Immunosuppression in toxoplasmosis: further studies on mice infected with louping-ill virus.

Mice were infected with an avirulent cyst-producing strain of Toxoplasma gondii and given injections of louping-ill virus 7 days later; control mice were given virus but not Toxoplasma. Test and control mice were then killed, in groups, 2, 4, 6, 8 and 10 days later. In the dually infected mice viraemia was later, greater and more prolonged; titres of virus recovered from brain and spleen were greater; production and haemagglutinating antibody to louping-ill virus was later and less, and inflammation in the brain was more severe, than in mice given virus alone. We suggest that T. gondii suppressed the immunity of mice, making them more susceptible to the virus, and that a significant proportion of the increased number of inflammatory cells observed in the brain could have been toxoplasma specific and not virus-specific and hence contributed to the increased susceptibility of the dually infected mice to louping-ill virus.

Immunoblotting analysis of the reaction of wildebeest, sheep and cattle sera with the structural antigens of alcelaphine herpesvirus-1 (malignant catarrhal fever virus).

Malignant catarrhal fever (MCF) is a disease of cattle and some other ruminants caused by alcelaphine herpesvirus-1 (AHV-1), a virus of wildebeest. The disease also occurs in the absence of wildebeest and is then thought to be caused by a viral agent harboured by the sheep. The structural proteins of AHV-1 have been used as antigens for the immunoblotting analysis of sera from wildebeest, sheep and cattle infected by either AHV-1 or the "sheep-associated" form of the disease. Wildebeest sera showed a uniform response reacting strongly with six polypeptides. Sheep sera also gave positive results but individual sera reacted with varying subsets of the antigens recognized by wildebeest. These results support the earlier suggestion that sheep harbour a herpesvirus related to AHV-1. A bovine serum from a case of MCF caused by AHV-1 also reacted only with a subset of the six wildebeest-reactive polypeptides. Sera from cattle affected with the "sheep-associated" form of the disease gave reactions in only two of the eight cases tested; both positive sera reacted to a few polypeptides only.

Transmission of wildebeest-associated and sheep-associated malignant catarrhal fever to hamsters, rats and guinea-pigs.

Wildebeest-derived malignant catarrhal fever (WD-MCF) was transmitted to hamsters, rats and guinea-pigs by inoculation of rabbit lymphoid cells infected with alcelaphine herpesvirus-1, strain C-500. Sheep-associated MCF (SA-MCF) was transmitted to hamsters by inoculation of lymphoid cells from rabbits affected with SA-MCF derived from deer. Mice were refractory to both forms of the disease. With both forms of MCF, the incubation period during initial transmission varied from 21 to 90 days and disease was readily passaged in rodents by inoculation of live lymphoid cells. Clinical signs in hamsters most closely resembled those described for naturally occurring MCF. Results given here and in two following papers indicate that rodents are useful models to study the aetiology and pathogenesis of both forms of MCF.

Antibody to alcelaphine herpesvirus-1 (AHV-1) in hamsters experimentally infected with AHV-1 and the 'sheep-associated' agent of malignant catarrhal fever.

Malignant catarrhal fever was induced in four groups of hamsters by the inoculation of cells infected with either the C/500 isolate of alcelaphine herpes-virus-1 (AHV-1) or the sheep-associated agent derived from cattle, red deer or Père David's deer. Using an indirect immunofluorescence assay, antibody to AHV-1 was detected in sera of clinically affected animals of all four groups. The reaction of sera from hamsters affected with malignant catarrhal fever induced by AHV-1 caused diffuse cytoplasmic staining while that from sera of hamsters with the sheep-associated form of the disease stained particulate nuclear antigens. Tests employing three other bovid herpesviruses were negative and no reaction was found with sera from normal hamsters. These studies provide convincing evidence that a virus antigenically related to AHV-1 is the cause of sheep-associated malignant catarrhal fever and that the same virus probably causes this form of the disease in both cattle and deer.

Transmission of a malignant catarrhal fever-like syndrome to sheep: preliminary experiments.

The transmission of a malignant catarrhal fever-like syndrome to sheep is reported. Fetal sheep between 40 and 66 days gestation were inoculated intravenously with viable cells either from a red deer with clinical malignant catarrhal fever or from rabbits with the disease. Of the 21 fetuses in the experiment only five were born live and of these four developed clinical signs similar to malignant catarrhal fever in other species and died or were killed 10, 16, 47 and 175 days after birth. The fifth lamb remained unaffected. Histology of the four affected lambs revealed a generalised lymphoproliferation, with T-dependent areas of lymphoid tissues being affected, and an overall paucity of immunoglobulin containing cells. In addition arteritis and interstitial infiltration of many organs by lymphoid cells was present. The infectious agent was not reisolated in rabbits from lambs or passed to red deer housed in the same pen and it is thus considered possible that gene expression of the putative virus was incomplete.

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

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 (Capreolus capreolus) 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.

Isolation and characterisation of lymphoblastoid cells from cattle and deer affected with 'sheep-associated' malignant catarrhal fever.

Cells with the histological and ultrastructural characteristics of large granular lymphocytes (LGL) have been obtained in culture from both cattle and red deer (Cervus elaphus) reacting with 'sheep-associated' malignant catarrhal fever (MCF). Such cells have been derived from thymus, lymph node and spleen suspensions as well as from cerebrospinal fluid cells and cultured cornea. On most occasions their presence was observed only transitorily but by providing the cells with feeder monolayers and, or, interleukin-2, several lines were maintained indefinitely, and some became independent of these factors after prolonged culture. A similar cell line was also derived from a Père David's deer affected with MCF at Whipsnade zoological park. Functionally, cultured LGL were cytotoxic to both primary cell cultures and cell lines and their cytotoxicity was not restricted to histocompatible target cells. These findings suggest that the cultured cells have natural killer cell-like activity and that they are important targets for the agent of MCF in cattle and deer. One cell line derived from a red deer transmitted the disease but none of the cells generated from cattle did.

Gamma herpesvirus carrier status of captive artiodactyls.

Between 1998 and 2000, 103 individuals of 19 species of the order Artiodactyla at Whipsnade Wild Animal Park were tested for evidence of infection with gamma herpesviruses in order to distinguish between species which are susceptible to malignant catarrhal fever (MCF), caused by alcelaphine herpesvirus-1 (AlHV-1) of wildebeest (Connochaetes sp.) or ovine herpesvirus-2 (OvHV-2) of domestic sheep, and species which carry related viruses sub-clinically. Gamma herpesvirus DNA was detected in the known, or suspected, carrier species: roan antelope (Hippotragus equinus), scimitar-horned oryx (Oryx dammah), gemsbok (Oryx gazella), musk ox (Ovibos muschatus) and mouflon (Ovis musimon). In six other species: lowland anoa (Bubalus depressicornis) yak (Bos grunniens), sitatunga (Tragelaphus spekei), greater kudu (Tragelaphus strepsiceros), waterbuck (Kobus ellipsiprymnus) and Nile lechwe (Kobus megaceros), DNA was present in some newborn calves and over 30% of adults, strongly suggesting a carrier state. In contrast five Père David's deer (Elaphurus davidianus) and two swamp deer (Cervus duvauceli) died of MCF during the study. A virus isolated from scimitar-horned oryx calves produced cytopathic effects in scimitar-horned oryx kidney cell-culture and caused MCF in a rabbit.

Pathogenesis of 'sheep-associated' malignant catarrhal fever in rabbits.

Pathogenesis studies of experimentally produced sheep-associated malignant catarrhal fever (MCF) in laboratory rabbits are described. Animals were examined at intervals after inoculation. The principal change was a proliferation of lymphoid cells which began as soon as three days and became quite pronounced by 13 days after inoculation. The appendix, mesenteric lymph node and spleen were most obviously affected. The reason for this was a progressive increase in T-lymphocytes, which appeared to be a hyperplasia rather than neoplasia in T-dependent areas of these organs. Lymphoid cells also accumulated in interstitial spaces of non-lymphoid organs. The use of cyclosporin-A suppressed the lymphoid proliferation but rabbits still developed clinical MCF after a similar incubation period. It is suggested that the agent of MCF might produce its effect by infecting and causing a dysfunction of lymphoregulatory cells, resulting in benign polyclonal T-lymphocyte proliferation. Terminal necrosis could be due to natural killer cell activity.

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

The pathogenesis of concurrent Cytoecetes phagocytophila and louping-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 louping-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 louping-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 the 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-ill 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.

Actions of bovine skin washings and sera on the motile zoospores of Dermatophilus congolensis.

Concentrated skin washings, even from vaccinated animals, failed to inhibit the motility of the infective zoospores of Dermatophilus congolensis, or to prevent them from germinating or infecting cattle; their constituent immunoglobulins did not attach to the flagella although IgA and IgG2 did bind to the cell bodies. It is concluded that the specific antibodies at the skin surface of ruminants are unlikely to have a role in zoospore immobilisation. Post vaccination sera rapidly immobilised and clumped the zoospores by means of a coat around the flagella, in which immunoglobulins, particularly IgM, were detected. IgM and IgG1 also attached to the cell bodies of the zoospores.

A cytotoxic T-lymphocyte line propagated from a rabbit infected with sheep associated malignant catarrhal fever.

The propagation of a cell line from a rabbit affected with the sheep associated form of malignant catarrhal fever is described. Immunological and morphological characteristics of the cell indicated that it was a T-lymphocyte and the presence of electron dense cytoplasmic granules suggested that the cell could be further classified as a large granular lymphocyte. The cell line required a feeder layer and was cytotoxic to both primary cell cultures and cell lines, a characteristic of large granular lymphocytes. No evidence of the nature of the agent could be detected but as few as 10(2) cells transmitted the disease. These findings are discussed and the possibility that infection and subsequent dysfunction of large granular lymphocytes may have a central role in the pathogenesis of malignant catarrhal fever is considered. That cells with similar characteristics have been derived from Herpesvirus saimiri and H ateles infected marmoset lymphocytes suggests that the lymphoproliferation associated with infection by these two simian herpes-viruses and malignant catarrhal fever may have a similar pathogenesis.

Genome re-arrangements associated with loss of pathogenicity of the gamma-herpesvirus alcelaphine herpesvirus-1.

The alcelaphine herpesvirus 1 (AlHV-1) causes malignant catarrhal fever in ruminants. Previous work had shown that serial passage of AlHV-1 in culture resulted in genome alterations that are associated with a loss in pathogenicity. Here we have analysed the re-arrangements that occur in more detail. None of the observed re-arrangements was entirely consistent. However, they did all involve translocation of a similar region of DNA from around the centre of the genome to areas either next to or in between terminal repeat elements at either end of the genome. There was also a concomitant loss of the wild-type locus. These re-arrangements appeared to be associated with the loss of virulence and the appearance of cell-free virus.

Outbreak of malignant catarrhal fever in Welsh Black cattle in Carmarthenshire.

An outbreak of malignant catarrhal fever (MCF) resulted in the deaths of 12 cattle in a herd of 77 animals during seven weeks in 1999; in addition, one cow developed a milder disease which was confirmed as MCF by PCR for ovine herpesvirus 2 DNA and an immunofluorescent antibody test for antibodies to the virus, but recovered. Further PCR and serological testing revealed the infection in three other animals, none of which developed clinical disease. Hypocuprosis and the possibility of a genetic predisposition were identified as factors which may have contributed to the outbreak.

Malignant catarrhal fever caused by ovine herpesvirus-2 in pigs in Norway.

This paper describes the first cases of malignant catarrhal fever (MCF) in pigs in which the diagnosis was verified aetiologically by polymerase chain reaction (PCR) and DNA analysis and by the demonstration of antibodies. Three pigs on two separate premises showed clinical signs, gross pathological and histopathological lesions which were in many respects similar to those of MCF in ruminants. The pigs were housed adjacent to sheep and DNA of ovine herpesvirus-2 (OHV-2) was detected by PCR in tissues of all the pigs. In addition, antibody to alcelaphine herpesvirus-1 was detected in the serum of the pigs and in five in-contact sheep. It is concluded that the disease described is MCF of pigs caused by OHV-2.

A disease resembling louping-ill in sheep in the Basque region of Spain.

A clinical syndrome resembling louping-ill which primarily affects lambs and yearlings in the Basque region of Spain is described. The disease has been observed for several years during May and June after the sheep flocks have been taken to the mountain grazings where the sheep tick (Ixodes ricinus) is known to occur. Examination of the brain from one of the affected animals revealed histological changes indistinguishable from those caused by louping-ill virus. In addition antibody that reacted with louping-ill virus antigen was detected in the serum of 57 per cent of the sheep tested from the affected flocks but in only 0.8 per cent of sera from flocks free of the disease. These preliminary results suggest that louping-ill or a related tick-borne virus is responsible for ovine encephalomyelitis in Spain.

Immunosuppression in toxoplasmosis: studies in sheep with vaccines for chlamydial abortion and louping-ill virus.

Toxoplasma gondii infection was established in sheep seven days (acute infection) or 28 days (chronic infection) before they were given enzootic abortion (EAE) vaccine alone or simultaneously with louping-ill (LI) vaccine. Uninfected controls received the same vaccination schedule. The serological response of acute toxoplasma-infected sheep to initial LI vaccination was significantly depressed but after revaccination four weeks later these sheep demonstrated a response equal to that of the revaccinated controls. Infected sheep also gave a significantly poorer response to EAE vaccine than control animals, the effect being more marked in those with chronic infection. Revaccination evoked no clear response in any group but on three of five sampling days antibody titres of controls were significantly higher than those of infected sheep.

Transmission of louping-ill virus in goat milk.

The course of louping-ill virus infection was examined in lactating goats. Seven goats were inoculated subcutaneously and titres of virus in blood and milk were monitored. All goats became viraemic with maximum titres of between 10(1.6) and 10(4.0) plaque forming units (pfu)/0.2 ml. Virus was also detected in the milk of all goats at maximum titres of between 10(0.6) and 10(5.7) pfu/0.2 ml. Only one of these goats exhibited clinical signs which were transient. In contrast, five of the 13 kids sucking these goats became infected and all showed marked clinical signs and one died and two were killed in extremis. It is considered that goats do not represent an efficient maintenance host for louping-ill virus but the excretion of virus in milk could represent a public health hazard.

Malignant catarrhal fever.

Malignant catarrhal fever is briefly reviewed and recent findings are described. Initially the disease was observed as a disease of cattle in Europe where, although no cause could be identified, circumstantial evidence implicated sheep as a source of infection and it was thus designated 'sheep-associated' malignant catarrhal fever. Subsequently the disease was observed in Africa where it became evident that a herpesvirus which normally infects wildebeest was the cause. It is now apparent that deer are highly susceptible to both forms of the disease, the sheep-associated form being a serious problem in farmed deer. The wide spectrum of clinical and pathological changes that occur in affected deer are described. A major constraint to studies of sheep-associated malignant catarrhal fever has been the absence of an experimental laboratory system. However, from affected deer it has been possible to transmit the disease to rabbits and thus has allowed detailed pathogenesis studies to be made which are summarised in this paper. It is suggested that the agent of sheep-associated malignant catarrhal fever is a virus and that when a particular subpopulation of T-lymphocytes is infected a profound immunological perturbation results; the lesions of malignant catarrhal fever being explained by a benign T-lymphocyte hyperplasia accompanied by a deregulation of cytotoxic natural killer lymphocytes that gives rise to tissue necrosis.

Isolation of a parapoxvirus from a grey seal (Halichoerus grypus).

A grey seal (Halichoerus grypus) developed cutaneous pocks which progressed to involve the skin extensively, necessitating euthanasia. Macroscopically and histologically the lesions resembled previous descriptions of parapoxvirus infections of seals and virus particles were observed in preparations of a scab and a skin lesion. Suspensions of the scab and skin lesion were prepared and inoculated on to monolayer cultures of grey seal kidney cells. After 25 days in culture and three passages, cytopathic effects were observed and parapoxvirus particles were detected by electron microscopy in the supernatant fluid. Both isolates were adapted to cultures of fetal lamb muscle cells and shown to be antigenically related to orf virus.