Literature review
LITERATURE REVIEW
2.1 INTRODUCTION AND AIM OF THE STUDY
Nervous disorders constitute an important threat to the livestock welfare and productivity, thus on the economy of the animal production industry. Sheep depend on locomotion to survive and to produce, as if their ability to move is inadequate they cannot graze appropriately. Also, it is extremely important to consider that most of these diseases affect young animals, which are the most important production resource for the farmers, as they are at the highest productive stage of their lives (Harper, 2009).
Among all the diseases affecting the central nervous system, viral encephalitides constitute a very important group. Although the prevalence of these conditions is not particularly high, they are extremely difficult to control, as there is no treatment available for almost all of them, and very few can be prevented by vaccination. That's why it is necessary to improve our knowledge about these diseases so that hopefully in the future they will be efficiently controlled (Callan and Van Metre, 2004).
Louping-ill is a major disease of sheep (although it can also infect other species), in which it causes locomotion disorders that lead to the leaping gait, the typical clinical sign shown by infected animals (Reid, 1991).
Literature review
It occurs in Scotland, Northern England, Ireland, Norway and perhaps elsewhere in Europe (McGuire et al., 1998).
Borna affects primarily horses and occasionally sheep (Summers et al., 1995), but recent studies have shown that its host spectrum includes many warm-blooded animals (Caplazi et al., 1994; Rott et al., 1995; Wormser et al., 1996; Reeves et al., 1998; Staeheli et al., 2000; Hagiwara et al., 2001; Helps et al., 2001; Dauphin et al., 2001; Okamoto et al., 2002; Kinnunen et al., 2007).
Infected animals show neurological clinical signs that vary from merely abnormal behaviour to severe CNS symptoms (Richt et al., 2001). This disease has a very wide geographic distribution (Kao et al., 1993; Bahmani et al., 1996; Berg et al., 1998; Reeves et al., 1998; Dauphin et al., 2001;
Taniyama et al., 2001; Helps et al., 2001; Hagiwara et al., 2001; Dauphin et al., 2002; Okamoto et al., 2002; Kamhieh et al., 2006; Miranda et al., 2006;
Kinnunen et al., 2007; Pisoni et al., 2007).
Visna only seems to occur in small ruminants (Cutlip et al., 1986), and it causes weakness of the limbs that eventually leads to paresis (Pétursson et al., 1990; Watt et al., 1992). Its geographic distribution is very wide (Pétursson et al., 1990; Watt et al., 1990), although the virus has never been found in Australia and New Zealand (Cutlip et al., 1986; Leginagoikoa et al., 2006).
Rabies virus causes a lethal encephalomyelitis and ganglionitis in humans, domestic and nondomestic animals and other warm-blooded vertebrates. It is world wide distributed (except Australia, Great Britain, New Zealand and Iceland) and it is transmitted from animal to animal or to man by bite inoculation of virus in saliva, as the agent resides in the salivary glands of infected carnivorous. Affected sheep are usually passive and
Literature review
anorectic, although they may show aggression to their handlers and to each other (Summers et al., 1995).
Aujeszky's disease is a common viral infection of the pig that occurs sporadically in other species, including ovine livestock (Henderson et al., 1995). In affected animals other then pigs, the infection is per-acute and fulminant, and in sheep it is associated with the "mad hitch", the typical clinical sign of Aujeszky's disease in ruminants (Henderson et al., 1995;
Summers et al., 1995).
West Nile virus is a mosquito-borne flavivirus of birds, but many mammals are also susceptible (Campbell et al., 2002). Several reports show that its host range includes sheep, in which it can cause a fatal meningoencephalitis (Tyler et al., 2003; Yaeger et al., 2004; Kecskeméti et al., 2007)
In this thesis, we have studied three of these viral encephalitides, Louping-ill, Borna and Maedi Visna, which have a common host, the sheep, but several differences, either in the pathogenesis or in the epidemiology. Carrying out a comparative study of these conditions could be of great help to further understand the mechanisms involved in the disease and the role of the host's immune response in the establishment of the encephalitis, which are key stages in the development of control strategies.
Furthermore, all these three diseases are of great comparative value to the study of human encephalitides as two of them, Louping-ill and Borna, are zoonosis and the pathogenesis in humans is yet not fully understood, and the aetiological agent of the third one, Maedi Visna Virus, is highly similar to Human Immunodeficiency Virus (HIV) with which it shares
Literature review
many of the pathogenetic mechanisms, as well as the pattern of the related CNS disorders. Therefore, the study of these diseases in sheep could also be a model to better understand human encephalitides.
This thesis has been carried out at Moredun Research Institute (Fig. 2.1), in Scotland, which has a long history in studying nervous conditions of sheep.
Figure 2.1: Moredun Research Institute, Edinburgh (Scotland).
Literature review
Samples from natural cases of the three diseases previously mentioned were available in this centre, so we considered carrying out a comparative study between them, with the following objectives:
Study, describe and classify the histological lesions observed in the brain of natural ovine cases of Louping-ill, Maedi Visna and Borna.
Compare the characteristics and distribution of these lesions in the three diseases.
Literature review
2.2 LOUPING-ILL 2.2.1 Aetiology
Louping-ill (LI) is caused by an RNA virus belonging to the Flaviviridae family and it is named after the leaping gait, which is the main clinical sign shown by the affected animals.
Louping-ill virus (LIV) belongs to the Flavivirus genus, in which there are three groups of viruses: the viruses for which there are no known vectors, the mosquito-borne and the tick-borne viruses. The last group is sub- divided into a mammalian group and a seabird group. LIV belongs to the tick-borne mammalian flaviviruses, together with eight more viruses (Tick- borne encephalitis virus, Langat virus, Powassan Virus, Omsk hemorrhagic fever virus, Kyasanur Forest disease virus, Kadam virus, Royal Farm Virus and Gadgets Gully virus). Although all of them are antigenically closely related (Stephenson et al., 1984; Reid, 1991; Gritsun et al., 2003), they have different hosts and environment. Louping-ill virus is the only one within the tick-borne encephalitis virus serocomplex that regularly causes disease in domestic livestock, while all the other occur primarily in men (except of Langat virus for which no naturally occurring disease has been described), and differs from the other members of the group for not being associated to a forest environment. (McGuire et al., 1998).
Literature review
2.2.2 Host Range
Louping-ill is a major disease of sheep (Reid, 1991) and red-grouse (lagopus scoticus) (Reid et al., 1974) but natural infection has also been sporadically reported in other species such as dog (MacKenzie et al., 1973), horse (Timoney et al., 1974; Timoney et al., 1976; Hyde et al., 2007), cattle (Twomey et al., 2001), red deer (Cervus elaphus) (Reid et al., 1978), roe deer (Capreolus capreolus) (Reid et al., 1976), goat (Gray et al., 1988), pig (Bannatyne et al., 1980; Ross et al., 1994), hare (Lepus timidus) (Jones et al., 1997), llama (Lama glama) (Macaldowie et al., 2005) and alpaca (Lama pacos) (Cranwell et al., 2008).
Although showing a low risk of infection and mainly linked to accidents in the laboratory, LIV is a dangerous zoonotic agent, causing potentially fatal encephalitis in man (Reid et al., 1972, Davidson et al., 1991).
2.2.3 Geographic distribution
Although LI was originally considered to only be present in Great Britain and Ireland and absent from continental Europe, in the last quarter of century, a disease of sheep indistinguishable from LI has been reported in Spain (Gonzalez et al., 1987), Bulgaria, Turkey (Reid 1991) and Norway (Gao et al., 1993). Although the virus involved are very similar and cannot be distinguished by conventional techniques, molecular analysis indicated that the Turkish and Spanish viruses are distinct, while the Norwegian viruses are identical to LIV (McGuire et al., 1998) (Fig. 2.2).
Literature review
Figure 2.2: Louping-ill geographic distribution.
2.2.4 Clinical signs
Animals affected by LI show neurological symptoms, mainly locomotor disorders. At initial stages these signs include hyperexcitability, muscular tremor particularly of the hind limbs, in-coordination, exaggerate movements in negotiating obstacles, which leads to the leaping gait characteristic of this disease (Fig. 2.3). Some affected animals recover after a few days, but in others severe neurological symptoms such as ataxia, posterior paresis followed by a complete flaccid paralysis, convulsions, coma, can be present after the initial phase. Most animals show a febrile response within a few days after infection, but the temperature then returns normal before rising up again in association to neurological dysfunction
Literature review
(Reid et al., 1971; Reid et al., 1981; Doherty et al., 1973; Reid 1991). Death occurs within 24 to 48 hours in the general course. All age sheep are susceptible to Louping-ill infection, although lambs and yearlings seem to be more frequently affected (Doherty et al., 1971; Reid et al., 1971).
Figure 2.3: Louping-ill affected sheep.
2.2.5 Transmission
The normal route of transmission of LIV is the bite of virus-infected sheep ticks (Ixodes ricinus). After the ingestion of viremic blood, the tick becomes infected and the virus localizes in the salivary gland so that it can be injected when the tick feeds on another host. Only sheep and red grouse show a high viremia, but the 80% of the birds die following infection, so
Literature review
they seem to only play a temporary role for the maintenance and the amplification of the virus (Reid 1991). However, Jones et al. in 1997 showed that the transmission of Louping-ill can also occur between infected and uninfected ticks co-feeding on vertebrate hosts without necessarily developing a viremia.
There are other possible routes of transmission of the virus, as the iatrogenic inoculation of the virus through sharp instruments and hypodermic needles (Reid et al., 1972; Reid 1991).
Several studies showed that the oral route of transmission could also be possible. The disease has been described in pigs following the ingestion of carcases of lambs suspected to have died from Louping-ill (Bannatyne et al., 1980).
Reid et al. in 1984 detected high titres of virus in the milk of goats inoculated subcutaneously with Louping-ill, even though no histopathological change attributable to virus was demonstrated in the mammary gland. The kids sucking milk from these infected dams became infected. They showed neurological signs, and widespread severe neuropathological changes due to Louping-ill were observed in their brains.
The kids also showed diarrhoea and small aggregates of neutrophils and other degenerated cells deep in the crypts of the small intestine were observed. The detection of pathological changes in the intestines of the kids is consistent with the possibility that the virus might cause cell damage outside the CNS.
Reid et al. (1985) reported the excretion of high titres of LIV in the milk of experimentally infected ewes, even when the viremic phase was over and the virus vas cleared from the blood, suggesting that the virus in the milk
Literature review
was probably derived from local replication in the mammary gland. Unlike the results shown on goats, none of the lamb sucking ewes excreting virus in their milk became infected. However, this route of transmission is of importance to public health since high titres of virus were excreted in the milk and it is possible that humans could become infected from this source (Reid 1991).
Experimental infection of rats with LIV via the olfactory route has been demonstrated and aerosol infection of man has been suggested to occur (Reid 1991).
2.2.6 Vectors and reservoirs
The vector of LIV is the sheep tick Ixodes ricinus. The tick needs a particular microclimate to survive, in which humidity is maintained close to saturation throughout the year. Such microclimate only is available in the mat of decaying vegetation close to the soil which can be found throughout the upland rough grazing of the British Isles, while in the south east of England and most of continental Europe it is only present in woodland areas. The other essential component for the maintenance of ticks is the availability of large mammalian hosts on which the adult ticks rely for their feeding. Immature larval and nymphal stages will feed on any vertebrate but the adult female will successfully engorge and mate only on the larger mammals. Thus the availability of the appropriate microclimate and host dictate the geographical distribution of the tick and hence Louping-ill distribution. The annual periodicity of the tick determines the time Louping-ill occurs: ticks are dormant during the winter and become active only when the mean maximum temperatures are around 7°C. The time of
Literature review
the year when this is reached varies considerably with latitude and altitude and as a consequence the period when Louping-ill occurs vary in different countries. Ticks quest for suitable hosts at the tips of the vegetation but as they are susceptible to desiccation they must return to the vegetational mat periodically to rehydrate. This process consumes the energy reserves of the tick, so a limited number of such cycles can be made. Thus, depending on the dryness of the weather a tick will quest for between four to eight weeks before becoming exhausted and dying. There is therefore a sharp peak of activity followed by a decline after which few ticks are found. Ticks that successfully feed return to the vegetation where they moult or lay eggs. The adult females die after depositing an egg mass up to 2000 eggs. The process of moulting and egg hatching occurs through the summer but the ticks remain dormant (diapause) until after a period of cold temperature is experienced, so a tick will feed only once a year and the whole life cycle takes three years to complete. In some areas there is also a period of tick activity during autumn season. Such ticks overwinter as engorged ticks and moult during August/September. These represent a distinct population of ticks and there is very little exchange with the spring feeding population (Arthur, 1973; Reid, 1991; Gritsun et al., 2002).
Several hypotheses have been made about the existence of a reservoir population for LIV but no evidence was found to show that small mammals are important in the epidemiology of Louping-ill (Gilbert et al., 2000).
As Louping-ill appeared to persist in some farms despite the long term use of vaccination protocols, a study has been made by Laurenson et al.
(2000) to clear the role of lambs in Louping-ill amplification. However, the
Literature review
study showed that lamb viremia is not an explanation for Louping-ill virus persistence.
Red grouse may contribute to the maintenance of the virus in the environment but may only act as temporary amplifying host due to their low survival rate and the short course of the disease after infection (Reid, 1991).
2.2.7 Diagnosis
In areas where ticks are present a diagnosis of Louping-ill should be considered when animals exhibit neurological dysfunction or die suddenly.
In areas where ticks are absent, Louping-ill may also affect sheep with a history of recent transportation. The wide range of clinical signs that might be presented makes diagnosis on clinical grounds alone unreliable, thus on most occasions laboratory confirmation is necessary. By the time clinical signs are apparent, virus has been eliminated from peripheral tissues including blood and is exclusively present in the CNS. Virus recovery can thus be made only from brain or spinal cord. Virus isolation can either be made by inoculation of mice or by tissue culture showing viral particles by electron microscopy (Reid, 1991).
From the histopathological point of view, the examination of haematoxilin and eosin-stained sections of the brain reveals a severe, non- suppurative meningoencephalitis affecting the cerebrum, midbrain, cerebellum, pons and medulla. The meningoencephalitis is characterized by the presence of numerous lymphoid perivascular cuffs, focal microgliosis,
Literature review
neuronal necrosis and neuronophagia but a definitive diagnosis relies on isolation of the virus (Doherty et al., 1972).
Virus detection can be enhanced through immunohistochemical labelling, which reveals widespread intraneuronal labelling in cerebellum, pons, midbrain and medulla (Krueger et al., 1994).
Serological examination can also be useful in reaching a diagnosis.
Several such tests have been described (complement fixation, gel precipitation, neutralization, ELISA) but the most convenient one to measure serum antibody to Louping-ill is haemoagglutination inhibition (HI) using gander red blood cells (Reid et al., 1971).
2.2.8 Tissue tropism
Louping-ill is a neurotropic virus and it is mainly localized in the pons, the medulla, the spinal cord and the cerebellar cortex. Secondary pneumonic lesions may develop at terminal stages of the disease (Doherty et al., 1971).
Some small aggregates of neutrophils and other degenerated cells deep in the crypts of the small intestine were observed in the kids sucking infected milk, which suggests that LIV might infect cells outside the CNS (Reid et al., 1984).
Literature review
2.2.9 Pathogenesis
Following inoculation either by the bite of the tick or injection, the virus initially replicates in the drainage lymph node and is subsequently disseminated in a cell-free form in the lymph and in the blood to the brain, spleen and retropharyngeal lymph nodes. During the viremic phase there are few clinical signs apart from an elevated rectal temperature. In sheep, high titres of virus are reached in the blood. Viremia rapidly declines in association with the appearance of serum antibodies, and virus becomes undetectable in the blood and in the lymphatic tissues six to seven days after infection and prior to the onset of overt clinical signs, but it persists in the brain for up to a further 5 days, during which clinical signs may develop. The initial humoral antibody is of the IgM class, and can be detected 5-6 days after infection. IgM antibodies are rapidly replaced by IgG over the following 7-10 days, before the onset of clinical disease. Once the virus has entered into the CNS it replicates in the neurons. In subclinical infections, only limited neuronal damage occurs before viral replication is interrupted by the immune response (Reid et al., 1971;
Doherty et al., 1971; Doherty et al., 1972).
2.2.10 Neuropahology
The principal neuropathological lesion in LI is widespread non- suppurative meningoencephalomyelitis. Neuronal necrosis and neuronophagia are most prominent in the motor neurons, cord, medulla, pons, midbrain and in the Purkinje cells of the cerebellum.
Immunohistochemical staining shows that the distribution of viral antigen
Literature review
corresponds to the damaged nerve cells. The viral antigen can only be observed in the neuronal cytoplasm, and occasionally in axons and dendrites but usually not far away from the cell body (Doherty et al., 1971a;
Doherty et al., 1971b).
The development of neurological signs is caused by a direct virus- induced dysfunction of many nerve cells resulting from virus replication.
The damage to the neuron is due to the recruitment of the cellular synthetic apparatus by the virus, which is a common pathway of the lytic neurotropic viruses. Active replication of the virus causes the necrosis of the neurons, which appear degenerate, often slightly shrunken and hypereosinophilic or vacuolated with indistinct Nissl bodies and pyknotic nuclei in routine paraffin sections (Doherty et al., 1971a). Necrotic neurons are phagocyted by microglial cells and are replaced by aggregates of these cells. Loss of Purkinje cells is characteristic, especially in intracerebrally infected animals where it can be observed mostly in the ventral areas of the cerebellum, while it is quite infrequent in natural infections (Doherty et al., 1971a;
Doherty et al., 1971b).
Other very important features of the LI-associated encephalitis are multifocal gliosis and lymphoid perivascular cuffs, which are more prominent in the reticular formation, hypoglossal, vestibular, trigeminal, and lateral cuneate nuclei, ventral columns of the spinal cord and the cerebellar cortex. The presence of glial foci is accentuated in the areas of neuronal necrosis. Glial nodules can be conspicuous in the normally acellular molecular layer of the cerebellum. The inflammatory cells involved in the focal gliosis and in the perivascular cuffs appear to be predominantly B-lymphocytes specific for Louping-ill virus antigens. Perivascular inflammation in the neuropile is largely lymphoplasmocytic, usually with a
Literature review
minor neutrophilic component, and extends to the leptomeninges, especially of the cerebellum. Cuffing in white matter is light. (Doherty et al., 1971a; Doherty et al., 1971c; Doherty et al., 1972).
Literature review
2.3 BORNA
2.3.1 Aetiology
Borna disease is caused by the Borna disease virus (BDV), an enveloped, negative single stranded (NNS), non-segmented RNA virus. Unlike all of the other NNS RNA viruses, BDV replicates and transcribes its genome in the nucleus (Cubitt et al., 1994). Moreover, unlike the majority of other RNA viruses, BDV genome sequence is extremely stable overtime (Kolodziejek et al., 2005). Because of these unusual aspects, BDV has been classified as the prototype of a newly established Bornaviridae family within the Mononegavirales order (Cubitt et al., 1994). Borna disease virus was named after the town of Borna in Saxony, Germany, where an epidemic of infectious encephalitis caused a large number of equine deaths in the 1890s.
2.3.2 Host range
Borna disease virus was believed to affect only sheep and horses, but more recent epidemiological data suggest that the host spectrum is wider than previously evaluated. In fact, BDV infection has been found in many warm blooded animal species such as cattle, dogs, cats, ostriches, rabbits, deer, goats, monkeys, alpacas, llamas, lynx, foxes, wild birds, small rodents and zoo animals (Caplazi et al., 1994; Rott et al., 1995; Wormser et al., 1996;
Reeves et al., 1998; Staeheli et al., 2000; Hagiwara et al., 2001; Helps et al., 2001; Dauphin et al., 2001; Okamoto et al., 2002; Kinnunen et al., 2007).
Literature review
Animals of all age groups can acquire the disease (Vahlenkamp et al., 2002).
The experimental disease is possible for almost all of these species, including primates. Moreover, antibodies against BDV have been detected in the blood of humans, showing the zoonotic character of the disease.
Because such antibodies were found more frequently in psychiatric patients then in the sera of healthy controls, several studies have been done to demonstrate that BDV infection was associated with human psychiatric disorders, but the results obtained were very different between different laboratories. The discrepant number of positive individuals in the different studies was explained by the fact that non-standardized systems were employed by the various laboratories, and that the titres of reactive antibodies were usually very low. However, the presence of serum antibodies is taken as a strong evidence for infection with BDV by some researchers, whilst an alternative possibility is that these antibodies were induced by infection with an antigenically related microorganism of unknown identity (Boucher et al., 1999; Bode et al., 2003; Chalmers et al., 2004; Bode et al., 2005; Wolff et al., 2006;).
2.3.3 Geographic distribution
Borna disease geographic distribution is not fully determined. Initially it seemed to be restricted to endemic regions (Germany, Switzerland, Austria and the Principality of Liechtenstein), but infections have been reported in Finland, Sweden, France, Italy, USA, UK, Turkey, Japan, Iran, China, Australia and Brazil (Kao et al., 1993; Bahmani et al., 1996; Berg et al., 1998;
Reeves et al., 1998; Dauphin et al., 2001; Taniyama et al., 2001; Helps et al., 2001; Hagiwara et al., 2001; Dauphin et al., 2002; Okamoto et al., 2002;
Literature review
Kamhieh et al., 2006; Miranda et al., 2006; Kinnunen et al., 2007; Pisoni et al., 2007) (Fig. 2.4). In Italy Patti et al. (2008) showed serological evidence of BDV infection in children, cats and horses in Sicily and in the population of Latium, whilst Pisoni et al. (2007) provided evidence for endemic BDV infection in healthy horses from different regions. In Australia Kamhieh et al. (2008) showed evidence of naturally-occurring infection in cats whilst according to a study made by the same author in 2006 there seems to be no evidence of endemic BDV infection in Australian horses.
Figure 2.4: Borna geographic distribution.
Literature review
2.3.4 Clinical signs
Clinical signs in naturally or clinically infected animals depend on the infected animal species, the age and immunological status of the animal, the viral strain and the pathway of infection, and vary from merely abnormal behaviour to severe CNS symptoms (Richt et al., 2001). The incubation period is variable, between two weeks and a few months (Rott et al., 1995;
Vahlenkamp et al., 2002). Animals of all age group can be affected (Vahlenkamp et al., 2002).
In infected sheep, mortality is higher than 50% (Rott et al., 1995).
Certain studies, however, have shown seropositivity without any apparent symptom (Hagiwara et al, 1997).
The death rate in horses is about 80-100%, but asymptomatic infections are also common in this species. Indeed, prevalence of serum antibodies between 30 and 50% have been observed in horses without any apparent symptom, and the virus may persist for five years without any visible symptom in its host (Rott et al., 1995; Staeheli et al., 2000).
Borna disease determines simultaneous or consecutive disorders in behaviour, sensitivity and motility. During the initial phase non-specific signs such as depression, hyperthermia, anorexia, alternation of colic and constipation are observed (Vahlenkamp et al., 2002).
During the acute phase, neurological signs resulting from meningoencephalitis are present: abnormal posture, ataxia, proprioceptive deficit, repetitive movements, somnolence, dysphagia (Caplazi et al., 1994;
Vahlenkamp et al., 2002). These signs can be associated with abnormal
Literature review
reactions to external stimuli such as hyperexcitability, aggressiveness, lethargy, somnolence and stupor.
If the animal survives the acute disease, then can show signs of chronic neurological disease (e.g. blindness, chronic apathy) (Rott et al., 1995; Richt et al., 2001).
After the onset of the clinical signs, the course of the disease in sheep is usually fatal and progressive over one to three weeks and affected animals are euthanized because of their poor prognosis (Vahlenkamp et al., 2002).
Some animals do not develop fatal encephalitis and exhibit significant behavioural abnormalities in a form of behavioural Borna disease (BBD).
Behavioural Borna disease is seen following BDV infection in some species or strains of immature animals (e.g., newborn Lewis rats) or adult animals with suppressed immune system (e.g. athymic or thymectomized or rats immunosuppressed via drug treatment). In neonatal BDV inoculated rats, the lack of significant immune cell infiltration in the brain is believed to stem from infection of the thymus during immune system maturation, leading to BDV-specific “immune tolerance”, although direct proof of this hypothesis is lacking (Carbone et al., 1991).
Behavioural abnormalities have been shown in all experimental animal models of BDV infection (summarized by Gonzalez-Dunia et al., 2005).
These abnormalities are linked in many cases to the damage caused by the prominent antiviral immune response in the CNS. However infection of newborn rats has shown that the virus itself has a direct impact on brain performance.
Literature review
2.3.5 Transmission
Borna transmission from an animal to another is not yet clear.
Vahlenkamp et al. in a study made in 2002, detected BDV (by RT-PCR) in saliva, nasal and conjunctival fluids of persistently infected asymptomatic sheep whilst BDV was not detected in urine.
2.3.6 Vectors and reservoirs
Several hypotheses have been made concerning the existence of a BDV vector or reservoir, due to the fact that sequence comparison showed that BDV strains from various host species seemed to lack species-specific signatures. Viruses from horses did not show a higher degree of similarity to each other than to viruses from sheep, donkeys or other hosts (Vahlenkamp et al., 2002; Kolodziejek et al., 2005). If BDV mainly spread from horse to horse and from sheep to sheep, species-specific patterns of neuclotide exchanges would be expected. Sequence comparisons of a limited number of field viruses from cases of classical BDV revealed that almost all strains are related closely to each other, as demonstrated in several studies (summarized by Staeheli et al., 2000). Their nucleic acid sequences differ by not more than 5%. The only exception so far is the divergent strain No/98 (Nowotny et al., 2000). For these reasons, and because transmission of BDV from horse to horse or from sheep to sheep has never been shown, the data seem to point towards a single source from which the various animals acquired the virus (Staeheli et al., 2000; Richt et al., 2001; Dürrwald et al., 2006).
Literature review
According to most scientists, rodents may be both the vectors and the reservoirs (Rott et al., 1995; Hilbe et al., 2006) and the transmission would occur by urine contamination of the pastures (Sauder et al.; 2003); for others, horses, domestic carnivores (cat in particular) cattle insects (Rott et al., 1995), wild birds (Berg et al., 2001) or shrews (Crocidura leucodon) (Hilbe et al., 2006) may be the vectors. The peak of incidence of disease seems to be situated between the beginning of the spring and the beginning of the summer (Vahlenkamp et al., 2002); this period coincides with the time of major activity of insects, but the hypothesis of insects being BDV vectors hasn’t been confirmed (Rott et al., 1995). The peak of rodent-borne viruses is usually late summer and autumn, when the population growth peak is (not spring and early summer). However, the higher frequency of BDV in spring could be related to mating activity of the host population, or to very a long incubation period in the final host (Dürrwald et al., 2006). Also, there might be more than one BDV reservoir population (Kolodziejek et al., 2005).
2.3.7 Diagnosis
The diagnosis of Borna disease is difficult, mainly because of low viral replication and excretion rates. Although a wide variety of tools have been developed for BDV diagnosis, standards have not been established yet, and large differences have been observed between the published results (particularly serological surveys) (Staeheli et al., 2000; Richt et al., 2001).
Borna disease can be diagnosed by serology, viral isolation, antigen detection, RT-nested-PCR, but none of these methods is yet sensitive and specific enough to be used alone (Staeheli et al., 2000).
Literature review
A clinical diagnosis alone cannot be sufficient to diagnose the disease, as animals show a big variety of non-specific symptoms.
Meningoencephalitis induces a slight alteration of protein concentration in the cerebrospinal fluid during the acute phase. However, these indicators are not specific of Borna Disease (Staeheli et al., 2000; Richt et al., 2001).
Serological diagnosis can be applied to living animals by antibody detection in blood and/or CSF. For this purpose, Western Blot, ELISA, and immunofluorescence assay (IFA) can be used. IFA is acknowledged to be the most reliable method for the detection of BDV-specific antibodies, even though inter assays have shown very variable results. Serological tests are mostly based on antibody detection directed against the most immunogenic BDV proteins, p24 and p40. Antibody titres are usually very low in BDV-infected animals and their detection requires particularly sensitive techniques. Antibodies are detectable in 100% of the cases during the acute phase of the disease but they are hardly detectable in the sub- acute or chronic stages (Staeheli et al., 2000).
Histologically, various degrees of encephalitis can be observed, in particular perivascular cuffs (mostly formed by lymphocyte infiltrations), and Joest-Degen inclusion bodies, located in the nuclei of infected neurons, have been used as BDV specific markers, but they are not systemically observed (Gosztonyi et al., 2000).
Sensitivity of virus detection can be enhanced by immunohistochemistry, which permits to visualize the major BDV antigens (nucleoproteins) and the p24 (phosphoprotein) using monoclonal or polyclonal antibodies. However, BDV-infected cells are not uniformly
Literature review
distributed in the brain tissue, so sometimes Borna disease can escape detection (Caplazi et al., 1997).
In situ hybridization can also complete histology by addictional RNA detection (Vahlenkamp et al., 2002).
The detection of viral genome by RT-PCR or RT-nested-PCR can be used for a virological diagnosis on brain or blood samples (Legay et al., 2000). However, this extremely sensitive technique is prone to cross contamination between samples, as well as laboratory contamination and is not able to detect variant strains that show altered sequences in the target gene. This was the case with the new genotype identified by Nowotny et al., in 2000.
RT-nested-PCR detects sequences encoding the two major BDV- proteins: p24 and p40. In order to avoid the use of a BDV-positive control, that could induce contamination, internal RNA standard molecules named
"mimics" have been produced. These fragments can be easily discriminated from the Borna fragments by agarose gel electrophoresis (Legay et al., 2000).
2.3.8 Tissue tropism
Borna Disease Virus is highly neurotropic, but it can persistently affect a broad range of non-neuronal cell-lines (Staeheli et al., 2000). In the early stage of the infection, virus specific antigens can be detected in the brain, all retinal layers, cerebrospinal fluid, peripheral nerves, adrenal glands (as summarized by Stitz et al., 1993). At later stages of the infection the virus disappears from the retina and the animals become blind, but a persistent
Literature review
productive infection is maintained in the rest of infected tissues. Viral antigens or infectious virus can be detected in peripheral blood mononuclear cells (Stitz et al., 1993; Vahlenkamp et al., 2002; Gosztonyi et al., 2008;).
Although the primary site of replication of the virus is the neuron, other cell populations of the nervous system are also permissive for BDV replication, such as astrocytes, oligodendrocytes, ependymal and plexus epithelial cells, Schwann cells and perineurial satellite cells of sensory and autonomic ganglia (Carbone et al., 1993). Nevertheless, the virus replicates primarily in neurons and secondarily in glial cells.
In persistently infected rats, 3 to 4 months after infection, visceral organs also become infected, as the virus travels centrifugally from infected neurons to the periphery. In these organs, multifocal areas of BDV antigen- positive cells develop, and the virus infection spreads from one neighbouring parenchymal cell to the other (Gosztonyi et al., 2008).
2.3.9 Pathogenesis
Following intranasal infection, the spread of BDV ensues along the connections of the olfactory system in a rostro-caudal direction to the limbic system, and later to almost all cortical areas, to the medulla, cerebellum, spinal cord and eye. The advancement of the virus is axonal and trans-synaptic and takes place mostly in the anterograde direction, except of the optic nerve and neurons and the Purkinje cells in which the virus reaches the neuronal perikarya by the retrograde axonal transport (Gosztonyi et al., 2008).
Literature review
Within a neuron viral antigen appears first in the perikarya, than in the nuclei, and later on in the axons and centrifugally in the dendrites and the finest arborizations (Gosztonyi et al., 2008).
With EM investigation, full virus particles could not be found in the brain, not even in the synaptic areas, so trans-neuronal transmission of viral nucleocapsid could be performed by an active transynaptic transport of viral nucleocapsid containing the viral RNA (Gosztonyi et al., 2008).
According to Gosztonyi et al. (2008), BDV only enters neurons that possess receptors of the glutamate receptor family; in fact its distribution is intimately related to certain constituents of the glutamanergic system. The widespread distribution of BDV infection within the CNS supports this postulation. The binding of BDV proteins to glutamate receptor enables the BDV RNA to enter the postsynaptic neuron. These proteins, at the same time, block the receptors for glutamate, hindering the excitatory neurotransmission. This elementary affinity of BDV to the glutamate system could explain its widespread distribution among vertebrates and possibly human beings (Gosztonyi et al., 2008).
The host’s immune response to BDV is central in the pathogenesis of BD. Morimoto et al., (1996) have performed an experiment in which they have treated a group of infected rats with dexamethasone or with saline solution. The dexamehasone treated animals did not show any clinical sign up to 30 days post infection. The brains of the dexamethasone treated BDV infected rats did not show any neuropathological lesions and absence of the activated macrophages normally seen in the CNS of BDV infected rats. However, virus specific antigen is found in immunocompromised animals in amounts comparable to fully immunocompetent rats.
Literature review
Carbone et al. in 1991 have shown the association of BD with the cellular immune response. This shows that BD is based on a virus induced immunopathological reaction rather than on a direct virus-cell interaction.
Several studies, summarized by Stitz et al., 1993 have excluded the possibility that antiviral antibodies play a significant role in the pathogenesis of BD, and have shown that the pathogenesis of BD is closely related to the cellular immune response. Immunohistological investigations into the quality of cells involved in the perivascular inflammatory reaction revealed the presence of CD4+ and CD8+ T cells in addiction to macrophages and B cells (Carbone et al., 1991).
2.3.10 Neuropathology
Borna Disease is a non-purulent polioencephalomyelitis that involves mostly the grey matter. Experimental BD in rats is characterized by severe lymphocytic encephalitis and by massive brain cells lesions finally leading to brain atrophy. Inflammation is accompanied by a moderate degeneration of the ganglion cells. The inflammation cells appear in the form of perivascular cuffs around venules and small veins and, rather infrequently, around small arteries (Stitz et al., 1993; Bilzer et al., 1993). The infiltrating cells consist mostly of lymphocytes and monocytes, and less frequently of plasma cells, especially present in cuffs of more than three layers (Caplazi et al., 1994). Polymorphonuclear leukocytes are not frequent within the infiltrate. The majority of the perivascular inflammatory infiltrates is found in the grey matter but the underlying white matter can also present cuffs of inflammatory cells (Caplazi et al., 1994). The inflammatory reaction is mainly localized in the frontal and temporo-basal cortex, in the amygdala,
Literature review
in the mesencephalon and in the hippocampus (Stitz et al., 1993;
Vahlenkamp et al., 2002; Gosztonyi et al., 2008), but also in the cerebellum, the spinal cord, and the eye (Gosztonyi et al., 2008). The fact that olfactory and limbic structures are usually involved suggests that the entry of the agent into the CNS is through the nasal neuroepithelium (Gosztonyi et al., 2008).
The pattern of inflammation that accompanies BD reflects a T-cell reaction rather than a direct virus-host cell interaction, and the immunopathological nature of the disease has been established in rats (Stitz et al., 1993).
BD can also occur without inflammation and still cause behavioural abnormalities and neurodevelopmental damage. This aspect has been studied on newborn rats, where the infection has been experimentally done when the brain is subjected to extensive tuning and shaping of neuronal connections. It has been proposed that BDV-linked CNS damage could be caused in part by viral interference with neuron plasticity. Neuron plasticity is the ability of neurons to adapt their functional and morphological characteristics to environmental influences. Although it is often related to changes in the efficacy of synaptic transmission, neuronal plasticity is also accompanied by morphological changes such as the formation of new synapses and the increase of dendritic arborizations. The molecular mechanism of neuron plasticity in BDV is likely to be neurotrophins linked, as neurotrophins and their specific receptors are expressed at high levels in areas of the brain subjected to intense neuronal plasticity, and BDV is linked to neurotrophin system dysregulation (Gonzalez-Dunia et al., 2005).
Literature review
BDV is a non-cytolitic virus; it replicates almost exclusively in neurons and has got a specific neurotropism for neurons of the limbic system (involved in memory and behaviour) and for those of the hippocampus (involved in the establishment of memory, in particular in acquiring orientation in a new environment, also called spatial learning) (Stitz et al., 1993; Gonzalez-Dunia et al., 2005). Infection of non-neuronal cells of the nervous system also occurs at later stages of infection (Carbone et al., 1993).
Neurons do not show any obvious change, and probably they work normally, but may contain intranuclear eosinophilic inclusion bodies, which are considered pathognomonic for BD (Gosztonyi et al., 2008). However, a protracted persistent infection determines a diminution of ergastoplasma and free ribosomes so that these neurons undergo a slow progressive decay (Gosztonyi et al., 2008).
Occasionally, microglial cells accumulate around diseased neurons forming neuronophagic nodules. However neuronophagia occurs less frequently in Borna than in other primary viral encephalitides.
In the neuropile a moderate gliosis can be found, and a slight leptomeningitis regularly accompanies the encephalitis (Caplazi et al., 1994).
The immunohistochemical examination shows diffuse staining of numerous neurons of the perikaryon and dendrites, and less frequently of intranuclear aggregates, corresponding partly to the inclusion bodies (Caplazi et al., 1994). The immunohistochemical staining detects only a few neurons positive to BDV p24 antigen in the frontal cortex, whilst numerous cells in the hippocampus and Purkinje cells in the cerebellum are found to be infected (Vahlenkamp et al., 2002).
Literature review
2.4 MAEDI VISNA
2.4.1 Aetiology
Maedi Visna (MV) is a chronic contagious disease of small ruminants caused by Maedi Visna Virus (MVV), a double-stranded RNA Lentivirus belonging to the Retroviridae family. It is closely related to the Caprine Arthritis Encephalitis Virus (CAEV), and together they are grouped as Small Ruminants Lentiviruses (SRLV) (Leginagoikoa et al., 2006). The lentivirus genus can be divided into five different serogroups according to the host they infect: primates, small ruminants, horses, cats and cows lentiviruses (Coffin et al., 1995).
The main characteristic of the retroviruses is their ability to integrate into the host's genome as a DNA provirus. At this purpose, the pol gene of the viral RNA codifies an inverse retrotranscriptase. Once integrated into the host's genome, the virus remains as a provirus in latent infection for a variable period of time, until it reactivates and begins to replicate, taking advantage of the host-cell synthesis mechanism (Murphy et al., 1999).
The history of Maedi Visna starts in 1933, when, due to a strong impact of the great worldwide economic depression on Icelandic sheep farmers, Icelandic government decided to import 20 Karakul sheep in attempt to help the farmers to increase their production. These central Asian breed sheep came from a flock kept for several decades at the Institut Für Tierzucht in Halle, in Germany. All the sheep were certified as free from all known infectious disease. At their arrival in Iceland, they were kept in a two
Literature review
months quarantine before being distributed to 16 farmers around the country. Unluckily, instead of bringing prosperity to the sheep farmers, this import caused their decline, as the imported sheep brought with them several infectious diseases previously unknown in Iceland. These diseases, subsequently referred to as the Karakul pests, spread insidiously in the local sheep breed, as they were extremely susceptible to these new diseases.
There were four diseases brought to Iceland through the Karakul sheep import. One was Johne's disease, also known as Paratuberculosis, a mycobacterical infection. Another was Jaagsiekte, a transmissible lung adenomatosis caused by a retrovirus. One more was Maedi, which is an Icelandic word meaning laboured breathing, a disease characterized by slowly progressive non-febrile interstitial pneumonia, initially confused with pulmonary adenomatosis. The last one was Visna, which in Icelandic means wasting. Visna is a slow progressive viral encephalomyelitis of adult sheep, characterized by weakness especially of the hind limbs, which gradually progresses to paresis. It has been observed in Icelandic flocks since 1940, and since the beginning it was only observed in flocks were Maedi was also known to be present. An association between these two diseases was therefore considered likely from the very beginning. This was later supported by animal experiments, and now Maedi and Visna are known to be two different manifestations of the same virus infection, and therefore referred to as Maedi-Visna disease (Pétursson et al., 1990).
Literature review
2.4.2 Host Range
MV has only been described in small ruminants (Cutlip et al., 1986). It affects mainly sheep, although natural and experimental transmission to goats has also been described. It is yet unclear how MVV can cross the species barrier. Probably the risk factors are the ingestion of infected sheep milk or colostrum by goats and vice versa as well as the close contact between the two species in overcrowded barns (Peterhans et al., 2004).
Although it seems that breed, sex or age do not clearly influence the susceptibility or resistance to MVV infections, several studies have demonstrated that a breed associated predisposition to the development of the disease can be shown (Cutlip et al., 1986; Pétursson et al., 1990;
Leginagoikoa et al., 2006).
2.4.3 Geographic distribution
The first description of MV was made in Iceland, in the 1930's, although previous reports of a similar disease, without known cause, were made in South Africa and Netherlands (Pétursson et al., 1990). Since then, several studies have shown a sero-prevalence of MVV worldwide, indicating the presence of the virus in the major sheep producing countries including United Kingdom (Watt et al., 1990). The virus has never been found in Australia and New Zealand (Cutlip et al., 1986; Leginagoikoa et al., 2006). The fact that Oceania is the only major geographical area of the world in which MVV has not been detected is probably due to the fact that all the sheep in this region descend from a small group of animals imported
Literature review
in the early 19th century which were probably MVV-free (Pétursson et al., 1990) (Fig. 2.5).
Figure 2.5: Maedi Visna geographic distribution.
2.4.4 Clinical signs
Maedi Visna is a slow disease, which means that although animals are mainly infected when they are young, the clinical signs are mainly observed in adult sheep (usually older than 2-3 years). MV onset is insidious and usually non-febrile. The symptoms begin with physical weakness and evolve into loss of weight, tachypnoea, hyperpnoea especially during and following exercise. The breath becomes laboured and the respiration rate is often increased. After the disease has reached the clinical stage the sheep usually don't survive longer than 3-8 months. Occasionally some animals
Literature review
can survive for a longer time (up to one or two years), especially if they are not exposed to any stress. Hypochromic anaemia can often be observed in advanced cases, in which lymphocytic leukocytosis can also be found.
Sometimes, due to the extremely slow progress of the disease, some sheep do not show any clinical signs, although some Maedi lesions are found during the post-mortem examination (Pétursson et al., 1990; Watt et al., 1992).
Visna is a progressive viral encephalomyelitis characterized by weakness especially of the hind limbs (Fig. 2.6), which eventually leads to paresis (Fig.
2.7). Its onset is insidious and only mature sheep seem to become affected.
The first sign is that affected sheep walk behind the flock especially on uneven or steep ground. Later on the affected animals can be seen resting the end of the metatarsus on the ground, and sometimes the head is tilted to one side and trembling of lips and facial muscles can be seen. The progression of the limb paresis is slow and the animals usually prefer to lie down even while they are grazing. At later stages of the disease the animal becomes paralytic and is not able to get on its feet. The course of the clinical disease can be protracted for several months until the stage of total paralysis is reached. Body temperature is usually normal, and clinical laboratory tests can show the presence of elevated γ-globulins levels in the cerebrospinal fluid (Pétursson et al., 1990; Watt et al.; 1992).
Literature review
Figure 2.6: Assaf sheep showing hind limb weakness (*).
Figure 2.7: Assaf sheep showing hind limb paresis (*).
Literature review
Some other symptoms have been reported in association with Maedi- Visna, as summarized by Pétursson et al. in 1990. These different clinical manifestations of the disease include chronic non-suppurative arthritis, especially affecting the carpal and the tarsal joints, and chronic non-febrile mastitis. The arthritis-affected sheep show swelling of the joints and associated synovial bursae, which appear thick with marked perivascular fibrosis. These animals become lame and emaciated. The mastitis determines a reduction in milk production and can therefore retard the growth of lambs born from affected dams, that appear small and weak. The udder palpation can show bilateral, diffuse, indolent indurations with firm nodules mostly distributed in the ventral part of the udder. The accessory lymph nodes are usually only slightly enlarged, and the milk appearance is not altered (Pétursson et al., 1990; Watt et al.; 1992).
2.4.5 Transmission
Maedi Visna virus can be isolated from the blood of infected animals in which it is associated with the white blood cells, especially monocytes. This might be the reason why organs containing a large number of macrophages, e.g. lung and udder, play an important role in MVV transmission. It is unlikely that the virus can remain infectious for a long time outside the host. Therefore, significant transmission is likely to take place if direct contact between infected and uninfected animals occurs (Dawson, 1980).
Aerogenic transmission through aerosol has been shown to be a very important route for viral spread, and this can explain why sheep housing in overcrowded barns together with infected animals can contract the disease more easily than those held in extensive rearing (Leginagoikoa et al., 2006).
Literature review
Secondary infections could therefore have an enhancing effect on MVV transmission, either by increasing the number of macrophages (thus the amount of virus) and by the induction of coughing (so that aerosols can be formed).
The other main route of transmission is trough the milk, especially colostrum, which is considered of prime importance in the transmission of MVV from mother to offspring. Contact between ewes and lambs also represent an important risk factor in viral transmission to the newborn, probably because of aerosol transmission from the respiratory tract (Cutlip et al., 1988).
Other routes of infection, as intrauterine or genital transmission, have been investigated. Although their importance is not clear, they are supposed to have very little impact in the transmission of the disease. The epidemiological role of rams is not important, as there is no evidence of the virus being transmitted by the semen or during mating, although semen has been demonstrated to contain virus. However, the role of rams can be important as they are often moved from one flock to another, thus this way they can determine horizontal transmission (De La Concha-Bermejillo, 1996).
Experimentally, the infection could be transmitted by drinking water contaminated with faeces of infected sheep although isolation of the virus in urine and faeces has never been reported. Only infrequently, the virus has been isolated from saliva and nasal swabs. However, it is unlikely that the virus can remain infectious for a long time outside the host. Therefore, significant transmission is likely to only take place if direct long contact between infected and uninfected animals occurs. Cases of iatrogenic
