Avian Pathology

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Avian encephalomyelitis: A review

G. A. Tannock & D. R. Shafren

To cite this article: G. A. Tannock & D. R. Shafren (1994) Avian encephalomyelitis: A review,
Avian Pathology, 23:4, 603-620, DOI: 10.1080/03079459408419031

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Avian Pathology (1994) 23, 603-620

REVIEW ARTICLE

Avian encephalomyelitis: a review

G. A. TANNOCK1 & D. R. SHAFREN

Faculty of Medicine, The University of Newcastle, Callaghan 2308, Australia

SUMMARY
Avian encephalomyelitis virus (AEV) is a picornavirus with a predilection for the
central nervous system and other parenchymous organs of chickens that is transmit-
ted by the oral-faecal route. The virus may be spread by the vertical and horizontal
routes and, because of its great stability, contaminated areas may remain infectious
for long periods. The egg-adapted Van Roekel strain is highly neurotropic and does
not grow efficiently in the enteric tract of the chicken. Despite this, the virion
polypeptides of both naturally-occurring strains and the Van Roekel strain are
antigenically identical, and there is only one virus serotype. Natural infection by
AEV induces a multi-organ disease with similarities in its pathogenesis for chickens
to that of Coxsackie B3 in neonatal and infant mice. Field isolates of AEV can be
adapted to grow in chicken embryo brain cultures, but virus growth is highly
cell-associated. Simple reliable antibody assays to determine the presence of recent
infections or to assess vaccine efficacy were not available until comparatively recently
when antibody detection assays, many involving the use of chick embryos, were
replaced by enzyme-linked immunosorbent assays. Effective vaccination pro-
grammes employing live, highly enterotropic strains of AEV, have greatly reduced
the incidence of the disease.

HISTORICAL OVERVIEW
Avian encephalomyelitis virus (AEV) is the aetiological agent of a widespread
disease of poultry that is transmitted via the oral-faecal route (Calnek et al.,
1960). In young chickens AEV induces paralysis, ataxia and muscular dystrophy
(Jungherr & Minard, 1942), while in older chickens infection is usually subclini-
cal, resulting in a decline in egg production and hatchability (Taylor et al, 1955).
Outbreaks of a disease causing neurotropic dysfunction in young chickens in
the New England region of the USA in 1930 were first reported by Jones (1932).
Jones termed this syndrome "encephalomyelitis in the chicken". Further examin-
ation of infected chickens, however, revealed the presence of unusual tremors and
vibrations of the head or neck, which prompted Jones (1934) to refer to the
disease as "epidemic tremor". At the time the incidence of the disease was
increasing in the New England region, but its prevalence elsewhere was unknown.
1
Present address: Department of Applied Biology and Biotechnology, Royal Melbourne Institute of
Technology, GPO Box 2476V, Melbourne, Victoria 3001, Australia.
Received 28 January 1994; Accepted 26 April 1994.

603
604 G. A. TANNOCK & D. R. SHAFREN

Van Roekel et al. (1938) considered the term "epidemic tremor" inappropriate, as
it neither emphasized the infectious nature of the aetiological agent nor its
predilection for the central nervous system (CNS) of the chicken, and proposed
the name "infectious avian encephalomyelitis", which was subsequently short-
ened to "avian encephalomyelitis". Concurrent outbreaks of avian en-
cephalomyelitis in chickens and of equine encephalomyelitis in both horses and
man in the New England region in 1938 aroused interest in a possible common
aetiology for both diseases. Olitsky (1939) concluded that the diseases were
caused by dissimilar agents and that AEV was unable to produce clinical disease
in guinea-pigs, mice, rabbits or monkeys.
The highly contagious nature of AEV was demonstrated from an outbreak of
disease in California in 1942, which occurred following the purchase of large
numbers of hatching eggs for stock replacement from the New England region in
1941 (Schaaf & Lamoreux, 1955) and by 1950 reports of sporadic outbreaks had
come from all parts of the USA (Taylor & Schelling, 1960). Over the next decade,
outbreaks of the disease were reported in Europe, Canada, Japan and Australia
(Hoekstra, 1964; Albiston, 1966).
Evidence for the existence of the disease in other avian species has come from
the isolation of the virus from turkeys and quail that exhibited neurological
symptoms similar to those described in chickens (Mathey, 1955; Hill & Ray-
mond, 1962; Hohlstein et al, 1970). For economic reasons, most studies on the
disease have been carried out in chickens. Comprehensive studies on the epidemi-
ology and pathogenesis of AEV have been hampered by the infrequent occurrence
of outbreaks of the disease and the difficulty of establishing, until comparatively
recently, a rapid, reliable means of laboratory diagnosis.
The following review includes descriptions of some properties of the virus, its
detection and pathogenesis that have been recently reported, and have been
derived from the application of newer experimental methodologies.

VIRUS PROPERTIES
Most studies with AEV have been carried out on the egg-adapted Van Roekel
strain which produces characteristic lesions in infected embryos (see below).
Earlier studies were largely concerned with resistance of the virus to heat and
chemical treatments. Infectivity was shown to remain unaffected by chloroform,
low pH (2.8), pepsin, trypsin and deoxyribonuclease (Butterfield et al., 1969;
Costing et al, 1980). Magnesium cations were shown to stabilise preparations of
the virus against heat inactivation (56°C, 1 h), while incubation with ribonuclease
and treatment at high pH (9.0 to 10.0) reduced its titre by 10 07 to 102 EID50/ml
(Butterfield et al, 1969; Gosting et al, 1980). Indirect evidence that the genome
of AEV consists of RNA comes from the observation that its growth in chicken
embryo brain cultures was not inhibited by concentrations of the nucleoside
analogue 5-bromo 2'-deoxyuridine that inhibit the growth of DNA viruses
(Shafren & Tannock, 1992).
The buoyant density of virions purified by different procedures has been
 AVIAN ENCEPHALOMYELjnS 605

estimated in caesium chloride density gradients, using embryo inoculation assays

to detect infectious virus in fractions of the gradients. Butterfield et al. (1969),
using a relatively crude AEV preparation, reported the density to be 1.33 g/ml,
while Gosting et al. (1980), using a more purified suspension, observed a major
peak of density of 1.31 to 1.32 g/ml with smaller viral peaks at 1.36 and 1.40 g/ml.
When a highly purified preparation of the Van Roekel strain was labelled with
Na123I and centrifuged to equilibrium in caesium chloride gradients, a single peak
of density 1.31 g/ml was observed (Tannock & Shafren, 1985) and subsequent
studies have confirmed this finding for two other non-egg adapted strains of the
virus (Shafren, 1990). The latter study revealed that all strains had sedimentation
coefficients identical to that of poliovirus which has been reported to be 160S
(Cooper et al, 1978).
The diameter of the virion was estimated by differential filtration through
Millipore membranes to be 16 to 25 nm (Butterfield et al, 1969). When exam-
ined under the electron microscope, purified virion preparations were shown to
possess a diameter of 26 ± 1 nm (mean ± standard deviation) after staining with
phosphotungstic acid (Tannock & Shafren, 1985). Shadow cast replicas of the
virion, formed by placing on freshly cleaved mica and spraying them with
platinum under vacuum, also measured 26 nm (Gosting et al, 1980). Crystalline
arrays of virions within the cytoplasm of Purkinje cells from the brain of experi-
mentally infected chickens were reported to have a diameter of 22 nm by Cheville
(1970) and 25 nm by Hishida et al. (1986). The centre-to-centre length of
particles in each array was estimated to be 33 nm (Hishida et al, 1986).
Electron microscopic examination has revealed the virion to be devoid of a lipid
envelope and to possess icosahedral symmetry (Krauss & Ueberschär, 1966;
Gosting et al, 1980; Tannock & Shafren, 1985). Gosting et al (1980), using a
technique developed by Markham et al (1963), which involved rotation of
enlarged images of virions from the original photographic plate, showed that the
AEV virion possesses 5-fold symmetry and comprises 32 to 42 capsomers.
The inability of AEV to grow efficiently in most cell cultures and the difficulties
encountered in producing highly purified preparations of the virus has probably
been responsible for the paucity of reports on the protein structure of the virus.
Tannock & Shafren (1985) labelled purified disrupted preparations of the egg-
adapted Van Roekel strain with Na125I and analysed the constituent polypeptides
under non-reducing conditions by polyacrylamide gel electrophoresis. Four
polypeptides were shown to be present with Mj of 43, 35, 32 and 14 kdal. These
studies were carried out on virus that was purified by sucrose-velocity gradient
centrifugation. Later studies, using virus that had been purified in caesium
chloride gradients, revealed the presence of three virion polypeptides with Mj of
35, 30 and 26 kdal, and that the larger protein previously reported was probably
contaminating ovalbumin (Shafren & Tannock, 1991). When purified prepara-
tions of two non egg-adapted strains and the Van Roekel strain were compared by
radioimmunoprecipitation, individual polypeptides within each strain were shown
to be antigenically identical (Shafren & Tannock, 1991).
606 G. A. TANNOCK & D. R. SHAFREN

PATHOGENESIS
Early studies on AEVs were performed on isolates from brain homogenates of
field chickens exhibiting neurological signs of infection. Field isolates of AEV can
be propagated by intracerebral (i.e.) inoculation of susceptible day-old chickens
or by yolk-sac inoculation of susceptible 6 to 7-day-old chickens embryos. Signs
of infection in embryos inoculated with field isolates are usually only evident after
hatching. Successful adaptation of field isolates to growth in susceptible embryos
has been reported by several groups (Van Roekel et al., 1938; Willis & Moulthrop,
1956; Sumner et al, 1957a; Butterfield et al, 1969). Viruses adapted for growth
in embryos induce gross symptoms of infection, which include stunting, an overall
haemorrhagic appearance, absence of skeletal muscle and splaying of the feet.
The number of serial passages required for adaptation varies with each strain. A
study by Butterfield et al, (1969) with 21 field isolates indicated that 8 to 20 serial
passages (mean of 12) are required. Egg-adapted strains are probably mutants of
non-egg-adapted strains and were probably selected as the dominant virus in
mixed virus populations during embryo passage (Miyamae, 1976). The most
widely used egg-adapted strain of AEV is the Van Roekel strain, which was
prepared after multiple passage of a field strain of AEV isolated from infected
chickens in the New England region of the USA (Van Roekel et al, 1938).

Egg-adapted strains
Infection of susceptible embryos and young chickens with egg-adapted strains has
been shown to induce histopathological changes in a number of tissues (Jungherr
et al, 1956; Van Roekel et al, 1938; Springer & Schmittle, 1968). Lesions in
chickens occur more frequently in the CNS than in other organs in the early
stages of infection. Initial changes to neural tissues are characterized by neuron
degeneration, mainly in the medulla and the anterior horn cells of the spinal cord.
As neuronal degeneration progresses, the Nissl substance gradually disappears
and perivascular infiltration by lymphoid cells is evident. This infiltration occurs
mainly in the cortex, cerebellum and medulla, while the grey matter of the spinal
cord is usually unaffected.
Gross changes in other organs are usually not evident, but histopathological
lesions can be observed. Lesions in the parenchymous organs and body viscera
are characterised by increased peri- and/or paravascular infiltration of
lymphocytes and focal lymphoid hyperplasia. Fine changes in muscular structure
are characterized by eosinophilic swelling, necrosis, fragmentation, loss of stria-
tion in fibres and heterophil infiltration. Lesions induced by AEV infection are
similar to those appearing in chickens infected with Newcastle disease and
Marek's disease viruses or experiencing vitamin E or A deficiency, and serological
tests are required to confirm a suspected infection.
As egg-adapted strains are probably laboratory mutants selected from naturally
occurring field strains, laboratory studies with these strains are probably not
representative of infections in the field (Willis & Moulthrop, 1956; Miyamae,
 AVIAN ENCEPHALOMYEUnS 607

1976). The majority of studies on the pathogenesis of egg-adapted strains have

been performed in susceptible embryos (Burke et al, 1965; Braune & Gentry,
1971; Willis & Moulthrop, 1956; Calnek & Jehnich, 1959a; Ikeda, 1976a).
Following yolk-sac inoculation, virus is usually detected first in the brain 3 to 5
days post-inoculation (p.i.) and then subsequently in most tissues of the embryos.
Maximum titres of virus occur in the brain 6 to 8 days p.i. (Burke et al, 1965;
Braune & Gentry, 1971; Ikeda, 1976a; Calnek & Jehnich, 1959a).
Egg-adapted strains, when administered to 1-to 2-day-old chickens by the oral
route, were shown to multiply to low titre in enteric tissues. No virus could be
detected in 110-day-old chickens inoculated in the same manner, but more acute
and widespread infection could be induced when egg-adapted strains were ad-
ministered by the subcutaneous (s.c.) route (Ikeda et al, 1976a,b). Ikeda et al
(1976a,b) also showed that virus could be detected at the site of inoculation (the
thigh muscle), the pancreas, liver, spleen and tissues of the CNS, and on
subsequent days in the remaining parenchymous organs. An age-related resist-
ance to infection was observed for egg-adapted strains in susceptible chickens
administered with the virus by either the s.c. or oral routes. Susceptible chickens
were shown to exhibit reduced age-related resistance to infection with egg-
adapted strains when they are administered by the i.e. route (Westbury &
Sinkovic, 1978a,c). When 6-months-old laying hens are infected by either the oral
or s.c. routes with egg-adapted virus, disease is more widespread and invasive
than that which occurs in 40- or 110-day-old chickens inoculated by the same
routes. The increased susceptibility of laying hens to infection by egg-adapted
AEVs is possibly due to the variation in hormone levels induced by the stress of
lay (Ikeda & Matsuda, 1976a,b).
Egg-adapted strains of AEV appear to lack the capacity to multiply in the
intestinal tract of susceptible chickens to sufficient levels to allow excretion of
virus in the faeces (Ikeda et al, 1976a,b; Calnek et al, 1960; Shafren & Tannock,
1991). Following oral inoculation of laying hens with egg-adapted strains, a
protective antibody response was shown to be evoked without the excretion of
virus in the faeces (Calnek et al, 1960). However, in a more recent study by
Shafren & Tannock (1991) in which AEV antigen was detected in the faeces and
antibody in the serum by sensitive ELISAs, there was neither evidence of
intestinal replication nor a serum antibody response. The decreased capacity of
egg-adapted strains to multiply in the chicken intestine reduces the likelihood of
viraemia and subsequent infection of the CNS (Ikeda et al, 1976a,b; Ikeda &
Matsuda, 1976a,b). Hence, egg-adapted AEVs appear to produce gross neural
disease in chickens only when directly administered by the i.c, intramuscular
(i.m.) and s.c. routes (Westbury & Sinkovic, 1978a; Miyamae, 1977).

Non-egg-adapted strains
Gross signs of infection induced by non-egg-adapted strains are usually limited to
young chickens. In adult chickens infection is usually subclinical, being detected
by drops in egg production and the appearance of clinical signs in infected
608 G. A. TANNOCK & D. R. SHAFREJST

progeny (Taylor et al, 1955; Calnek et al, 1960). The characteristics of infection
in young chickens inoculated with non-egg-adapted strains have been described
by Jones (1934), Van Roekel et al (1938), Olitsky (1939) and Hoekstra (1964).
Infected chickens usually exhibit tremors of the head or neck, ataxia, immobility,
muscular dystrophy and abnormal gait, which often prevent the chicken from
obtaining sufficient nourishment for survival. Lesions that are evident during
infection by non-egg-adapted strains are very similar to those induced by egg-
adapted strains. However, non-egg-adapted strains induce greater numbers of
lesions in the tissues of parenchymous organs of susceptible chickens than
egg-adapted strains (Miyamae, 1977; Ikeda et al, 1966).
Generally, non-egg-adapted strains of AEV do not induce gross signs of
infection in susceptible embryos (Butterfield et al, 1969; Van Roekel et al, 1938;
Hoekstra, 1964; Shafren & Tannock, 1990), although high titred strains may
induce some mortality in embryonated eggs. Six to 7-day-old embryos inoculated
with non egg-adapted strains by the yolk-sac exhibit clinical signs of the disease
1 to 9 days after hatching, indicating that virus multiplication occurs during
embryogenesis (Hoekstra, 1964; Springer & Schmittle, 1968; Braune & Gentry,
1971; Calnek et al, 1960). Hoekstra (1964) reported that maximum virus titres
could be detected in the brain, liver and intestines of 20-day-old chicken embryos
inoculated at 5 days.
Non-egg-adapted strains of AEV are more enterotropic than egg-adapted
strains and multiply in the intestines of susceptible chickens to levels that allow
excretion of virus in the faeces (Butterfield et al, 1969; Ikeda et al, 1976a,b;
Westbury & Sinkovic, 1978a,d; Shafren & Tannock, 1991). The concentration of
virus excreted in the faeces has been reported to be sufficient to induce clinical
disease in susceptible chickens when administered by the oral route (Calnek et al,
1961; Westbury & Sinkovic, 1976). Following oral administration of non-egg-
adapted strains to susceptible chickens, virus can be first detected in the pancreas,
liver and spleen within 3 days p.i., and on subsequent days in the CNS and
enteric tissues such as the proventriculus, small intestine and caecum (Springer &
Schmittle, 1968; Ikeda et al, 1976a,b). Similar patterns of virus distribution have
been observed in the organs of laying hens inoculated with non-egg-adapted
AEVs by the s.c. or oral routes, with highest titres of virus being detected in the
liver, spleen, pancreas and ovarian follicle. Virus in the ovarian follicle was shown
to remain at high titre for approximately 2 weeks, enabling potential transmission
of AEV to large numbers of eggs (Ikeda & Matsuda, 1976a,b). Inoculation of
susceptible chickens with non-egg-adapted strains by the oral, s.c, i.m. and i.e.
routes can induce neural symptoms that are indistinguishable from chickens
naturally infected with AEV (Calnek et al, 1960; Westbury & Sinkovic, 1978a).
Shafren & Tannock (1991) determined levels of antigen in various organs of
chickens infected with the egg-adapted Van Roekel strain, a non-adapted field
and a non-adapted vaccine strain by a sensitive antigen ELISA. Higher antigen
levels were detected in the heart, liver, pancreas and small intestines of birds
infected with the field and vaccine strains than for the Van Roekel strains 3 to 10
days p.i., but antigen levels in the brain were comparable for all three strains. The
 AVIAN ENCEPHALOMYELITIS 609

predilection of non-adapted strains for the intestine, liver and pancreas and the
growth of all three strains in chicken embryo heart muscle, suggests a pathogen-
esis similar to that of Coxsackie B3 viruses, which induce forebrain anomalies in
neonatal and infant mice and infect a range of other gut-associated organs (Gaunt
et al, 1984).

Mode of transmission
Studies on the route of transmission of AEVs have been largely concerned with
naturally-occurring non-egg-adapted field strains. Such strains have been shown
to be transmitted to susceptible chickens or embryos by either the vertical (Calnek
et al, 1960) or the horizontal (Calnek et al, 1961; Springer & Schmittle, 1968;
Miyamae, 1976, 1977) route. Excretion of the virus in faeces has been reported
to occur after 3 days (Calnek et al, 1961) following oral administration of
non-egg-adapted AEVs to adult chickens. The oral-faecal route of transmission is
the accepted mode of transmission of AEVs, but infection by the ocular route was
also suggested by Calnek et al (1960). The ocular route is an efficient means of
delivering vaccines, due possibly to its capacity to facilitate replication of the virus
in the upper enteric tract (Shafren et al, 1992). As with other enteroviruses,
infections due to AEV occur through ingestion of faecal contaminated food and
water, with initial replication probably taking place in columnar epithelial cells of
the small intestine. Thereafter, the virus probably enters the blood stream via
Peyer's patches and lymphatics, spreading to other organs and to the CNS.

The role of antibody in resistance to infection

The presence of neutralizing antibodies in the serum of chickens has long been
recognized as a major determinant of resistance to infection by AEV (Jungherr &
Minard, 1942; Schaaf & Lamoreux, 1955; Sumner et al, 1957b; Calnek &
Jehnick, 1959a,b; Cheville, 1970; Matsukura, 1970; Westbury & Sinkovic,
1978b). The failure of chickens to produce protective antibody following surgical
removal (Cheville, 1970) or chemical inactivation (Westbury & Sinkovic, 1978b)
of the bursa and thymus of developing chickens has been reported. An age-related
resistance to AEV, related to the ability of the chicken to produce neutralizing
antibodies against the virus, has been observed by many investigators (Cheville,
1970; Ikeda et al, 1976a,b; Westbury & Sinkovic, 1978a). However, the im-
munoglobulin class (es) involved in the neutralization of AEV have not been
described. As with human polioviruses, virus-specific IgG in the serum probably
prevents virus reaching the CNS and the development of neural symptoms.
Neutralizing antibodies, when passively transferred to susceptible chickens by
intraperitoneal inoculation, are able to inhibit AEV infection in susceptible
chickens challenged orally (Westbury & Sinkovic, 1978d). Passive transfer of
maternal antibody from hen to progeny has been shown to result in decreased
susceptibility to clinical signs of avian encephalomyelitis following challenge
(Westbury & Sinkovic, 1978d; Shafren et al, 1992). The duration of the immu-
610 G. A. TANNOCK & D. R. SHAFREN

nity has been shown to vary from 3 weeks after hatching (Westbury & Sinkovic,
1978d; Shafren et al, 1992) to 10 weeks (Calnek et al, 1960). However, the
presence of neutralizing antibody in the intestine, either as a component of
ingested maternal yolk or, indirectly, following the i.p. administration of immune
serum, has been shown to limit the development of clinical signs of infection and
reduce excretion of virus in faeces (Westbury & Sinkovic, 1978d). The role of
gut-mediated cellular immunity to AEV infection is uncertain.

ANTIBODY DETECTION ASSAYS

There have been many attempts over the years to develop rapid, reliable in vitro
assays for the detection of AEV. A neutralization test for AEV antibody was first
reported by Sumner et al. (l'957b), in which dilutions of an egg-adapted virus
were incubated with either undiluted serum or a 1:1 dilution of yolk in phosphate-
buffered saline and inoculated to 11-day-old embryos. After incubation for 7
days, the embryos were examined for signs of infection.
Calnek & Jehnich (1959a) developed a modified serum neutralization (SN) test
to that reported by Sumner et al. (1957b), in which the virus-serum mixtures were
administered to the yolk-sac of 6-day-old embryos. After incubation for 12 days,
embryos were examined for signs of infection that had been described by Jungherr
et al. (1956). Antibody levels were expressed as neutralization indices (NIs) from
the logarithmic difference in titre between a control virus mixture and virus-serum
mixtures. For these tests, the egg-adapted Van Roekel strain of AEV was used and
the results indicated that sera with NIs greater than 1.1 contained significant
levels of neutralizing antibody.
Due to the cost and time-consuming nature of the SN test, its application has
been limited to evaluation of single or pooled serum samples. In order to
overcome this, Sumner et al. (1957b) devised an assay, in which a standard
preparation of the Van Roekel strain was titrated in embryos from test flocks.
Embryos, in which virus growth was reduced by the presence of maternal
antibody in the yolk by at least 10 18 EID50/ml, were considered to come from a
positive flock. Embryos from 119 flocks were tested by the procedure and only
those from four were classed as susceptible. Arising from this work, two further
procedures were developed for assessing the susceptibility of flocks to outbreaks
of AEV, which were commonly used until comparatively recently:-

The embryo titration method

Here the Van Roekel strain is inoculated to 6-day-old embryos of a test flock at
a dilution that slightly exceeded the endpoint of the titration (usually a dilution of
10 ~6 to 10 ~ 7 ), in parallel with embryos from a known susceptible flock. The test
flock is considered antibody-positive, and therefore protected against AEV infec-
tion, where there is a difference of 102 EID 50 between the titres. When there is
no difference, the flock is classed susceptible; where the difference is less than
 AVIAN ENCEPHALOMYEIiTIS 611

lO2EIDso immunity is considered questionable, possibly indicating recent ex-

posure to the virus.

The embryo susceptibility (ES) test

At least 20 6-day-old embryos are inoculated via the yolk-sac with 100 EID 50 of
the Van Roekel strain. Following a 12-day incubation period, the embryos are
examined for signs of infection. The flock is considered to be fully susceptible if
all embryos were infected; if 0 to 50% were infected, the flock is considered
immune. A recent exposure to AEV is indicated when 50 to 100% are infected.
Development of in vitro antibody detection assays was hindered by difficulties
in preparing large amounts of concentrated purified virus antigen, problems in
obtaining specific-pathogen-free embryos and difficulties in growing AEV in cell
culture. As AEVs do not agglutinate red blood cells from known animal species
(Butterfield et al, 1969), simple haemagglutination-inhibition assays for antibody
detection are not possible.
An attempt to develop a complement-fixation (CF) assay using antigen from
chicken embryo brains infected with the Van Roekel strain was reported by Sato
et al. (1969). They showed that in only four out of 10 preparations of brain
antigen could low complement-fixing activity be detected in sera that had been
previously shown to be positive by an SN test. The performance of the CF test
was disappointing due to the presence of anti-complementary and haemolytic
activity in low dilutions of serum (Sato et al., 1969). Due to deficiencies in the
capacity of chicken sera to bind guinea-pig complement, CF tests are rarely used
for avian disease diagnosis. Choi & Miura (1972) reported the development of an
indirect fluorescent antibody technique for the detection of antibody to AEV,
using smears of brains from embryos infected with the Van Roekel strain as the
source of detector antigen. Of 40 sera from chickens in either early or late stages
of infection with NIs of less than 1.1 (i.e. negative), 19 were shown to be negative
at a 1:5 dilution and 21 were positive at either a 1:5 or a 1:10 dilution. These
results indicated that the test was more sensitive than the SN test although,
because the procedure is relatively time consuming, its potential for screening of
large numbers of sera is limited.
Numerous attempts to produce agar gel precipitin (AGP) tests for AEV
antibody detection have been reported (Lukert & Davis, 1971; Ikeda, 1976b]
Ahmed et al, 1982; Girshick & Crary, 1982), using preparations of the Van
Roekel strain as the detector antigen. Conflicting results for these tests have been
reported, which relate to the quality of the AEV antigen isolated from the brains
and gastrointestinal tracts of infected embryos. Virus concentrated from an
infected brain suspension by polyethylene glycol precipitation was reliably used to
detect AEV antibody by Ikeda (1976b) and Girshick & Crary (1982). However,
other reports suggested that, while gastrointestinal preparations of infected em-
bryos performed well, infected brain preparations were unsatisfactory (Lukert &
Davis, 1971; Ahmed et al, 1982). Ikeda (1976b) proposed that these discrepan-
cies were due to differences in viral antigen concentration and that a virus titre of
612 G. A. TANNOCK & D. R. SHAFREN

at least 10 50 EID50/0.1 g was required to produce precipitin lines. However, again

this proposal differed from the findings of Ahmed et al. (1982) who used a
concentrated brain homogenate with a titre 106-5EID50/ml without success.
The reported sensitivity of AGP tests for the detection of AEV antibody has
also varied considerably (Ikeda 1976b; Ahmed et al, 1982; Lukert & Davis, 1971;
Girshick & Crary, 1982), but, overall, there has been a reasonable correlation
with results obtained by the SN and ES tests. It is unknown whether the AGP test
detects the same types of antibody that is detected by the SN and ES tests.
However, the test may be useful for monitoring for the presence of AEV in flocks
and does not require specialized equipment.
A passive haemagglutination (PHA) test has been described for AEV antibody
detection which gave results that were more similar to results obtained with ES
and SN tests rather than AGP tests (Ahmed et al., 1982).
In recent years enzyme-linked immunosorbent assays (ELJSAs) for AEV anti-
body have virtually replaced SN, ES and AGP and other tests (Garrett et al,
1984; Malkinson et al, 1986; Richter et al, 1985; Smart & Grix 1985; Smart et
al, 1986; Shafren & Tannock, 1988; Shafren et al, 1989a, b) and a good
correlation between levels of antibody detected by EOSA, and VN and ES tests
has been reported. These tests have the combined advantages of being sensitive
and specific, as well as being rapid, relatively cheap and amenable to large-scale
screening for antibody in flocks and for assessing the effectiveness of vaccination
programmes.

INFECTrvTTY ASSAYS
The Van Roekel and other egg-adapted strains of AEV can be readily titrated by
inoculation of susceptible embryos and observing the embryo macroscopically for
signs of infection (Jungherr et al, 1956). However, titration of field isolates and
non-egg-adapted strains of AEV is more difficult, as these strains do not usually
induce clinical signs in embryos. Infection may be observed after allowing
inoculated embryos to hatch or after i.e. inoculation of chickens and observing for
a 28-day period et al, (Van Roekel et al, 1938; Jungherr et al, 1956). Hoekstra
(1964) reported the development of a chicken hatchout infectivity assay, whereby
embryos were inoculated via the yolk-sac with dilutions of virus and, after
hatching, the chickens were examined for signs of infection over a 28-day period.
This assay is often difficult to perform because embryos inoculated with high
concentrations of virus frequently do not hatch. Furthermore, as hatched unin-
fected chickens occasionally exhibit signs of abnormal gait, head tremors and can
be difficult to distinguish from infected chickens. The assay is cumbersome and
specialized facilities are required to hatch and brood infected chickens. Further-
more, as a procedure in which the endpoint is determined from the production of
neurological signs, the test is not viewed favourably by Animal Welfare authori-
ties. Berger (1982) reported the establishment of a cell culture infectivity assay for
all strains of AEV. Cultures of CEB cells in 96-well tissue culture plates were
inoculated with 10-fold dilutions of AEV and endpoints were determined by the
 AVIAN ENCEPHALOMYEUnS 613

detection of antigen in cells by immunofluorescence. Titres obtained by this

method were comparable with others obtained by the conventional embryo
inoculation or chicken hatchout assays. There have been further reports on the
use of chicken embryo brain cell culture assays for the titration of AEV based
upon the procedure, which confirm the findings of Berger (Hyde, 1986; Nicholas
et al, 1986a,b, 1987).
Shafren & Tannock (1990) reported the development of an egg infectivity assay
in which endpoints were determined according to the presence of AEV antigen in
specific-pathogen-free chicken embryo brains by an ELISA. This assay, which can
be used for both egg-adapted and non-egg-adapted strains, is particularly useful
for vaccine standardization.

Propagation of AEV in cell culture

Over the years there have been numerous attempts to propagate AEV in cell
cultures. In the first report by Hwang et al. (1960), egg-adapted AEV was shown
to induce cytopathic changes in monolayers of CEK cell cultures. However, in a
subsequent report, Burke et al. (1965; quoting unpublished data from R. E.
Luginbuhl) indicated that Hwang and others had, in fact, propagated chicken
embryo lethal organ (CELO) virus and not AEV.
Mancini & Yates (1967) reported multiplication of the egg-adapted Van Roekel
strain in cultures of chicken embryo brain (CEB) cells, using inoculation of
susceptible chicken embryos to detect cultured virus. Titres of up to 10 50
EIDso/ml were obtained 15 days p.i. without the appearance of a cytopathic effect
(c.p.e.), inclusion bodies or the formation of plaques in monolayer cultures. Virus
could be detected up to 20 days p.i. Because of the difficulties in producing
confluent monolayers of CEB cells due to contamination by fibroblasts and the
12-day incubation period required to reach confluence, alternative cell cultures
were investigated.
Chicken embryo fibroblast (CEF) and chicken embryo kidney (CEK) cultures
were shown to support multiplication of AEV to low titre (Mancini & Yates,
1968, 1986). Virus was detected for up to 25 days in CEF and 7 days in CEK
cultures, and maximum titres were obtained that were approximately lO2EID5o
less than that reported for CEB cell cultures (Mancini & Yates, 1967) with an
eclipse phase of 3 days. Despite growth of the virus, no visible c.p.e. or plaque
formation were observed in these cells.
Many other attempts have been made to propagate non-egg-adapted vaccine
strains of AEV in cell cultures (Sato et al., 1971; Halpin, 1966; Kodama et.al,
1975; Berger, 1982; Nicholas et al, 1986a,b; Shafren & Tannock, 1991). Cul-
tures of CEB and chicken pancreatic (CP) cells were shown to support the growth
of some non egg-adapted strains to produce titres of approximately 10 35 TCID 50 /
ml, but titres greater than that of the inoculum (lO3oEID5o/ml) were seldom
achieved. Eclipse periods of up to 4 days were evident in the above cell cultures
but plaque formation, inclusion bodies and a c.p.e. were not observed. AEV
614 G. A. TANNOCK & D. R. SHAFREN

antigen could not be detected by immunofluorescence in CEB or CP cells that

had been shown to support multiplication of AEV to low titre.
Throughout the 1980s there have been several reports of the successful appli-
cation of immunofluorescence for determining infectivity endpoints of egg-
adapted and non-egg-adapted strains of AEV in cultures of CEB cells (Berger,
1982; Nicholas et al, 1986a,b; Hyde, 1987). The replication of the Van Roekel
strain was also studied in CEB cell cultures by Nicholas et al. (1987). They
demonstrated an eclipse phase of 4 days and reported that five serial passages
were required to obtain titres of 10 20 TCID 50 /ml and an additional 10 passages to
achieve a titre of 10 50 TCID 50 /ml. Titres of approximately 10 50 TCID 50 /ml were
obtained in 10 subsequent passages of the virus in CEB cell cultures. Replication
of AEV in cell cultures of non-avian origin was first reported by Halpin (1966),
who reported that a brain homogenate from chickens exhibiting clinical signs of
AEV infection produced a c.p.e. in a monkey kidney cell line. However, the
serological identity of the infectious agent was not given and no further reports
have appeared. Abe (1968) compared the capacity of primary cell cultures
prepared from chicken, swine, and monkey tissues with many continuous cell
lines for their ability to support AEV replication following initial passage in CEB
cultures of an egg-adapted strain of AEV. Replication could not detected in any
of the cell cultures, according to the appearance of a c.p.e., gross signs of infection
in embryos inoculated with the cell culture material or the presence of viral
antigen by immunofluorescence. Shafren & Tannock (1991) studied the replica-
tion of the Van Roekel strain, a field strain and a vaccine strain in CEB cultures,
using an antigen ELISA to detect viral growth. They demonstrated an eclipse of
approximately 2 days for the Van Roekel strain, which produced antigen titres 8
to 10 times those produced by either of the non-adapted strains. Virus growth was
highly cell-associated.

VACCINATION
Live vaccines
Early attempts to produce effective AEV vaccines were undertaken by Jungherr &
Minard (1942) and Schaaf & Lamoreux (1955), using vaccine preparations
consisting of crude suspensions of brains from chickens exhibiting signs of
infection which were administered by the i.m. and wing-web (w.w.) routes.
Schaaf & Lamoreux (1955) observed clinical signs in approximately 1% of
400,000 chickens inoculated with live virus by the w.w. route 2 to 4 weeks p.i.
Administration of 25ID 5 0 of live virus by the w.w. route to susceptible chickens
afforded twice the level of protection from i.e. challenge with 250 ID 50 of virus,
than was observed in unvaccinated controls.
Schaaf (1958) compared the efficiency of the oral, i.m. and w.w. routes of virus
administration in inducing resistance to i.e. challenge with virulent strains.
Intramuscular inoculation was shown to induce a faster and more protective
response than after either w.w. or oral inoculation. However, a greater incidence
 AVIAN ENCEPHALOMYEIJTIS 615

of clinical signs of infection were noted in chickens following i.m. inoculation

compared with that observed after w.w. inoculation. Resistance to infection by
the progeny of vaccinated parents was considered to occur, either by vertical
transmission or by the passive transfer of neutralizing antibodies in the yolk
(Calnek et al, 1960).
The development of a vaccine that was both highly immunogenic and of low
neurovirulence was reported by Calnek et al, 1960, 1961, who compared its use
by a number of routes of virus administration. They showed that, although either
i.m. or w.w. vaccination could induce an acceptable level of protection, clinical
disease still occurred in approximately 10 to 20% of the flock. Virus doses of
15 X 103 ID 50 induced NIs of > 2.0 when administered by the i.m. or w.w. routes
and lower titres by the oral route. All birds with NIs of > 1.1 were refractory to
i.e. challenge, although some birds with NIs of < 1.1 were resistant.
Subsequent studies have shown that highly enterotropic strains of AEV admin-
istered orally are able to induce neutralizing antibody (Calnek et al, 1961;
Hoekstra, 1964; Westbury & Sinkovic, 1978) and that infectious virus was
excreted in faeces of inoculated birds for a period of 1 week (Calnek et al, 1961;
Hoekstra, 1964; Westbury & Sinkovic, 1978a,c,d; Shafren & Tannock, 1991).
The rapid spread of AEV through susceptible flocks following ingestion of
infected faeces enables small numbers of chickens to be administered vaccine
virus in order to confer protection on their contacts within the flock (Calnek et al,
1960, 1961; Hoekstra, 1964). These studies were confirmed by Shafren &
Tannock (1992), using an antibody EOSA to measure responses to vaccination.
Vaccination of 5 to 10% of a flock by the ocular route was also shown by the latter
workers to at least as efficient as inducing protective levels of antibody as that
achieved orally in the drinking water.

Inactivated vaccines
Betapropiolactone-inactivated preparations of the Van Roekel stain have been
studied as alternatives to live vaccines by several groups (Schaaf, 1959; Calnek &
Taylor, 1960; Butterfield et al, 1961). Approximately 102 to 103 times the initial
dose of virus was required in inactivated vaccines to induce antibody levels
comparable to those induced by oral administration with live vaccines. Inacti-
vated vaccines may be useful for inducing protection in susceptible flocks that are
close to or currently in lay without causing drops in egg production which
sometimes occur with live vaccines (Glisson & Fletcher, 1987).

• CONCLUDING REMARKS
AEV has long been recognised as the aetiological agent of one of the most
significant diseases of poultry. Despite its importance, relatively little progress had
been made towards an understanding of its basic biology and pathogenesis until
comparatively recently. Progress has been hindered by the incapacity of the virus
to grow efficiently in cell cultures and the absence, until comparatively recently,
616 G. A. TANNOCK & D. R. SHAFREN

of satisfactory in vitro assays for the detection of viral antigen and antibody.
Virtually nothing is known about the structure of the viral genome or details of
viral replication which, in comparison with other picornaviruses, appears to be
relatively inefficient and restricted to certain cells. Why clinical disease only
occurs in young chickens and causes a decline in egg production occurs is also
unknown. Details of the sequence of its RNA genome may provide information
as to why naturally occurring strains are usually enterotropic while egg-adapted
strains are usually neurotropic. Despite these limitations, satisfactory vaccines
against avian encephalomyelitis are available which can be standardized more
satisfactorily than was previously possible by older techniques. Furthermore,
vaccine efficacy and disease diagnosis can be monitored by antibody ELISAs
which afford reliable results within hours. Vaccination of flocks with live vaccines
is usually carried out at 11 to 12 weeks and the flock monitored for immunity to
AEV by an antibody ELISA after 3 to 4 weeks. If antibody levels are unsatisfac-
tory, there is usually sufficient time for revaccination before the time of lay.

ACKNOWLEDGEMENT
We thank Dr C. J. Morrow of the Victorian Institute for Animal Sciences for
reviewing the manuscript prior to submission.

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RESUME
Encéphalomyélite aviaire: une synthèse
Le virus de l'encéphalomyélite aviaire (AEV) est un picornavirus ayant une prédilection pour le
système nerveux central et d'autres organes parenchymateux du poulet, transmis par voie
orale-fécale. Le virus peut se disséminer d'une manière verticale ou horizontale du fait de sa grande
stabilité, les zones contaminées pouvant rester infectantes pendant de longues périodes. La souche
adaptée à l'oeuf, Van Roekel, est hautement neurotropique et se développe peu dans le tractus
digestif du poulet. Malgré cela, les polypeptides du virion des souches terrain et Van Roekel sont
antigéniquement identiques et il n'existe qu'un sérotype viral. L'infection naturelle par l'AEV
induit une maladie dans divers organes avec une pathogénie chez le poulet, similaire à celle
observée avec le virus Coxsackie B3 chez la souris nouveau-né. Les isolements du terrain d'AEV
peuvent être adaptés en cultures de cerveau d'embryon de poulet mais la croissance du virus est
très liée aux cellules. Des essais simples et fiables d'évaluation du taux des anticorps, pour
déterminer la présence d'infection récente ou étudier l'efficacité des vaccins, n'étaient pas
disponibles jusqu'à ce qu'ils soient récemment remplacés par la technique ELISA, alors
qu'auparavant ces essais nécessitaient l'utilisation d'embryons de poulet. Des programmes de
vaccination efficace utilisant les souches vivantes hautement entérotropiques de l'AEV ont
grandement réduit l'incidence de la maladie.
620 G. A. TANNOCK & D. R. SHAFREN

ZUSAMMENFASSUNG
Aviäre Enzephalomyelitis: Eine Übersicht
Das Virus der aviären Enzephalomyelitis (AEV) ist ein Picornavirus mit einer Prädilektion für das
Zentralnervensystem und andere paranchymatöse Organe des Huhnes, das über den Kot oral
übertragen wird. Das Virus kann vertikal und horizontal verbreitet werden, und wegen seiner
großen Stabilität können kontaminierte Areale für lange Zeit infektiös bleiben. Der eiadaptierte
Stamm Van Roekel ist hochgradig neurotrop und vermehrt sich nicht effizient im Darmtrakt des
Huhnes. Trotzdem sind sie Strukturproteine der natürlich vorkommenden Virusstämme und des
Van Roekel-Stammes antigenmäßig identisch, und es gibt nur einen Serotyp des AE-Virus. Die
natürliche Infektion mit AEV führt zu einer viele Organe erfassenden Erkrankung, die in ihrer
Pathogenese für Hühner der von Coxsackie B3 bei neugeborenen und jungen Mäusen ähnlich
ist. AEV-Feldisolate können an Hühnerembryo-Gehirnkulturen adaptiert werden, aber die
Virusvermehrung in Zellkulturen ist hochgradig zellassoziert. Einfache zuverlässige Anti
körpertests zur Ermittlung von frischen Infektionen oder zur Überprüfung der Wirksamkeit von
Vakzinen waren nicht verfügbar, bis vor relativ kurzer Zeit die vielfach auf der Verwendung von
Hühnerembryonen basierenden Methoden zum Antikörpernachweis durch den ELISA ersetzt
wurden. Wirksame Impfprogramme mit Einsatz von lebenden, hochgradig enterotropen
AEV-Stämmen haben das Vorkommen der Krankheit stark reduziert.

RESUMEN
Encefalomielitis aviar: Revisión
El virus de la encefalomielitis aviar (AEV) es un picornavirus con una predilección por el sistema
nervioso central y otros órganos parenquimatosos de los pollos y es transmitido por la ruta
orofecal. Este virus puede transmitirse horizontal y verticalmente y, debido a su gran estabilidad,
las áreas contaminadas pueden permanecer infecciosas durante largos períodos de tiempo. La
cepa Van Roekel adaptada al huevo es altamente neurotrópica y no crece bien en el tracto entérico
del pollo. A pesar de ello, los polipéptidos virales de esta cepa y de las cepas de casos espontáneos
de AEV son antigénicamente idénticos y existe un sólo serotipo. La infección natural con AEV
produce una enfermedad multisistémica con similitudes en su patogenia a la infección con el virus
Coxsackie B3 en ratones neonatos y muy jóvenes. Los aislamientos de campo de AEV pueden
ser adaptados para crecer en cultivos de cerebro de embrión de pollo aunque el crecimiento del
virus está muy asociado a la célula. Hasta hece poco tiempo no existían métodos sencillos y de
confianza que detectaran anticuerpos para diagnosticar infecciones recientes o para determinar
la eficacia de las vacunas, pero en la actualidad los métodos clásicos, mucho de los cuales
empleaban embriones de pollo, han sido substituidos por métodos de enzimoinmunoensayo. Los
programas de vacunación empleando cepas vivas muy enterotrópicas de AEV han reducido la
incidencia de la enfermedad.