Response of sheep to experimental concurrent infection with tick-borne fever (Cytoecetes phagocytophila) and louping-ill virus

Research in Veterinary Science /986, 41, 56-62

Response of sheep to experimental concurrent infection with tick-borne fever (Cytoecetes phagocytophila) and louping-lll virus H. W. REID, D. BUXTON, I. POW, Moredun Research Institute, 408 Gilmerton Road, Edinburgh, EH17 7JH, T. A. BRODIE, P. H. HOLMES, G. M. URQUHART, Glasgow University Veterinary School, Bearsden Road, Glasgow, G61 lQH

The pathogenesis of concurrent Cytoecetes phagocytophila and lou ping-ill virus infection was studied in two experiments. In the first experiment 18 four- to seven-year-old rams were used. Ten were infected with C phagocytophila and five days later eight of these animals and the remaining eight sheep were infected with lou ping-ill virus. The two rams infected with C phagocytophila alone developed no clinical signs apart from a transient pyrexia, while only three of the eight rams infected with lou ping-ill virus alone showed mild clinical signs. In marked contrast, all eight dually infected sheep developed severe clinical signs with pronounced depression and dysentery and three died and five were killed in extremis. They developed higher titres of viraemia and the antibody response was depressed while necrotising lesions affecting a variety of organs were detected at post mortem examination. Rhizomucor pucillus was recovered from these lesions in seven of the eight sheep. A second experiment using 10 sheep, five aged seven months and five aged two to three years, confirmed th'e findings of the first experiment indicating that the age of the animal had not significantly influenced the initial result. It was concluded that C phagocytophila infection could enhance the pathogenicity of louping-lll virus and that, operating together, the two pathogens facilitated fungal invasion. It is postulated that sudden deaths in sheep recently transferred to tick-infested pastures may be due to this newly described syndrome.

(Zlotnik et al 1970, Doherty and Reid 1971a,b) did not support this hypothesis. However, increased susceptibility to lou ping-ill virus has been demonstrated in mice concurrently infected with Trypanosoma brucei (Reid et al 1979) and in mice and lambs infected with Toxoplasma gondii (Buxton et a11980, Reid et al 1982) and attributed to the immunosuppressive effects of these protozoa. Since TBF infection of sheep has been shown to depress immune responses (Batungbacal and Scott 1982a, b, Gilmour et al 1982), the increase in susceptibility to lou ping-ill virus of sheep with concurrent TBF could arise from a suboptimal immune response. The present study was designed to examine the interaction between TBF and louping-i11 virus following experimental infection of sheep.

WORKERS responsible for the initial isolation of lou ping-ill virus experienced difficulty in reproducing the disease in sheep by parenteral inoculation of virus. Limited evidence suggested that sheep infected with Cytoecetes phagocytophila, the cause of tick-borne fever (TBF), regularly developed typical signs of lou ping-ill and it was proposed that TBF facilitated invasion of the central nervous system by lou ping-ill virus (Macleod and Gordon 1932). Subsequent studies which indicated that virus invasion of the central nervous system following peripheral inoculation with lou ping-ill virus invariably occurred

In experiment 1, 10 of the rams were inoculated intravenously with 2 ml of a whole blood stabilate of TBF which had been stored in liquid nitrogen with a cryoprotectant (12' 5 per cent glycerol). The strain of TBF had been isolated originally from a sheep in Argyll. Five days later eight of these rams, together with the remaining eight, were inoculated subcutaneously with 106 " plaque forming units (pfu) of the SB526 isolate of louping-ill virus prepared from infected suckling mouse brains, as previously described (Reid and Doherty 1971a). The remaining two TBF infected

Materials and methods

Experimental animals In experiment 1, 18 four- to seven-year-old Cheviot or Cheviot cross rams which had been reared in an environment free of Ixodes ricinus, the vector of both TBF and louping-i11 virus, were employed. Five two- to three-year-old rams and five seven-month-old Cheviot lambs which had been similarly reared were used in experiment 2.

Experimental procedure

56

Concurrent

TBF

and louping-ill infection

rams were similarly injected with a clarified suspension of normal brain. In the second experiment, all 10 sheep were inoculated intravenously with the same dose and strain of TBF as the animals in experiment 1, five days before infecting all of the sheep subcutaneously with 106 ' 4 pfu of the SB526 isolate of the virus. In both experiments animals were observed at approximately 12 hourly intervals and rectal temperatures were recorded daily. Animals which died or became severely affected were necropsied and survivors killed on day 21. At post mortem examination, portions of approximately I ern! of cerebrum, cerebellum, brain-stem, representative lymph nodes and spleen were collected and stored at - 80°C until homogenised and tested for virus as previously described (Reid et aI1982). When it became apparent following histological examination that systemic mycosis had affected a number of animals, samples of selected organs were cultured on Sabauraud's medium (Oxoid). Small fragments of tissue were aspirated into a pasteur pipette and embedded in the agar plates which were incubated aerobically at 37°C and examined daily for evidence of fungal growth. The remainder of the brain together with other organ samples were placed in 10 per cent Baker's formol saline for histological examination. Blood was collected daily in experiment I for virus and antibody assays (Reid and Doherty 1971b) and for the preparation of blood films. Virus titres were determined using a plaque method and antibody was assayed by a haemagglutination inhibition test (in). Blood films were stained with Giemsa to assess the intensity of the TBF parasitaemia. To assess the severity of histopathological changes in the brain, coronal slices through the anterior pole of cerebrum, corpus striatum, thalamus, hippocampus, midbrain (anterior end of the red nucleus and another at the posterior end of the red nucleus), cerebellar peduncles and three levels of medulla were taken together with a sagittal section through the cerebellar vermis. These were then processed in paraffin wax and sections 6 /Am thick were cut and stained with haematoxylin and eosin and selected sections were stained by the Grocott-Gomori methenamine silver method or by the periodic acid Schiff technique (Bancroft and Stevens 1977).

Results CLINICAL OBSERVA TlONS

Experiment 1 The two rams infected with TBF alone showed no obvious clinical signs apart from a raised rectal temperature while three of the eight rams infected with

57

lou ping-ill virus alone showed only transient clinical signs and made uneventful recoveries. In contrast, all eight dually infected animals developed clinical signs and three died and five were killed in extremis. First signs were observed on day 6 when two of these animals appeared depressed, and by day 7 six were depressed and four ataxic; two of the latter became comatose and were killed. Over the next two days, the remaining six animals having developed severe dysentery, trembling and ataxia, died or became recumbent and were killed.

Experiment 2 Nine of the 10 dually infected animals died or were killed in extremis on days 5 to II. Three had developed dysentery or profuse diarrhoea before death and six showed signs of ataxia or tremor while one died suddenly having shown no obvious clinical signs. The surviving ram had mucoid diarrhoea on day 8 and thereafter made an uneventful recovery and was killed on day 21.

GROSS PATHOLOGY

Experiment I No gross lesions were observed in any of the animals given either TBF or lou ping-ill virus alone. In contrast, all dually infected tups showed marked congestion of the meningeal vessels. Haemorrhage into the intestinal lumen was apparent in six cases and varied from mild leakage, with associated petechiation of the mucous membranes, to very severe with blood clots 10 mm in diameter in the ileum and up to 50 mm in diameter in the large bowel. In half the necropsies, haemorrhagic necrosis was present in the retropharyngeal, prescapular, popliteal and mediastinal lymph nodes. Infarcts were found in the livers or kidneys of three animals and cardiac petechiation was also present in three cases. In the abomasal mucosa of one there were striking rings of haemorrhage up to 15 mm in diameter with pale centres.

Experiment 2 Marked lesions were present in seven of the nine animals that died or were killed in extremis. Haemorrhage of the small and large intestine were present in two and enlarged lymph nodes, frequently oedematous and haemorrhagic, were seen in six; the retropharyngeal node was most frequently involved. Infarcts of the liver and kidney were seen in three and four of the sheep respectively. In most sheep, the meningeal blood vessels appeared congested.

58

H. W. Reid, D. Buxton, I. Pow, T. A. Brodie, P. H. Holmes, G. M. Urquhart

MICROSCOPIC PATHOLOGY

Experiment 1 In the sheep given TBF only, a few large lymphoid cells were present in the cerebral and cerebellar meninges with mild scattered accumulations around blood vessels. No other abnormalities were found. In the sheep which received lou ping-ill virus alone mild to moderate non-suppurative meningoencephalitis was present in seven brains with mild accumulations of lymphoid cells in the cerebral and cerebellar meninges. Lymphoid vascular cuffing was present in the cerebrum, cerebellum and brainstem of all these animals. Focal gliosis, although mild, was present in the cerebral white matter, thalamus, midbrain, cerebellar white matter, pons and medulla. Neurone necrosis was observed only in four animals and was confined to the pons and medulla and neuronophagia was seen in only one brain. The remaining tup in the group had only very mild encephalitis composed of a few perivascular cuffs in the medulla. Pathological changes in the group injected with TBF and louping-ill virus were more complex with lesions also being present in non-neural tissues. Five showed neuropathological changes characteristic of lou ping-ill virus infection to varying degrees, ie, a non-suppurative meningoencephalitis with lymphoid meningitis and perivascular cuffing, with focal gliosis, neurone necrosis and neuronophagia in the brainstem. The other three brains had, in addition to changes characteristic of louping-ill virus infection, inflammatory exudate composed primarily of polymorphonuclear cells and, in one case, infarction of meningeal blood vessels in the forebrain and malacia in the adjacent cortex and white matter. Special staining revealed in this area heavy infiltrations of non-septate branching fungal hyphae within infarcted blood vessels, vessel walls and meninges. In seven dually infected tups there was a mild, focal, interstitial lymphoid nephritis. In two of these, infarction had occurred and the resulting 'cone' of dead tissue was heavily infiltrated by non-septate branched fungal hyphae. There was a clear line of demarcation between the infarct and normal tissue, with the latter not infiltrated by fungal hyphae (Fig I). Fungi were not found in the liver but in six animals there were mild periportal accumulations of lymphoid cells and, in one, thrombosis and focal necrosis. Morphologically similar hyphae associated with severe necrosis were present in various lymph nodes of four of the rams although haemorrhages were present in seven. Pulmonary oedema and congestion were present in seven cases with purulent exudative foci of pneumonia in two. Histological changes in the gastrointestinal tract were not very marked. The abomasal

lesions noted macroscopically in one sheep also showed non-septate branching fungal hyphae extending into the lamina propria and adjacent blood vessels. In all, non-septate branching fungi were demonstrated histologically in five animals of this group although changes characteristic of fungal infection were found in seven.

Experiment 2 The neuropathological changes observed were not typical of lou ping-ill meningoencephalitis. In the first animals to succumb inflammatory changes were not present but there was widespread vascular haemorrhage and extensive areas of necrosis of lymph nodes, lung and liver in which many bacteria were observed. Of the remaining animals all had a severe nonsuppurative meningoencephalitis with vascular cuffing by large lymphoblast-like cells which was particularly severe in the cerebral cortex. In addition, macrophages containing necrotic debris were present in the cuffs. Five of the sheep to succumb had necrotic lesions in organs other than the brain and these were seen to be associated with fungal invasion. No lesions could be detected in the surviving ram that was killed on day 21. MICROBIOLOGY

Experiment 1 Louping-i11 virus was isolated from the plasma of all but one (13) of the tups given the virus. Evidence of infection in this animal relied on the detection of serum HI antibody from day 6 and the presence of mild neuropathological changes at necropsy. In the remainder, viraemia was detected from day 1 and rose rapidly in titre through the ensuing three days (Fig 2). Compared to animals infected with louping-ill virus alone, the viraemia in dually infected animals was greater and more persistent, maximum titres in this group ranging from 1()6'19 to 108 ' 53 pfu per 0'2 ml of plasma compared to 10°'7 to 1()6'19 in the group infected with louping-ill virus alone. Viraemia in the dually infected group persisted until day 7 in seven of the animals while in the group infected with virus alone, virus was present in only five on day 4 and persisted until day 5 in only one. Virus was recovered from the brains of all the dually infected animals (Table I) but from none of the animals infected with the virus alone that were killed on day 21. Virus was also present in most of the lymph node and spleen specimens taken from the dually infected animals. From tissues of seven of the eight dually infected animals cultured on Sabauraud's agar numerous white colonies with extensive aerial

Concurrent

TBF

and louping-ill infection

59

FIG 1: Dark staining fungal hyphae within a renal infarct from a sheep infected with TBFand louping-ill virus. Grocott-Gomori methenamine silver. a I x 145) and b I x 680)

mycelial growth were recovered. These moulds were subcultured and identified by Dr L. Milne of the Western General Hospital, Edinburgh as Rhizomucor pucillus. HI antibodies were not detected in any of these sheep before day 6 when all eight of those infected with virus alone were positive (Table 2). Thereafter titres in this group rose rapidly with the exception of two animals in which viraemia was low or not detected. In contrast, in the dually infected group,

antibody was detected in only four of the eight on day 7 and, even by day 8, one of the surviving animals had failed to seroconvert. On day 0 (day 5 following infection with TBF), seven of the eight dually infected rams and both TBF control animals had patent TBF infections and by the following day the eighth ram was also parasitaemic. The percentage of parasite-infected neutrophils in both of the above groups ranged from 20 per cent to 60 per cent (mean 39 per cent) on day 0 and rickettsial bodies

H. W. Reid, D. Buxton, I. Pow, T. A. Brodie, P. H. Holmes, G. M. Urquhart

60 8

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23456789 Day after inoculation

FIG 2: Mean l± SE) titre of louping-ill virus in the plasma of sheep given louping-ill virus alone ( - - ) or virus five days following inoculation with TBF (- - -I. Group means compared using the Student's t test and P values expressed as NS= >0,05, "= <0,05, "" = <0·01 and""" = <0'001

were visible in neutrophils for four to seven days (mean 4· 8 days) after TBF infection.

Experiment 2 On day 0 all 10 animals had patent TBF infections, the percentage of infected neutrophils ranging from 37 per cent to 68 per cent (mean 58 per cent). Virological studies were not conducted in this experiment. Discussion The response of the sheep to infection with lou pingill virus alone was variable (Pool et a1I930, Reid and Doherty 197Ib), thus the mild reaction of the group of eight rams given virus alone was not exceptional; the absence of clinical signs in the rams infected only with TBF was also in accordance with the benign nature of this disease (Woldehiwet 1983). In contrast, in two separate experiments, sheep infected with TBF

before exposure to louping-ill virus experienced a very much more severe reaction from which 17 out of 18 died or were killed in extremis. In the first experiment old male sheep were used and the second experiment was therefore performed to compare the response of mature animals and seven-month-old lambs. Both categories of animal proved equally susceptible, indicating that the advanced age of the animals employed in the first experiment did not influence the result. The mortality that occurred in the dually infected group was, however, not entirely due to an increased susceptibility to louping-ill virus but was also apparently associated with a profound depression of the animals' normal defence mechanisms. In a previous study of the effect of toxoplasmosis on lou ping-ill virus infection in mice and sheep (Buxton et a11980, Reid et a11980, 1982) the mortality in dually infected animals compared to those infected with lou ping-ill virus alone was considerably greater. However, the clinical course was extended and the deaths occurred later. This pattern of delayed but increased mortality has been observed in numerous studies of virus encephalitis in immunosuppressed hosts (eg, Camenga et al 1974, Semenov et al 1975, Bhatt and Jacoby 1976); it was concluded that the toxoplasma infection exerted on immunosuppressive effect which enhanced the pathogenicity of lou ping-ill virus. In contrast, in the present study deaths in dually infected animals commenced on day 6 and continued to day II, with most dying earlier than would be expected for animals given virus alone (Reid and Doherty 1971b). However, the viraemias in the dually infected lambs were markedly greater and more prolonged while the HI antibody response either did not develop or was delayed, both features suggesting an immunosuppressed response to lou ping-ill virus. In addition, high titres of virus were found in nonneuronal tissues, whereas virus is normally restricted to the central nervous system in sheep that succumb to infection with lou ping-ill virus alone (Reid et aI1982). Elimination of virus from plasma and tissues is

TABLE 1: Isolation of louplng-ill virus and R pucillus from tissues of sheep infected with TBFand 10uping-i11 virus

Sheep number

1 2 6 9 11 15 16 17

"

t

Day of death

Cerebrum

Cerebellum

Brainstem

Spleen

Prescapular lymph node

Mediastinal lymph node

8 9 7 7 8 9 8 9

5·7" -t 4'6t 3-9 5·5 4·8 3'3:1: 6·9

6'5'1= 2·3 5·2 4·7 6-4 6·5 5·2 6·7

6·8 4·1 5·7 4·4 6·7 6·2 4·8 7·0

5·2 -t 6·8

6'3:1: 3·2t 6·9t

6'2:1:

6'7:1: 3·5

6·7t 4'8t

5·0

6·7:1:

5·8t 4·0 4'4:1: 6·6t

Log 10 plaque forming units per 0·2 g of tissue Tissues from which R pucil/us was also recovered or :l:could not be recovered

3·6:1:

Concurrent

TBF

and louping-ill injection

TABLE 2: HI antibody response to louping-ill virus in sheep infected withvirus alone and with TBF and louping-i11 virus Sheep Treatment number 1 2 6

9

Li + TBF Li + TBF Li + TBF Li + TBF

11 15 16 17

Li + TBF Li + TBF Li + TBF Li + TBF

5 7 8 10 12 13 14 18

Li Li Li Li Li Li Li Li

Li

Louping-ill

< Titre of less than

6

7

< < < < < < < <

<

1110

Animal dead " Reciprocal of titre D

5

< < < < < < < < < < < < < < < <

160" 160 320 20 80 40 40 80

160 10 10

< <

320

<

9

8 D 2560 D D 320 320 1280

<

10

5120 D 1280 D D

2560 1280 1280 640 80

10,240 5120 5120 10,240 2560 2560 640 320 80 80

640 640

5120 10,240 1280 640

80

80

80

D

D

2560 2560 1280 640 80 40 5120 160

recorded

mediated through antibody to the virus (Reid and Doherty 1971 b, Reid et al 1971)so that the persistence of virus in extraneural tissue further supports the concept that the immune response of the TBF-infected animals to lou ping-ill virus was severely compromised. In accordance with previous studies of sheep infected with lou ping-ill virus alone (Doherty and Reid 197Ia,b), evidence that virus entered the central nervous system of all surviving animals was confirmed by the consistent presence of mild to moderate encephalitis detected in brains collected on day 21. The wider distribution of this lesion in the sheep infected with both TBF and louping-ill virus suggests that viral invasion of the central nervous system was augmented. It is probable that this reflected a greater intensity of viral replication in these animals, as indicated by the magnitude of the viraemia. However, the cause of the increased mortality in the dually infected sheep was not solely attributable to increased susceptibility to louping-ill virus, the lesions of which are restricted to the central nervous system. In both experiments the dually infected sheep developed dysentery and the majority had extensive haemorrhagic lesions in the intestine and, or, elsewhere. Histopathological examination revealed widespread necrotic'lesions and in some of these the presence of fungal hyphae was demonstrated. The isolation of R pucillus from the tissues of seven of the eight dually infected rams in experiment I suggests that this fungus was specifically involved. Although neutropenia and immunosuppression are known to be associated with TBF (reviewed by Scott 1984), the fact that this fatal syndrome occurred only

61

in the dually infected sheep indicates that its aetiology is dependent on concurrent infection with TBF and louping-ill, That there was no evidence of overwhelming bacterial infection implies either that the pertubation of the host's defences operates very locally at sites where R pucillus is present or that it selectively abrogates homeostatic mechanisms responsible for maintaining equilibrium with mycotic symbiots. An apparently similar syndrome has been described in British sheep on two previous occasions. Haernorrhagic enteritis was reported in sheep following experimental inoculation of TBF (Foster et al 1968) and intestinal mycosis was reported in a lamb (Angus et al 1971) also given TBF. In the light of the present findings, it is possible that on these two occasions blood used for the TBF infection also, incidentally, contained lou ping-ill virus. The probability of the syndrome described in this paper occurring naturally will depend on the duration of the enhanced susceptibility to lou ping-ill virus infection following exposure to TBF. In the current experiments lou ping-ill virus was given only five days after injection of TBF and thus, if this interval is critical, the syndrome may occur only infrequently. A further feature militating against the natural occurrence of the syndrome is that colostral antibody is very efficient at protecting lambs from louping-ill virus (Reid and Boyce 1976) but does not normally provide any protection from TBF. Thus the interval between lambs becoming infected with TBF and louping-ill is likely to be considerable. However, sheep introduced to tick-infested pasture for the first time are most likely to become infected with both agents together and the consequences of dual infection reported here could explain the very high mortality attributed to louping-ill virus in this category of animal. Acknowledgements

The financial assistance of the Wellcome Trust is gratefully acknowledged. One of the authors (T .A.B.) was supported by the Agricultural and Food Research Council. We are grateful to Dr L. Milne of the Western General Hospital, Edinburgh, for identification of the R pucillus. References

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62

H. W. Reid, D. Buxton, I. Pow, T. A. Brodie, P. H. Holmes, G. M. Urquhart

Comparative Pathology 92, 415-428 BUXTON, D., REID, H. W., FINLAYSON, J. & POW, I. (1980) Journal of Comparative Pathology 90, 331-338 BHATT, P. N. & JACOBY, R. O. (1976) Journal of Infectious Diseases 134, 166-173 CAMENGA, D. L., NATHANSON, N. & COLE, G. A. (1974) Journal of Infectious Diseases 130, 634-641 DOHERTY, P. C. & REID, H. W. (l97Ia) Journal of Comparative Pathology 81,331-337 DOHERTY, P. C. & REID, H. W. (197Ib) Journal of Comparative Pathology 81,531-537 FOSTER, W. N. M., FOGGIE, A. & NISBET, D. I. (1968) Journal of Comparative Pathology 78, 255-258 GILMOUR, N. J. L., BRODIE, T:A. & HOLMES, P. H. (1982) Veterinary Record 111,512 MACLEOD, J. & GORDON, W. S. (1932) Journal of Comparative Pathology and Therapeutics 45, 240-256 POOL, W. A., BROWNLEE, A. & WILSON, D. R. (1930)Journal of Comparative Pathology and Therapeutics 43,253-290 REID, H. W. & BOYCE, J. B. (1976) Journal of Hygiene 77, 349-354 REID, H. W. & DOHERTY, P. C. (l97Ia) Journal of Comparative

Pathology 81, 291-298 REID, H. W. & DOHERTY, P. C. (l97Ib) Journal of Comparative Pathology 81,521-529 REID, H. W., DOHERTY, P. C. & DAWSON, A. McL. (1971) Journal of Comparative Pathology 81, 537-543 REID, H. W., BUXTON, D., FINLA YSON, J. & HOLMES, P. H. (1979) Infection and Immunity 23, 192- I96 REID, H. W., BUXTON, D., POW, I. & FINLAYSON, J. (1980) Journal of Medical Microbiology 13, 313-318 REID, H. W., BUXTON, D., GARDINER, A. c., POW, I., FINLAYSON, J. & MACLEAN, M. J. (1982) Journal of Comparative Pathology 92,181-190 SCOTT, G. R. (1984) Veterinary Annual, 24th issue. Eds C. S. G. Grunsell, F. W. G. Hill. Bristol, Scientechnica. pp 100- 106 SEMENOV, B. F., VARGIN, V. V., ZOCHIESCHE, W. & VECKENSTEDT, A. (1975) Intervirology 5,220-224 WOLDEHIWET, Z. (1983) Veterinary Research Communications 6, 163-175 ZLOTNIK, I., CARTER, G. B. & GRANT, D. P. (1970) Veterinary Record 86, 659-660

Accepted July 4, 1985