containing 108 , 107 , 106 or lOS ml : I were prepared and I ml aliquots of each inoculated into single rabbits. All except the lOS cell dose caused typical MCF. Three bovine calves inoculated with tissues from affected red deer failed to react as did another five bovine calves inoculated with tissues from rabbits reacting with D/1. Tissue suspensions from rabbits reacting with MCF derived from deer were inoculated into single red deer on 12 occasions and three developed MCF. Two reacted 41 and 42 days respectively following inoculation with DI2 and one reacted on day 99 following inoculation with D/I. One uninoculated red deer developed MCF 78 days after it had been in contact with a deer reacting with experimentally induced disease. The roe deer inoculated with tissue from a rabbit infected with DI2 developed MCF 19 days after inoculation.

Transmission ojD/1 through rabbits Rabbits reacted at all passages between I and 125 with typical signs of MCF which consisted of an abrupt rise in temperature coinciding with the cessation of drinking and eating (Buxton and Reid 1980). During the subsequent days catarrhal nasal discharges developed and diarrhoea, either profuse and fluid or thick and mucoid, was frequently present. Of the 366 rabbits inoculated 12 failed to react but all were shown to be susceptible when rechallenged. In addition 13 rabbits reacted atypically. They did not develop a febrile reaction although other clinical signs including profuse diarrhoea were present. All rabbits were confirmed as having MCF by histological examination. All rabbits were killed on day I or 2 of reaction except for 56 which died. Eight of these had shown no clinical signs while the others died between one and three days after the onset of clinical disease. The interval between inoculation and reaction ranged

from two to 33 days and was bimodal with modes of three and II days respectively. The results of 77 pairs of rabbits that received either 106 or lOS cells in the course of passaging D/I are shown in Table I. The time to reaction tended to be earlier in those that received 108 cells and while most rabbits which received this dose reacted at nine days or later there was a distinct sub population (14 per cent) that reacted on days 2 to 5. This bimodal response was less evident in the rabbits given 106 cells but three did react on or before day 9 while the remainder reacted on or after day II. Discussion Inconsistent success in the transmission of sheepassociated MCF from cattle to cattle is a feature of most previously reported attempts (Gotze 1932, Magnusson 1940, Duncan and Pearson 1956, Pearson 1956, Roderick 1959, Blood et al 1961, Horner et al 1975, Pierson et al 1979). In contrast Selman et al (1978) serially passaged the disease 10 times in cattle before the isolate was lost. On initial isolation from their field case three of five calves reacted and subsequent subpassages resulted in consistent reactions until the 10th which failed to transmit. However they stated that '-certainly, we have experienced failures before' implying that in their investigations transmission from field cases was not regularly achieved. In addition, attempts by numerous investigators to transmit the sheepassociated agent from cattle to rabbits have failed or proved inconclusive (Magnusson 1940, Duncan and Pearson 1956, Roderick 1959, Blood et al 1961, Pierson et al 1974, 1979, Horner et al 1975, Straver and van Bekkum 1979). Pattison (1946) reported transmission of MCF to rabbits in Palestine although the accompanying pathological description does not support his assertion. Daubney (1959), in a brief letter, claimed to have transmitted the disease to rabbits from cattle in Syria and Lebanon but presented no supportive evidence. Likewise, Kock and Neitz (1950), working in South Africa, mention the transmission of snotsiekte to rabbits from cattle

80

H. W. Reid, D. Buxton, I. Pow, J. Finlayson

that developed the disease following contact with sheep but give no further information. The present report therefore represents the first substantive evidence of transmission of sheep-associated MCF from cattle to laboratory animals. Of the 16 attempts to transmit MCF from cattle only three were successful. In one case (C/l), disease was transmitted to a bovine calf only and further passage was not achieved while in another (CI3) disease was transmitted to a calf and it then proved possible to subpassage infection to another calf and to a rabbit although rabbits that received inocula from the original field case failed to react. Transmission from a third case (CI2) was successful in rabbits while a calf and a deer that each received lO-fold more cells failed to react as did a deer and a calf injected with cells from the reacting rabbits. Following initial isolation both CI2 and C/3 could be readily passaged in series through rabbits and in addition CI2 was passed to two of 23 Syrian hamsters and subsequently could be passaged readily in this species. In these studies the irregular transmission of MCF from field cases to cattle is consistent with previous reports while transmission of disease to laboratory animals provides a novel tool for future studies of the disease. In accordance with previous reports disease was readily transmitted from affected red deer to other red deer (Oliver et al 1983) and rabbits (Buxton and Reid 1980) as well as to a single roe deer but could not be passaged from deer to cattle. Transmission of disease with tissues from rabbits reacting with D/l or DI2 to red deer was achieved on only three of 12 occasions after relatively long incubation periods, while the single roe deer reacted after only 19 days suggesting that this species may be highly susceptible to infection. Neither of these rabbit-passaged isolates from deer produced disease when inoculated into cattle. One red deer developed MCF 78 days after being in contact with a deer reacting with the disease, which accords with observations of natural disease outbreaks in Australia (Denholm and Westbury 1982) and New Zealand (McAllum et al 1982) where it is suggested that some lateral transmission of disease in deer herds may have occurred. However, on seven other occasions that deer reacted following inoculation in-contact animals remained unaffected. Thus, contagion would not appear to be a usual form of transmission. Transmission of the D/l isolate through 125 serial rabbit passages produced no evidence of adaptation to rabbits: the incubation period, clinical signs and pathological changes remained essentially unchanged. However, the clinical reaction tended to be more regular when standard doses of lymph node cell suspensions were administered by the intravenous route and a bimodality to the reaction time became

apparent, suggesting that the pathogenesis of the disease in these two populations could be distinct. It has been proposed that MCF represents a profound dysfunction of large granular lymphocytes that give rise to both lymphoid hyperplasia and autoimmune tissue destruction (Reid et al 1983, 1985, Reid and Buxton 1984). It is therefore possible that with the early reactors the cells in the inoculum may themselves be the effectors of the disease while in those animals reacting later the agent was transferred to the recipient's cells. Relatedness of the donor and recipient did not apparently influence the outcome as early reactors occurred in both New Zealand White and Lop rabbits irrespective of the breed of the donor. The relatively high infectivity of isolates D/l and DI2 for both rabbits and other deer but not for cattle is difficult to explain if the sheep-associated agent of MCF in cattle and deer is the same. However, the epidemiology of MCF in both deer (Reid et al 1979, Denholm and Westbury 1982) and cattle (Plowright 1968, Selman et al 1974) strongly implicates sheep as the source of infection and therefore at present it would appear likely that only one agent is responsible for the disease in both species. Furthermore, in these studies assessment of transmission relied on the detection of gross and microscopic lesions characteristic of MCF (Buxton and Reid 1980, Buxton et aI1984). Although slight quantitative variations in the lesion profiles produced by the different isolates from deer and cattle in rabbits and hamsters were detected the fundamental changes produced by them and the herpesvirus of wildebeest appeared to be the same (D. Buxton et ai, unpublished data). The pathogenesis of MCF is thus likely to be similar whether produced by the herpesvirus of wildebeest or the sheep-associated agent. The transmissibility of the sheep-associated agent from affected animals clearly varies both between species and between different individuals of that species. That the interval between collecting material from the donor animal to inoculation of the recipient should be minimal has been suggested to be an important factor when transmission from cattle has been achieved (Blood et al 1961, Selman et al 1978) and in the present studies this was invariably ensured. Selman et al (1978) also suggested that transmission from cattle may be achieved more readily if it is performed early in the course of clinical reaction but this was not apparent in these studies. Furthermore, Oliver et al (1983) could transmit disease from both chronically and acutely affected red deer. To explain the absence of viral particles in cattle infected with the wildebeest herpesvirus Hunt and Billups (1979) have suggested that viral DNA is either incorporated into the cellular genome or is present in an episomal form. The absence of detectable virus in

Malignant catarrhal fever transmission cattle with sheep-associated MCF may also be explained on this basis and it is tempting to suggest that transmissibility may be related to the extent of integration with the host cell genome, those animals from which transmission is achieved having more episomal viral genome. Lateral transmission in deer suggests that at least a limited amount of complete virus replication occurs in this species and this may explain the relative ease with which infection may be transmitted from them, although it does not explain why disease was not transmitted from deer to cattle. However, it is possible that the agent primarily infects a specific subpopulation of cells and that for successful transmission interaction between a similar subset of cells in the recipient animal is required. This latter suggestion would be in accord with the proposal that MCF arises from a dysfunction of large granular lymphocytes which represent an important target cell for the agent (Reid et al1983, 1985, Reid and Buxton 1984). Acknowledgements We are most grateful to members of Scottish, English and Welsh veterinary investigation services and to the veterinary practitioners without whose assistance in identifying clinical cases this work could not have been performed. References BERKMAN, R. N., BARNER, R. D., MORRILL, C C & LANGHAM. R. F. (1960) American Journal of Veterinary Research 21,1015-1027 BLOOD, D. C, ROWSELL, H. C & SAVAN, M. (1961) Canadian Veterinary Journal 2, 312-325 BUXTON, D. & REID, H. w. (1980) Veterinary Record 106, 243-245 BUXTON, D., REID, H. W., FINLAYSON, J. & POW, I. (1984) Research in Veterinary Science 36, 205-211 DAUBNEY, R. (1959) Veterinary Record 71,493 DENHOLM, L. J. & WESTBURY, H. A. (1982) Australian Veterinary Journal 58, 81-87 DUNCAN, D. W. & PEARSON, I. G. (1956) Australian Veterinary . Journal 32, 156-161

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GOTZE, R. (1932) Berliner Tierarztliche Wochenschrift 53.848-855 HORNER, G. W., OLIVER, R. E. & HUNTER, R. (1975) New Zealand Veterinary Journal 23, 35-38 HUNT, R. D. & BILLUPS, L. H. (1979) Comparative Immunology, Microbiology and Infectious Diseases 2, 275-283 KOCK DE G. & NEITZ, W. O. (1950) South African Journal of Science 46, 176-180 LIGGITT, H. D. & DEMARTINI, J. C (1980) Veterinary Pathology 17, 73-83 LIGGITT, H. D., DEMARTINI, J. C, McCHESNEY, A. E., PIERSON, R. E. & STORZ, J. (1978) American Journal of Veterinary Research 39, 1249-1257 McALLUM, H. J. F., MAVOR, N. M., HEMMINGSEN, P. (1982) New Zealand Veterinary Journal 30, 99-101 MAGNUSSON, H. (1940) Veterinary Bulletin (1942) 12,213-214 OLIVER, R. E., BEATSON, N. S., CATHCART, A. & POOLE, W. S. (1983) New Zealand Veterinary Journal 31, 209-212 OLIVER, R. E., BEATSON, N. S., CATHCART, A. & POOLE, w. S. (1985) Biology of Deer Production. Eds P. F. Fennessy, K. R. Drew. Royal Society of New Zealand Bulletin 22, 143-146 PATTISON, I. H. (1946) Journal of Comparative Pathology 56, 254-265 PEARSON, I. G. (1956) Australian Veterinary Journal 32, 77-88 PIERSON, R. E., HAMDY, F. M., DARDIRI, A. H., FERRIS, D. H. & SCHLOER, G. M. (1979) American Journal of Veterinary Research 40, 1091-1095 PIERSON, R. E., STORZ, J., McCHESNEY, A. E. & THAKE, D. (1974) American Journal of Veterinary Research 35,523-525 PLOWRIGHT, W. (1968) Journal of the American Veterinary Medical Association 152,795-804 REID, H. W. & BUXTON, D. (1984) Proceedings of the Royal Society of Edinburgh 828, 261-293 REID, H. W., BUXTON, D., BERRIE, E., POW, I. & FINLAYSON, J. (1985) Deer Production. Eds P. F. Fennessy, K. R. Drew. Royal Society of New Zealand Bulletin 22,139-142 REID, H. W., BUXTON, D., CORRIGALL, W., HUNTER, A. R., McMARTIN, D. A. & RUSHTON, R. (1979) Veterinary Record 104, 120-123 REID, H. W., BUXTON, D., POW, I., FINLAYSON, J. & BERRIE, E. L. (1983) Research in Veterinary Science 34, 109-113 RODERICK, L. M. (1959) Veterinary Medicine 54,509-532 SELMAN, I. E., WISEMAN, A., MURRAY, M. & WRIGHT, N. G. (1974) Veterinary Record 94,483-490 SELMAN, I. E., WISEMAN, A., WRIGHT, N. G. & MURRAY, M. (1978) Veterinary Record 102, 252-257 STRAVER, P. J. & VAN BEKKUM, J. G. (1979) Research in Veterinary Science 26,165-171 WESTBURY, H. A. & DENHOLM, L. J. (1982) Australian Veterinary Journal 58, 88-92

Accepted September 19, /985