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ADVANCES IN UNDERSTANDING MALIGNANT CATARRHAL FEVER 

Sir. Hugh W. Reid

Courtesy : Festschrift - Dr. S. Ramachandran

When Ramachandran investigated Malignant Catarrhal Fever (MCF) in Indonesia (1), little was known about the aetiology or the causal virus nor was there any satisfactory explanation for the extraordinary pathological changes that characterised the disease. Although circumstantial evidence at that time suggested that contact with sheep was a pre-requisite before cattle and other species developed the disease, very little else was known. In the intervening years major advance has been made in our understanding of this condition and that will be described in this dedication.

Introduction
Malignant Catarrhal Fever (MCF) is a generally fatal disease affecting many ruminant species including cattle, water buffalo, bison, many species of deer as well as domestic pigs (2). 

Particularly susceptible to the disease are the native cattle of India, the gaur (Bos gaurus) (3) and Banteng cattle (Bos javanicus) (1) of Indonesia in which losses can be high following contact with sheep. In addition to these naturally susceptible species, infection can be transmitted experimentally to laboratory rabbits though disease in wild rabbits has never been reported (4). A complicating aspect of this disease is that there are two forms: one which follows contact with wildebeest (Connochaetes spp.) known as wildebeest associated (WA) MCF and another which occurs following contact with domestic sheep termed as sheep associated (SA) MCF. In Africa and some zoological collections WA-MCF occurs, often affecting several animals following direct or indirect contact with wildebeest particularly calves less than 6 months of age. SA-MCF tends to occur sporadically affecting only one animal though severe outbreaks can occur particularly in the more susceptible species of deer, Banteng cattle and North American bison.

Both WA-MCF and SA-MCF cause similar clinical syndromes though dependent on the susceptibility of the host there can be considerable variation with the more acutely affected animals showing limited manifestations before death while in the more chronic cases the signs can be florid (5). In addition, the incubation period can vary from as little as two weeks to up to seven or nine months. When clinical signs do develop the response may be peracute to chronic. The peracute disease is characterised by the rapid onset of depression and, high fever followed by diarrhoea, which may become haemorrhagic with death occurring within 24 hours of onset. The onset of signs is associated with the development of fever, inappetence, photophobia, lachrymation and serous nasal exudate which progresses to profuse mucopurulent discharge. Bilateral corneal opacity develops which starts at the periphery and progresses to involve the whole cornea accompanied by ulceration and hypopyon. Salivation is commonly accompanied by erosion of the oral epithelium particularly of the gums, hard palate and tips of the buccal papillae. In some cases exanthema, exudation and encrustation affects areas of skin, particularly in the perineum, udder, teats and at the base of horns and hooves while the muzzle often becomes encrusted, necrotic and can slough. The superficial lymph nodes are frequently swollen and the limb joints puffy. Nervous signs may be present with few other clinical signs or as part of a typical clinical case and include hyperaesthesia, incoordination, nystagmus and head pressing.

Although the usual outcome of MCF is fatal, recovery does occur both from mild and severe cases (6). However the frequency with which this occurs is unclear.

At post-mortem macroscopic lesions may be widespread and involve most organs. External lesions reflect the clinical signs. The mucous membranes of the respiratory tract are congested, haemorrhagic, often with erosions and accumulation of mucopurulent material. The nasal passages may be blocked and pneumonia may be present. The mucosa of the mouth may be hyperaemic with erosions and the formation of diphtheritic deposit particularly around the gums and pharynx. Similar lesions may be found in the oesophagus and less frequently affecting the rumen and abomasum. The large intestine is affected with erosions and haemorrhage, often longitudinally orientated along the ridges of the mucosa. Lymph nodes are generally enlarged and oedematous and may be necrotic and haemorrhagic. The surface of the kidney is often affected with numerous greyish raised foci of 1 to 5 mm. diameter and the wall of the urinary bladder frequently contains petechiae and ecchymosis.

Histological lesions are characterised by a widespread massive lymphoid cell hyperplasia in lymphoid tissues as well as non-lymphoid organs, vasculitis and necrosis of epithelial surfaces.

Aetiology
From cases of WA-MCF and wildebeest a herpesvirus Alcelaphine herpesvirus-1 (AlHV-1) (named after the subfamily Alcelaphinae to which wildebeest belong) has been isolated and shown to be the cause of the disease (7). This virus has been characterised and the whole nucleic acid sequence published (8). In contrast, no aetiological virus has been isolated from sheep or from cases of SA-MCF. Experimental transmission using large volumes of blood or tissue suspensions has however been achieved on a number of occasions both to susceptible ruminants and laboratory rabbits which has facilitated the advances made in our understanding of this form of MCF over the last 20 years (1,9). These advances will be the principle focus of the remainder of this article.

Following the isolation of AlHV-1 it was generally assumed that the SA-MCF would be caused by a very similar virus though veterinary researchers consistently failed to isolate it. Nor could neutralising antibody to the wildebeest virus be identified in sheep sera. However, using AlHV-1 infected tissue culture cells, antibody in sheep serum was identified using an Indirect Immunofluorescence Assay (10). Antibody was present in virtually every domestic sheep serum from all countries examined. The specificity of this antibody was further confirmed using the structural proteins of AlHV-1 in immunoblotting studies with sheep serum (11). Thus it was apparent that most if not all domestic sheep were infected with a virus antigenically related to the wildebeest virus.

Attempts however to identify a tissue culture system to isolate the virus either from sheep or clinically affected animals consistently failed. Even experimental transmission from bovine cases of MCF were only infrequently successful. It was however discovered that infection from cases of MCF in the more susceptible species such as farmed red deer Cervus elaphus and Banteng cattle could be transmitted to other susceptible animals and laboratory rabbits consistently (9). Thus for the first time a laboratory model was available for studying this form of the disease.

It was further discovered that from SA-MCF affected animals lymphoblastoid cell lines (LCL) could be cultured (12). This was to have a profound effect both in identifying the aetiology but also in elucidating the pathogenesis of the disease.

Epidemiology of ovine herpesvirus - 2
The LCL derived from affected animals appeared homogeneous and when inoculated back into animals caused MCF. Even as few as 100 cells could transmit the disease. Thus there was good evidence that these LCL carried the aetiological agent. Meanwhile studies to characterise the genome of AlHV-1 had provided clones of much of this virus (13). Such clones could be used to probe for related viral sequences in the LCL. Thus by extracting the DNA of the LCL and employing AlHV-1 cloned DNA in southern blots it was possible to confirm the presence of related DNA in the cells. Accordingly, a genomic library from an LCL was prepared and probed with clones of AlHV-1 DNA. In this way specific viral DNA clones were isolated from the cell lines and their nucleic acid sequence determined and compared with AlHV-1 and other herpesviruses (14).
In this way it was possible to confirm that the cause of SA-MCF was a gammaherpesvirus related to, but distinct from, AlHV-1 which was designated ovine herpesvirus-2 (OvHV-2). Within one of the clones, a unique nucleotide sequence was identified which allowed the design of primers to form the basis of a polymerase chain reaction (PCR) for the detection of viral DNA in the natural host and clinically affected animals (15). Because of the low virus copy number normally found in infected animals the diagnostic PCR is based on a primary amplification followed by secondary amplification of a smaller internal fragment. This provides an extremely sensitive test for detecting OvHV-2 DNA - as little as one viral genome per cell in the test sample.
This test has now been used to detect virus in clinical cases of MCF, around the world - Europe (16), America (17), Indonesia (18), Australia (19). It has proved to be a specific and robust diagnostic test and provides for the first time a methodology for confirming a clinical diagnosis in the live animal because it can be used to detect virus in peripheral blood leucocytes. Previously, diagnosis could only be confirmed by the histological examination of tissues collected post mortem.

The application of the test is providing new insights into the epidemiology of the virus in both the natural host and the MCF susceptible species. Viral DNA has been detected in the nasal secretions of neonatal lambs thus providing evidence that probably the principle transmission route is through infection of the upper respiratory tract of lambs in their first few months of life (20). In addition it is now possible to confirm cases of MCF in live animals which has provided evidence that in a proportion of animals the infection is clinically mild and that animals can recover following typical clinical diseases (6). The PCR has been used to confirm that the disease is the cause of significant losses in Indonesia not only in the Banteng cattle but also water buffalo (18). In North America it has been used to confirm massive losses in farmed bison (21) while in Norway and Finland, typical MCF caused by OvHV-2 has been confirmed in pigs (2). Why the disease has only been observed to affect pigs in Scandinavian countries remains obscure because the virus appears to be identical to that found elsewhere and the breeds of pigs involved are the same as found in other countries.
It has also been used to confirm that outbreaks of disease affecting several animals do periodically occur in normal domestic cattle. The reason for this is not understood but the evidence supports the concept that on occasion a mutant virus emerges which is very much more contagious for cattle than is the normal virus (22).
Above all what is clear is that, over the last 20 years since Ramachandran investigated MCF in Indonesia, molecular biology has provided the technology to rapidly advance our understanding of the complex epidemiology of SA-MCF. There are many aspects, which have still to be resolved. However, now that the technology is available it will be possible to resolve these and develop a comprehensive understanding of this enigmatic disease.

Pathogenesis of MCF
The other aspect of MCF that has confounded veterinary research workers is the pathogenesis of the infection. In the natural host, infection is apparently completely benign with no clinical signs having been attributed to infection. In a longitudinal study of suckling lambs using the PCR it became apparent that all become infected during the first eight weeks of life with viral DNA being detectable in the oro-pharynx and lymphoid tissues (20). From this and other studies it is apparent that in a normal flock of sheep virus is transmitted amongst lambs highly efficiently probably through productive viral replication in the oro-pharynx. Thereafter a latent infection is established in B-lymphocytes where it is maintained for life with persistent high levels of antibody (20). There is evidence that sheep periodically excrete virus. Thus presumably, the latent virus can give rise to productive viral excretion possibly associated with “stress” such as adverse weather conditions, pregnancy, inadequate nutrition, etc. At no point have any clinical or pathological changes been identified in sheep due to natural infection.

In contrast to these benign events, infection of the MCF-susceptible host is clinically and pathologically dramatic, normally resulting in death. Such infections are associated with very little evidence of virus replication and the production of cell-free infectious virus. Thus there is no spread by contagion from affected animals. The lesions are characterised by a dramatic T-lymphocyte hyperplasia and accumulation in non-lymphocyte organs, vasculitis and degeneration of epithelial tissues particularly of the gastro-intestinal and upper respiratory tracts (23). Virus, viral antigen and even viral DNA is either absent or present in very low amounts in these dramatic lesions (24). It is thus difficult to reconcile these changes with conventional viral pathogenesis and a variety of novel mechanisms have been proposed all of which identified some immuno-pathological event - none of which were entirely satisfactory. It was not until the discovery that LCL carrying viral genomes could be cultured from MCF affected animals that a hypothesis which largely satisfies the observed features of the disease was developed (25). This proposes that these cells have a central role in the pathogenesis of the disease.

The LCL in culture are large lymphocytes with abundant granules in their cytoplasm and carry surface markers of T-lymphocytes. Functionally they are highly cytotoxic for a variety of cells and there is no major histocompatibility restriction to this property. The cells thus have the morphological, phenotypic and functional characteristics of lymphokine Activated Killer (LAK) cells and it is proposed that these cells have a pivotal role in the in vivo pathogenesis.

It is thus hypothesised that when OvHV-2 transmits to MCF-susceptible species, it infects T-lymphocytes, little or no complete viral replication occurs but limited viral transcription occurs. These transcripts are almost certainly those, which are transcribed, in the ovine B-lymphocyte during latency. When, however, these same transcripts are expressed in the T-lymphocyte of a susceptible animal there is a resulting cytokine cascade, which drives lymphoproliferation and terminally tissue destruction through the equivalent of LAK cells operating in vivo. Such an explanation explains the absence of productive virus replication and both the lymphoproliferative and degenerative components of the disease. The challenge now is to characterise the actual molecular pathway for this immunological catastrophy and identify strategies that have the capacity to interrupt it and hence facilitate recovery.

There has thus been significant progress in our knowledge of both the epidemiology and pathogenesis of MCF but there remain many questions still to be answered before the multiple facets of this disease are fully understood. Though the application of molecular biology to the study of MCF has made a major contribution to our understanding of MCF (26) it is the spirit of enquiry which comprehensively embraces many aspects of veterinary science, as exemplified by the lifetime work of Ramachandran which will be required if the remaining questions about this fascinating disease are finally to be resolved.

References

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Authors Corresponding address: 

Dr. Hugh W. Reid 
Head, Virology Division, Moredun Research Institute, Pentlands Science Park, Edinburgh EH26 0PZ
Email: reidh@mri.sari.ac.uk 

The views expressed in this article are of the author(s), and any clarifications can be obtained from the author(s).