- Email: [email protected]
Picornaviridae
CHAPTER
29
Richard E. Gough and M. Stewart McNulty
Picornaviridae The family Picornaviridae contains small, 22–30 nm, non-enveloped, icosahedral, singlestranded RNA viruses and is divided into nine genera. No virus isolated from avian species has been assigned to these genera, although avian encephalomyelitis has tentatively been placed in the Hepatovirus genus. A number of viruses that are pathogenic for chickens, turkeys and ducks have been provisionally identified as picornaviruses and, on the basis of their biological characteristics, are considered to be most like viruses of the Enterovirus genus. However, these viruses are poorly characterized and their true taxonomic status has not been determined. They include the causal viruses of duck virus hepatitis type 1, turkey viral hepatitis and others.
AVIAN ENCEPHALOMYELITIS (EPIDEMIC TREMOR) VIRUS The first record of avian encephalomyelitis was published in 1932 and concerned an outbreak in baby chicks in the USA 2 years earlier. The disease in chickens is now worldwide and was recognized in turkey poults in 1968. The natural disease has also been recognized in pheasants, Japanese quail and pigeons. Its economic significance stems from disease in chicks (paralysis, ataxia and muscular dystrophy), reduced egg production of a temporary nature in laying hens and an accompanying lowered hatchability of fertile eggs.
EPIDEMIOLOGY Cause Avian encephalomyelitis virus (AEV) has physical and chemical properties consistent with those of enteroviruses of the family Picornaviridae. However, recent characterization of its genome indicates that it is more closely related to Hepatitis A virus than to enteroviruses, and it has now been provisionally classified as a tentative species in the genus Hepatovirus in the family Picornaviridae. All strains seem to be antigenically uniform but there are variations in neurotropism and 350
Picornaviridae
virulence. Field strains are mainly enterotropic while fowl-embryo-adapted strains such as the Van Roekel (VR) strain are mainly neurotropic and are much more likely to kill embryos. The virus is relatively resistant to physical and chemical agents but there is little published information on its susceptibility to disinfectants.
Hosts The natural host range is limited to chickens, turkeys, Japanese quail, pheasants and pigeons. In addition, ducklings and guinea fowl are susceptible to experimental infection, while mice, guinea pigs, rabbits and monkeys are refractory. It seems to be of no significance to public health although other species of virus belonging to the Hepatovirus genus cause disease in mammals.
Spread The humoral immune status of the host appears to be the main factor influencing the outcome of infection. Maternal antibody can protect young birds against systemic infection and it generally becomes increasingly difficult to produce disease in chicks as they become older. This is attributed to increasing immunocompetence with age and the development of a protective humoral response. However, recent field observations have shown that chickens vaccinated by the oral route at 14 weeks of age may develop typical central nervous system lesions and clinical signs within 2–5 weeks of vaccination. It is considered that immunosuppression may have predisposed the birds to the clinical signs of disease in this instance. In birds infected or vaccinated during the rearing period, the immune response to natural infection or vaccination prevents subsequent egg transmission of the virus and provides protective maternal immunity that lasts through the highly susceptible period of the life of the young bird. When susceptible birds are infected after they come into lay, transmission of the virus occurs through the egg, and spreads horizontally from vertically infected to susceptible stock housed together and through fomites. Egg transmission occurs during the period from the infection of susceptible laying hens to the development of flock immunity: a period of 3–4 weeks. Transmission of infection from vertically infected to susceptible chicks can occur in the hatchery and during brooding and in older stock. It has been suggested that some infected birds become enteric carriers of the virus and excrete virus in their droppings for extended periods. The comparative resistance of the virus to physical inactivation also supports indirect spread through fomites. In the pathogenesis of the condition the virus enters the tissues from the infected egg in vertically infected chicks, while the oral route is most probable after hatching. In very young chicks the virus may be disseminated over a variety of tissues, including the brain, viscera and muscles. A proportion of infected embryos may be killed during the last few days of incubation; most of those that hatch show signs at 1–7 days of age. For those infected after hatching, the incubation period is at least 11 days. Thus the disease pattern may involve a reduction in hatchability, clinical signs in vertically infected chicks, which appear during the first 10 days of life, and signs in those infected after hatching, which are seen at 2–5 weeks. Although infection may be generalized following entry of the virus at any age, disease of the nervous system is normally seen only in the first few weeks of life.
DIAGNOSIS The clinical signs in young birds, the absence of gross lesions and the histological lesions in the brain, spinal cord and viscera, together with the absence of other virus infections and nutritional 351
3
| VIRAL DISEASES
deficiencies affecting the nervous system, are strongly suggestive of avian encephalomyelitis and are frequently used for routine presumptive diagnosis. In differential diagnosis it is necessary to consider other causes of neurological disorders, such as nutritional encephalomalacia and virus infections such as Newcastle disease, Marek’s disease, and equine encephalomyelitis and infectious meningoencephalomyelitis of turkeys, both of which are restricted geographically. A definitive diagnosis of avian encephalomyelitis requires demonstration of the virus by isolation or by other means.
Signs Clinical signs include depression, ataxia and tremors in chicks and some depression in egg production in layers, with reduced hatchability. Opacity of the lens, either unilateral or bilateral, occurs in relatively few survivors of a diseased flock and is thought to be caused by the virus. The nervous signs may be seen at or soon after hatching but are more commonly first seen at 1 week of age. The ataxia varies from slight incoordination to sitting on the hocks and lateral recumbency, when death results from inanition or trampling by other members of the flock. Very mildly affected birds may recover completely. Tremors may be absent from some outbreaks or may occur in only a few birds in an affected flock. They usually follow the initial stages of incoordination and may be confined to the head and neck, or be observed over the whole body. Some affected birds emit a plaintive cheep. Morbidity may be as high as 60% but is more commonly 15% of the flock, but mortality in affected birds is high. Fresh cases rarely occur after 5–6 weeks of age but avian encephalomyelitis has been reported in birds of more than 17 weeks. They showed paralysis of the legs and increased flock mortality of 0.2–1% per week. In layers the fall in egg production is about 5–10% and lasts for 5–14 days, with return to full potential production at the end of this time. The fall in hatchability accompanying the depression in production is about 5% of fertile eggs. In turkey poults and turkey hens the corresponding clinical signs are usually less severe. Clinical signs associated with natural infection of pigeons include paralysis of the wings, opisthotonos, torticollis and head tremor, particularly in birds up to 3 months of age. In adult birds diarrhoea also features.
Lesions There are no gross lesions in the young or older bird apart from rare pale areas in the gizzard muscle of chicks and opacity and fixation of the lens in a small proportion of survivors. Histologically there are lesions in the brain, spinal cord (but not the peripheral nerves) and viscera. Gliosis occurs in the molecular layer of the cerebellum as nodular aggregates. More diffuse gliosis is seen in the brainstem and midbrain and these changes, together with central chromatolysis of neurones in these areas, are considered by some to be almost pathognomonic. Neuronal degeneration is seen in Purkinje cells and neurones throughout the brain and spinal cord, especially in the pons, medulla oblongata and ventral horn cells of the cord. Perivascular cuffing with small lymphocytes is seen throughout the central nervous system and foci of infiltrating lymphocytes are seen in the dorsal root ganglia and in the proventriculus, gizzard, pancreas and other viscera. Similar lesions have been described in pigeons naturally infected with AEV.
Virus isolation/detection For virus isolation a suspension of brain, pancreas or duodenum from affected chicks is inoculated into the yolk sac of 5–6-day-old susceptible chick embryos. Eggs are candled daily and 352
Picornaviridae
12 days after inoculation some of the embryos are examined for gross signs of AEV infection, consisting of inertia, muscular dystrophy and occasional mortality. Virus neutralization tests in embryonated eggs, using extracts of brain suspension from affected embryos and AEV monospecific antiserum, have been used to confirm the identity of the virus. The remaining embryos are hatched and during the first 10 days of life the chicks are observed for typical clinical signs of avian encephalomyelitis. Examination of smears from the brain or cryostat sections stained by direct immunofluorescence may also be used to demonstrate virus; positive results are confirmatory but negative results are often unreliable. Increased sensitivity and specificity have been obtained by including an AEV monoclonal antibody in the assay. A nested polymerase chain reaction (PCR) has been described for the detection of AEV RNA in chicken embryos and in vaccinated chickens. However, the PCR is not yet routinely used in the diagnosis of AEV infection.
Serology A number of serological tests are available for determining the presence of infection. A virus neutralization (VN) test involves the inoculation of dilutions of an egg-adapted strain of virus alone and virus together with serum into susceptible chick embryos. By this means the neutralizing index of the serum can be determined and an index of log10 1.1 or greater is considered positive. The test may be made on acute and convalescent paired sera from birds showing a fall in egg production. A rise in titre indicates recent infection. Another method of demonstrating specific antibody is the embryo susceptibility test: 36–48 eggs from a flock are incubated for 6 days and then each live embryo is inoculated into the yolk sac with 100 EID50 (infective dose for 50%) of VR virus. The embryos are incubated for a further 12 days and examined. If fewer than 50% of them show muscular dystrophy the flock is considered to be immune. An indirect immunofluorescence test, immunodiffusion and an enzyme-linked immunosorbent assay (ELISA) have also been developed and compare favourably with the VN test. Because of its specificity and sensitivity, rapidity of performance and amenability to large-scale screening, the ELISA has replaced other tests for antibody, including the assessment of efficiency of vaccination.
CONTROL Drug therapy is of no value and under commercial conditions it is impracticable to attempt the elimination of infection by high standards of hygiene. Thus control is dependent on the vaccination of stock. Live and inactivated vaccines are available. Live vaccines are more commonly used; these can be given by mass administration, usually in the drinking water, and stimulate a durable and adequate degree of protection. Vaccination is undertaken to protect primarily against egg transmission of the virus and consequent disease in the progeny, and against a fall in egg production and hatchability. Commercial layers are often unvaccinated, since the total fall in egg production is small. The age at which birds are vaccinated with live vaccine should be chosen so that the virus will not produce adverse effects or fail to stimulate immunity. During the first 6 weeks of life live vaccine may produce disease, and during the first 8 weeks maternal antibody may interfere with the immune response. Live vaccine given to birds in lay may cause egg transmission and disease in the progeny. Thus, such vaccines are usually given between 8 and 16 weeks of age. A period of at least 2 weeks on either side of vaccination is allowed between this and the administration of other vaccines. 353
3
| VIRAL DISEASES
Vaccines similar to those used for chickens can be used for turkeys. In an outbreak in chicks or poults, affected birds are best destroyed, since most will succumb. Inactivated vaccines may be used for protecting susceptible laying flocks that are close to or currently in lay without causing a fall in egg production as might occur with live vaccines. Although certain features of the spread of this infection are not understood, hygienic precautions such as cleaning and disinfection between flocks and disposal of carcasses and litter must be practised in order to limit, at least, the build-up and spread of virus.
FURTHER READING Calnek B W 1993 Picornaviridae. In: McFerran J B, McNulty M S (eds) Virus infections of vertebrates, vol 4. Virus infections of birds. Elsevier, Amsterdam, p 465–478 Calnek B W 2003 Avian encephalomyelitis. In: Saif Y M (ed) Diseases of poultry, 11th edn. Iowa State Press, Ames, p 271–278 Calnek B W, Fabricant J 1981 Immunity to infectious avian encephalomyelitis. In: Rose M E et al (eds) Avian immunology. Poultry Science Symposium No. 16. British Poultry Science, Edinburgh, p 235–254 Marvil P, Knowles N J, Mockett A P A et al 1999 Avian encephalomyelitis virus is a picornavirus and is most closely related to hepatitis virus. J Virol 80: 653–662 Tannock G A, Shafren D R 1994 Avian encephalomyelitis virus: a review. Avian Pathol 23: 603–620 Todd D, Weston J H, Mawhinney K A et al 1999 Characterisation of the genome of avian encephalomyelitis virus with cloned cDNA fragments. Avian Dis 43: 219–226
DUCK VIRUS HEPATITIS Duck virus hepatitis was first described in 1950, causing severe losses in ducklings in Long Island, New York, then the major duck-growing area of the USA. It has since been described in most important duck-growing areas of the world. The disease has usually become endemic in these regions. Duck virus hepatitis is an acute, highly infectious viral disease of ducklings aged from 2 days to 3 weeks. Older ducklings may be diseased, particularly if affected by toxic substances or suboptimal nutrition, but adult stock are resistant. Age resistance to disease is essentially complete from 7 weeks of age.
EPIDEMIOLOGY Cause Three antigenically distinct viruses have been described that cause clinical signs and lesions sufficiently similar that they have been called Duck hepatitis virus (DVHV) type I, type II and type III. The originally described classic duck hepatitis (type I) agent is probably a picornavirus, while type II virus is an astrovirus (see Chapter 33 ). Type III virus, described in the USA, is also probably a picornavirus but it has not been fully characterized. The type I virus is more widespread and more virulent than the type II and III viruses. The viruses belonging to types I and III do not cross-protect. DVHV type I is highly resistant to physical and chemical conditions and the virus can remain viable in the environment for long periods of time. Variant strains of the virus have been reported from several countries. 354
Picornaviridae
Hosts While experimental infection has been described in other poultry species, natural infections with type I virus have been reported only in ducks.
Spread Type I and type III viruses remain viable for many weeks in faeces, etc., and it is therefore probable that infection follows the ingestion by susceptible ducklings of virus from the environment. Spread between sites is probably by means of contaminated equipment, vehicles and personnel. Egg transmission is not thought to occur. Within a flock the disease spreads rapidly to all susceptible ducklings.
DIAGNOSIS Signs Signs of type I infection are peracute and death usually follows within an hour of their onset. Affected birds are often in good condition but start to lose contact with the main flock. Soon they fall over on their sides and, after a short struggle, with paddling movements of the legs, the birds die. The head is usually stretched upwards and backwards (opisthotonos; Fig. 29.1). The mortality rate may be over 90% of the flock, although in the endemic situation a 5–10% loss is more common. The highest losses occur in ducklings less than 7 days of age. Signs of type III virus infection are similar, but less severe, with mortality usually below 30%. Any disease causing sudden death in young ducklings must be considered for differential diagnosis, the main ones being duck virus enteritis, bacterial septicaemia, coccidiosis and mycotoxicosis.
Lesions The main lesions are in the liver (Fig. 29.2), which is enlarged and has a number of petechial and ecchymotic haemorrhages. In older birds, a bacterial septicaemia may be superimposed,
Fig. 29.1 A young duckling that has died from duck virus hepatitis. Note the typical position of opisthotonos and the cleanliness of the down and feet.
Fig. 29.2 Young duckling showing typical liver lesions of duck virus hepatitis.
355
3
| VIRAL DISEASES
although the liver haemorrhages should still be evident. In addition, fatty kidneys described as duck fatty kidney syndrome may be caused by DVHV.
Virus isolation The sudden onset of a disease causing high mortality in young ducklings, the opisthotonos of the dead bird and the characteristic liver haemorrhages are together sufficient to justify the diagnosis of duck virus hepatitis. Laboratory diagnosis of type I virus infection is based on virus isolation following the inoculation of blood or organ suspension from affected ducklings into the allantoic sac of 9-day-old embryonating chicken eggs or 10- to 14-day-old duck embryos. Most embryos die within 6 days of infection. Rapid diagnosis using direct immunofluorescence may be made on the livers of affected ducklings. Type III virus can be isolated following inoculation of liver suspensions on to the chorioallantoic membrane (CAM) of duck embryos. Susceptible 1-day-old ducklings and duck cell cultures have also been used for virus isolation. Molecular techniques have not been described for the detection of DVHV type 1 in clinical material.
Serology Serological tests have not proved useful in diagnosis of type I DVHV. Neutralization tests in embryonated chicken eggs can be used to detect antibodies in convalescent serum samples and to determine the response to vaccination.
CONTROL Immunization of breeders with live attenuated vaccines provides maternally derived antibodies that prevent high mortality in young ducklings due to DVHV types I and III. Multiple vaccinations of breeders with attenuated type I virus are often required, both during the rearing period and continuing throughout lay. In some cases, breeder vaccination is augmented by passive immunization of their progeny using convalescent duck sera or egg antibodies derived from hyperimmunized fowl; this procedure has also been used to control outbreaks in the progeny of unvaccinated breeders. Experimental inactivated, adjuvanted type I vaccines for breeders have also been used successfully; priming with live vaccines gave best results. Vaccination of newly hatched ducklings with a single dose of live attenuated type I vaccine has also been used. These vaccines have been administered by a variety of routes, including foot web stab, subcutaneous, intramuscular, aerosol spray and drinking water. This strategy is dependent on the ducklings being fully susceptible (i.e. free from maternal antibodies) and on their being exposed to fairly low doses of virus following development of active immunity. Live vaccines readily revert to virulence following passage in susceptible ducklings. In theory, the disease can be prevented by rearing ducklings in strict isolation; in practice this is very difficult to achieve.
FURTHER READING Crighton G W, Woolcock P R 1978 Active immunization of ducklings against duck virus hepatitis. Vet Rec 102: 358–361 Woolcock P R 2003 Duck virus hepatitis. In: Saif Y M (ed) Diseases of poultry, 11th edn. Iowa State Press, Ames, p 343–354
356
Picornaviridae
OTHER AVIAN ENTEROVIRUS-LIKE VIRUSES A number of other enterovirus-like viruses have been detected in samples from several avian species, including chickens, turkeys, guinea fowl, pheasants, partridge, ostriches and psittacine species. In most cases virus particles have been observed by electron microscopic examination of intestinal contents or faeces and attempted propagation of the viruses has not generally been successful. However, several enterovirus-like viruses from chickens and turkeys have been isolated and propagated in the yolk sac and CAM of embryonated chicken and turkey eggs and partially characterized. They include isolates from the faeces of a broiler chicken, designated 84-700, from the meconium of dead-in-shell chicks from flocks with runting stunting syndrome, designated FP3, and from broilers with respiratory disease, designated 612. These isolates are antigenically unrelated to each other by cross-immunofluorescence or to AEV and duck hepatitis viruses. Antibodies to 612 and FP3 viruses are widely distributed in chickens in the UK. However, although 84-700 was isolated from chicken faeces, serological studies have shown that antibodies to 84-700 are widespread in turkeys. Turkey enterovirus-like viruses have been isolated from turkey poults with enteric problems in the USA and France. The US viruses are the best characterized and some of these are now known to be astroviruses. Enterovirus-like viruses from chickens and turkeys have been associated with a variety of conditions, including transmissible enteritis, runting and stunting syndrome and baby chick nephropathy. Experimental transmission studies with some of these isolates resulted in histopathological lesions of varying severity in the small intestine and the kidneys. Horizontal spread occurs readily, probably through ingestion of faecally contaminated material and vertical transmission through the egg probably occurs with most of these viruses. With the development and availability of molecular techniques it is probable that more enteroviruslike viruses will be detected and fully characterized in the future. Recent molecular investigations have already resulted in several viruses, formerly referred to as ‘enterovirus-like viruses’ (Avian nephritis virus, turkey small round viruses and entero-like viruses), being classified as species of Astrovirus. Indeed, it may transpire that more of the enterovirus-like viruses detected by electron microscopy in the past are in fact astroviruses. There is no evidence suggesting that these viruses are transmissible from avian species to humans or other mammals. Their transmissibility between avian species is also unknown. Vaccines and other specific control measures are not available.
TURKEY VIRAL HEPATITIS Turkey viral hepatitis is an acute, highly contagious disease of turkeys. It has been recognized in the USA, Canada, Italy and the UK. Outbreaks are usually seen in turkeys under 6 weeks of age. The condition is usually diagnosed only at post-mortem examination, when lesions are seen in the liver and sometimes the pancreas. Liver lesions are macroscopic pale foci 1–2 mm in diameter, occurring on the surface and in the parenchyma of the liver. Foci represent focal necrosis of hepatocytes and infiltration with mononuclear cells. Electron microscopic examination of degenerating hepatocytes has revealed the presence of intracytoplasmic aggregates of enterovirus-like viruses. The causal virus is currently classified as an unassigned virus in the family Picornaviridae. Turkey viral hepatitis is often subclinical but disease may be precipitated by stress. Depression, anorexia and increased mortality are the main signs. Morbidity can be very 357
3
| VIRAL DISEASES
high but mortality is normally low and occurs for only 1–2 weeks. Infection of laying turkeys may impair reproductive performance but deaths are not seen in birds over 6 weeks of age. Vertical transmission probably occurs. The causal virus can be grown in the yolk sac of chicken and turkey embryos, but has not yet been grown in cell cultures. Infected embryos die, with subcutaneous oedema and congestion, and may be stunted. As these signs are nonspecific, confirmation that turkey viral hepatitis is present requires intraperitoneal inoculation of day-old turkey poults with yolk sac material harvested from dead embryos. If the virus is present, necropsy of the inoculated poults 6–9 days later should reveal typical liver lesions. There are no effective treatments or control measures.
FURTHER READING Guy J S 1998 Virus infections of the gastrointestinal tract of poultry. Poult Sci 77: 1166–1175 Guy J S 2003 Turkey viral hepatitis. In: Saif Y M (ed) Diseases of poultry, 11th edn. Iowa State Press, Ames, p 399–403 Macdonald J W, Randall C J, Dagless M D 1982 Picorna-like virus causing hepatitis and pancreatitis in turkeys. Vet Rec 111: 323 McFerran J B 1993 Avian enterovirus infections. In: McFerran J S, McNulty M S (eds) Virus infections of vertebrates, vol 4: Virus infections of birds. Elsevier Science, London, p 497–503 McNeilly F, Connor T J, Calvert V M et al 1994 Studies on a new enterovirus-like virus isolated from chickens. Avian Pathol 23: 313–327 McNulty M S, Guy J S 2003 Avian enterovirus-like viruses. In: Saif Y M (ed) Diseases of poultry, 11th edn. Iowa State Press, Ames, p 326–332 McNulty M S, Connor T J, McNeilly F et al 1990 Biological characterization of avian enteroviruses and enterovirus-like viruses. Avian Pathol 19: 75–87
358
29
Richard E. Gough and M. Stewart McNulty
Picornaviridae The family Picornaviridae contains small, 22–30 nm, non-enveloped, icosahedral, singlestranded RNA viruses and is divided into nine genera. No virus isolated from avian species has been assigned to these genera, although avian encephalomyelitis has tentatively been placed in the Hepatovirus genus. A number of viruses that are pathogenic for chickens, turkeys and ducks have been provisionally identified as picornaviruses and, on the basis of their biological characteristics, are considered to be most like viruses of the Enterovirus genus. However, these viruses are poorly characterized and their true taxonomic status has not been determined. They include the causal viruses of duck virus hepatitis type 1, turkey viral hepatitis and others.
AVIAN ENCEPHALOMYELITIS (EPIDEMIC TREMOR) VIRUS The first record of avian encephalomyelitis was published in 1932 and concerned an outbreak in baby chicks in the USA 2 years earlier. The disease in chickens is now worldwide and was recognized in turkey poults in 1968. The natural disease has also been recognized in pheasants, Japanese quail and pigeons. Its economic significance stems from disease in chicks (paralysis, ataxia and muscular dystrophy), reduced egg production of a temporary nature in laying hens and an accompanying lowered hatchability of fertile eggs.
EPIDEMIOLOGY Cause Avian encephalomyelitis virus (AEV) has physical and chemical properties consistent with those of enteroviruses of the family Picornaviridae. However, recent characterization of its genome indicates that it is more closely related to Hepatitis A virus than to enteroviruses, and it has now been provisionally classified as a tentative species in the genus Hepatovirus in the family Picornaviridae. All strains seem to be antigenically uniform but there are variations in neurotropism and 350
Picornaviridae
virulence. Field strains are mainly enterotropic while fowl-embryo-adapted strains such as the Van Roekel (VR) strain are mainly neurotropic and are much more likely to kill embryos. The virus is relatively resistant to physical and chemical agents but there is little published information on its susceptibility to disinfectants.
Hosts The natural host range is limited to chickens, turkeys, Japanese quail, pheasants and pigeons. In addition, ducklings and guinea fowl are susceptible to experimental infection, while mice, guinea pigs, rabbits and monkeys are refractory. It seems to be of no significance to public health although other species of virus belonging to the Hepatovirus genus cause disease in mammals.
Spread The humoral immune status of the host appears to be the main factor influencing the outcome of infection. Maternal antibody can protect young birds against systemic infection and it generally becomes increasingly difficult to produce disease in chicks as they become older. This is attributed to increasing immunocompetence with age and the development of a protective humoral response. However, recent field observations have shown that chickens vaccinated by the oral route at 14 weeks of age may develop typical central nervous system lesions and clinical signs within 2–5 weeks of vaccination. It is considered that immunosuppression may have predisposed the birds to the clinical signs of disease in this instance. In birds infected or vaccinated during the rearing period, the immune response to natural infection or vaccination prevents subsequent egg transmission of the virus and provides protective maternal immunity that lasts through the highly susceptible period of the life of the young bird. When susceptible birds are infected after they come into lay, transmission of the virus occurs through the egg, and spreads horizontally from vertically infected to susceptible stock housed together and through fomites. Egg transmission occurs during the period from the infection of susceptible laying hens to the development of flock immunity: a period of 3–4 weeks. Transmission of infection from vertically infected to susceptible chicks can occur in the hatchery and during brooding and in older stock. It has been suggested that some infected birds become enteric carriers of the virus and excrete virus in their droppings for extended periods. The comparative resistance of the virus to physical inactivation also supports indirect spread through fomites. In the pathogenesis of the condition the virus enters the tissues from the infected egg in vertically infected chicks, while the oral route is most probable after hatching. In very young chicks the virus may be disseminated over a variety of tissues, including the brain, viscera and muscles. A proportion of infected embryos may be killed during the last few days of incubation; most of those that hatch show signs at 1–7 days of age. For those infected after hatching, the incubation period is at least 11 days. Thus the disease pattern may involve a reduction in hatchability, clinical signs in vertically infected chicks, which appear during the first 10 days of life, and signs in those infected after hatching, which are seen at 2–5 weeks. Although infection may be generalized following entry of the virus at any age, disease of the nervous system is normally seen only in the first few weeks of life.
DIAGNOSIS The clinical signs in young birds, the absence of gross lesions and the histological lesions in the brain, spinal cord and viscera, together with the absence of other virus infections and nutritional 351
3
| VIRAL DISEASES
deficiencies affecting the nervous system, are strongly suggestive of avian encephalomyelitis and are frequently used for routine presumptive diagnosis. In differential diagnosis it is necessary to consider other causes of neurological disorders, such as nutritional encephalomalacia and virus infections such as Newcastle disease, Marek’s disease, and equine encephalomyelitis and infectious meningoencephalomyelitis of turkeys, both of which are restricted geographically. A definitive diagnosis of avian encephalomyelitis requires demonstration of the virus by isolation or by other means.
Signs Clinical signs include depression, ataxia and tremors in chicks and some depression in egg production in layers, with reduced hatchability. Opacity of the lens, either unilateral or bilateral, occurs in relatively few survivors of a diseased flock and is thought to be caused by the virus. The nervous signs may be seen at or soon after hatching but are more commonly first seen at 1 week of age. The ataxia varies from slight incoordination to sitting on the hocks and lateral recumbency, when death results from inanition or trampling by other members of the flock. Very mildly affected birds may recover completely. Tremors may be absent from some outbreaks or may occur in only a few birds in an affected flock. They usually follow the initial stages of incoordination and may be confined to the head and neck, or be observed over the whole body. Some affected birds emit a plaintive cheep. Morbidity may be as high as 60% but is more commonly 15% of the flock, but mortality in affected birds is high. Fresh cases rarely occur after 5–6 weeks of age but avian encephalomyelitis has been reported in birds of more than 17 weeks. They showed paralysis of the legs and increased flock mortality of 0.2–1% per week. In layers the fall in egg production is about 5–10% and lasts for 5–14 days, with return to full potential production at the end of this time. The fall in hatchability accompanying the depression in production is about 5% of fertile eggs. In turkey poults and turkey hens the corresponding clinical signs are usually less severe. Clinical signs associated with natural infection of pigeons include paralysis of the wings, opisthotonos, torticollis and head tremor, particularly in birds up to 3 months of age. In adult birds diarrhoea also features.
Lesions There are no gross lesions in the young or older bird apart from rare pale areas in the gizzard muscle of chicks and opacity and fixation of the lens in a small proportion of survivors. Histologically there are lesions in the brain, spinal cord (but not the peripheral nerves) and viscera. Gliosis occurs in the molecular layer of the cerebellum as nodular aggregates. More diffuse gliosis is seen in the brainstem and midbrain and these changes, together with central chromatolysis of neurones in these areas, are considered by some to be almost pathognomonic. Neuronal degeneration is seen in Purkinje cells and neurones throughout the brain and spinal cord, especially in the pons, medulla oblongata and ventral horn cells of the cord. Perivascular cuffing with small lymphocytes is seen throughout the central nervous system and foci of infiltrating lymphocytes are seen in the dorsal root ganglia and in the proventriculus, gizzard, pancreas and other viscera. Similar lesions have been described in pigeons naturally infected with AEV.
Virus isolation/detection For virus isolation a suspension of brain, pancreas or duodenum from affected chicks is inoculated into the yolk sac of 5–6-day-old susceptible chick embryos. Eggs are candled daily and 352
Picornaviridae
12 days after inoculation some of the embryos are examined for gross signs of AEV infection, consisting of inertia, muscular dystrophy and occasional mortality. Virus neutralization tests in embryonated eggs, using extracts of brain suspension from affected embryos and AEV monospecific antiserum, have been used to confirm the identity of the virus. The remaining embryos are hatched and during the first 10 days of life the chicks are observed for typical clinical signs of avian encephalomyelitis. Examination of smears from the brain or cryostat sections stained by direct immunofluorescence may also be used to demonstrate virus; positive results are confirmatory but negative results are often unreliable. Increased sensitivity and specificity have been obtained by including an AEV monoclonal antibody in the assay. A nested polymerase chain reaction (PCR) has been described for the detection of AEV RNA in chicken embryos and in vaccinated chickens. However, the PCR is not yet routinely used in the diagnosis of AEV infection.
Serology A number of serological tests are available for determining the presence of infection. A virus neutralization (VN) test involves the inoculation of dilutions of an egg-adapted strain of virus alone and virus together with serum into susceptible chick embryos. By this means the neutralizing index of the serum can be determined and an index of log10 1.1 or greater is considered positive. The test may be made on acute and convalescent paired sera from birds showing a fall in egg production. A rise in titre indicates recent infection. Another method of demonstrating specific antibody is the embryo susceptibility test: 36–48 eggs from a flock are incubated for 6 days and then each live embryo is inoculated into the yolk sac with 100 EID50 (infective dose for 50%) of VR virus. The embryos are incubated for a further 12 days and examined. If fewer than 50% of them show muscular dystrophy the flock is considered to be immune. An indirect immunofluorescence test, immunodiffusion and an enzyme-linked immunosorbent assay (ELISA) have also been developed and compare favourably with the VN test. Because of its specificity and sensitivity, rapidity of performance and amenability to large-scale screening, the ELISA has replaced other tests for antibody, including the assessment of efficiency of vaccination.
CONTROL Drug therapy is of no value and under commercial conditions it is impracticable to attempt the elimination of infection by high standards of hygiene. Thus control is dependent on the vaccination of stock. Live and inactivated vaccines are available. Live vaccines are more commonly used; these can be given by mass administration, usually in the drinking water, and stimulate a durable and adequate degree of protection. Vaccination is undertaken to protect primarily against egg transmission of the virus and consequent disease in the progeny, and against a fall in egg production and hatchability. Commercial layers are often unvaccinated, since the total fall in egg production is small. The age at which birds are vaccinated with live vaccine should be chosen so that the virus will not produce adverse effects or fail to stimulate immunity. During the first 6 weeks of life live vaccine may produce disease, and during the first 8 weeks maternal antibody may interfere with the immune response. Live vaccine given to birds in lay may cause egg transmission and disease in the progeny. Thus, such vaccines are usually given between 8 and 16 weeks of age. A period of at least 2 weeks on either side of vaccination is allowed between this and the administration of other vaccines. 353
3
| VIRAL DISEASES
Vaccines similar to those used for chickens can be used for turkeys. In an outbreak in chicks or poults, affected birds are best destroyed, since most will succumb. Inactivated vaccines may be used for protecting susceptible laying flocks that are close to or currently in lay without causing a fall in egg production as might occur with live vaccines. Although certain features of the spread of this infection are not understood, hygienic precautions such as cleaning and disinfection between flocks and disposal of carcasses and litter must be practised in order to limit, at least, the build-up and spread of virus.
FURTHER READING Calnek B W 1993 Picornaviridae. In: McFerran J B, McNulty M S (eds) Virus infections of vertebrates, vol 4. Virus infections of birds. Elsevier, Amsterdam, p 465–478 Calnek B W 2003 Avian encephalomyelitis. In: Saif Y M (ed) Diseases of poultry, 11th edn. Iowa State Press, Ames, p 271–278 Calnek B W, Fabricant J 1981 Immunity to infectious avian encephalomyelitis. In: Rose M E et al (eds) Avian immunology. Poultry Science Symposium No. 16. British Poultry Science, Edinburgh, p 235–254 Marvil P, Knowles N J, Mockett A P A et al 1999 Avian encephalomyelitis virus is a picornavirus and is most closely related to hepatitis virus. J Virol 80: 653–662 Tannock G A, Shafren D R 1994 Avian encephalomyelitis virus: a review. Avian Pathol 23: 603–620 Todd D, Weston J H, Mawhinney K A et al 1999 Characterisation of the genome of avian encephalomyelitis virus with cloned cDNA fragments. Avian Dis 43: 219–226
DUCK VIRUS HEPATITIS Duck virus hepatitis was first described in 1950, causing severe losses in ducklings in Long Island, New York, then the major duck-growing area of the USA. It has since been described in most important duck-growing areas of the world. The disease has usually become endemic in these regions. Duck virus hepatitis is an acute, highly infectious viral disease of ducklings aged from 2 days to 3 weeks. Older ducklings may be diseased, particularly if affected by toxic substances or suboptimal nutrition, but adult stock are resistant. Age resistance to disease is essentially complete from 7 weeks of age.
EPIDEMIOLOGY Cause Three antigenically distinct viruses have been described that cause clinical signs and lesions sufficiently similar that they have been called Duck hepatitis virus (DVHV) type I, type II and type III. The originally described classic duck hepatitis (type I) agent is probably a picornavirus, while type II virus is an astrovirus (see Chapter 33 ). Type III virus, described in the USA, is also probably a picornavirus but it has not been fully characterized. The type I virus is more widespread and more virulent than the type II and III viruses. The viruses belonging to types I and III do not cross-protect. DVHV type I is highly resistant to physical and chemical conditions and the virus can remain viable in the environment for long periods of time. Variant strains of the virus have been reported from several countries. 354
Picornaviridae
Hosts While experimental infection has been described in other poultry species, natural infections with type I virus have been reported only in ducks.
Spread Type I and type III viruses remain viable for many weeks in faeces, etc., and it is therefore probable that infection follows the ingestion by susceptible ducklings of virus from the environment. Spread between sites is probably by means of contaminated equipment, vehicles and personnel. Egg transmission is not thought to occur. Within a flock the disease spreads rapidly to all susceptible ducklings.
DIAGNOSIS Signs Signs of type I infection are peracute and death usually follows within an hour of their onset. Affected birds are often in good condition but start to lose contact with the main flock. Soon they fall over on their sides and, after a short struggle, with paddling movements of the legs, the birds die. The head is usually stretched upwards and backwards (opisthotonos; Fig. 29.1). The mortality rate may be over 90% of the flock, although in the endemic situation a 5–10% loss is more common. The highest losses occur in ducklings less than 7 days of age. Signs of type III virus infection are similar, but less severe, with mortality usually below 30%. Any disease causing sudden death in young ducklings must be considered for differential diagnosis, the main ones being duck virus enteritis, bacterial septicaemia, coccidiosis and mycotoxicosis.
Lesions The main lesions are in the liver (Fig. 29.2), which is enlarged and has a number of petechial and ecchymotic haemorrhages. In older birds, a bacterial septicaemia may be superimposed,
Fig. 29.1 A young duckling that has died from duck virus hepatitis. Note the typical position of opisthotonos and the cleanliness of the down and feet.
Fig. 29.2 Young duckling showing typical liver lesions of duck virus hepatitis.
355
3
| VIRAL DISEASES
although the liver haemorrhages should still be evident. In addition, fatty kidneys described as duck fatty kidney syndrome may be caused by DVHV.
Virus isolation The sudden onset of a disease causing high mortality in young ducklings, the opisthotonos of the dead bird and the characteristic liver haemorrhages are together sufficient to justify the diagnosis of duck virus hepatitis. Laboratory diagnosis of type I virus infection is based on virus isolation following the inoculation of blood or organ suspension from affected ducklings into the allantoic sac of 9-day-old embryonating chicken eggs or 10- to 14-day-old duck embryos. Most embryos die within 6 days of infection. Rapid diagnosis using direct immunofluorescence may be made on the livers of affected ducklings. Type III virus can be isolated following inoculation of liver suspensions on to the chorioallantoic membrane (CAM) of duck embryos. Susceptible 1-day-old ducklings and duck cell cultures have also been used for virus isolation. Molecular techniques have not been described for the detection of DVHV type 1 in clinical material.
Serology Serological tests have not proved useful in diagnosis of type I DVHV. Neutralization tests in embryonated chicken eggs can be used to detect antibodies in convalescent serum samples and to determine the response to vaccination.
CONTROL Immunization of breeders with live attenuated vaccines provides maternally derived antibodies that prevent high mortality in young ducklings due to DVHV types I and III. Multiple vaccinations of breeders with attenuated type I virus are often required, both during the rearing period and continuing throughout lay. In some cases, breeder vaccination is augmented by passive immunization of their progeny using convalescent duck sera or egg antibodies derived from hyperimmunized fowl; this procedure has also been used to control outbreaks in the progeny of unvaccinated breeders. Experimental inactivated, adjuvanted type I vaccines for breeders have also been used successfully; priming with live vaccines gave best results. Vaccination of newly hatched ducklings with a single dose of live attenuated type I vaccine has also been used. These vaccines have been administered by a variety of routes, including foot web stab, subcutaneous, intramuscular, aerosol spray and drinking water. This strategy is dependent on the ducklings being fully susceptible (i.e. free from maternal antibodies) and on their being exposed to fairly low doses of virus following development of active immunity. Live vaccines readily revert to virulence following passage in susceptible ducklings. In theory, the disease can be prevented by rearing ducklings in strict isolation; in practice this is very difficult to achieve.
FURTHER READING Crighton G W, Woolcock P R 1978 Active immunization of ducklings against duck virus hepatitis. Vet Rec 102: 358–361 Woolcock P R 2003 Duck virus hepatitis. In: Saif Y M (ed) Diseases of poultry, 11th edn. Iowa State Press, Ames, p 343–354
356
Picornaviridae
OTHER AVIAN ENTEROVIRUS-LIKE VIRUSES A number of other enterovirus-like viruses have been detected in samples from several avian species, including chickens, turkeys, guinea fowl, pheasants, partridge, ostriches and psittacine species. In most cases virus particles have been observed by electron microscopic examination of intestinal contents or faeces and attempted propagation of the viruses has not generally been successful. However, several enterovirus-like viruses from chickens and turkeys have been isolated and propagated in the yolk sac and CAM of embryonated chicken and turkey eggs and partially characterized. They include isolates from the faeces of a broiler chicken, designated 84-700, from the meconium of dead-in-shell chicks from flocks with runting stunting syndrome, designated FP3, and from broilers with respiratory disease, designated 612. These isolates are antigenically unrelated to each other by cross-immunofluorescence or to AEV and duck hepatitis viruses. Antibodies to 612 and FP3 viruses are widely distributed in chickens in the UK. However, although 84-700 was isolated from chicken faeces, serological studies have shown that antibodies to 84-700 are widespread in turkeys. Turkey enterovirus-like viruses have been isolated from turkey poults with enteric problems in the USA and France. The US viruses are the best characterized and some of these are now known to be astroviruses. Enterovirus-like viruses from chickens and turkeys have been associated with a variety of conditions, including transmissible enteritis, runting and stunting syndrome and baby chick nephropathy. Experimental transmission studies with some of these isolates resulted in histopathological lesions of varying severity in the small intestine and the kidneys. Horizontal spread occurs readily, probably through ingestion of faecally contaminated material and vertical transmission through the egg probably occurs with most of these viruses. With the development and availability of molecular techniques it is probable that more enteroviruslike viruses will be detected and fully characterized in the future. Recent molecular investigations have already resulted in several viruses, formerly referred to as ‘enterovirus-like viruses’ (Avian nephritis virus, turkey small round viruses and entero-like viruses), being classified as species of Astrovirus. Indeed, it may transpire that more of the enterovirus-like viruses detected by electron microscopy in the past are in fact astroviruses. There is no evidence suggesting that these viruses are transmissible from avian species to humans or other mammals. Their transmissibility between avian species is also unknown. Vaccines and other specific control measures are not available.
TURKEY VIRAL HEPATITIS Turkey viral hepatitis is an acute, highly contagious disease of turkeys. It has been recognized in the USA, Canada, Italy and the UK. Outbreaks are usually seen in turkeys under 6 weeks of age. The condition is usually diagnosed only at post-mortem examination, when lesions are seen in the liver and sometimes the pancreas. Liver lesions are macroscopic pale foci 1–2 mm in diameter, occurring on the surface and in the parenchyma of the liver. Foci represent focal necrosis of hepatocytes and infiltration with mononuclear cells. Electron microscopic examination of degenerating hepatocytes has revealed the presence of intracytoplasmic aggregates of enterovirus-like viruses. The causal virus is currently classified as an unassigned virus in the family Picornaviridae. Turkey viral hepatitis is often subclinical but disease may be precipitated by stress. Depression, anorexia and increased mortality are the main signs. Morbidity can be very 357
3
| VIRAL DISEASES
high but mortality is normally low and occurs for only 1–2 weeks. Infection of laying turkeys may impair reproductive performance but deaths are not seen in birds over 6 weeks of age. Vertical transmission probably occurs. The causal virus can be grown in the yolk sac of chicken and turkey embryos, but has not yet been grown in cell cultures. Infected embryos die, with subcutaneous oedema and congestion, and may be stunted. As these signs are nonspecific, confirmation that turkey viral hepatitis is present requires intraperitoneal inoculation of day-old turkey poults with yolk sac material harvested from dead embryos. If the virus is present, necropsy of the inoculated poults 6–9 days later should reveal typical liver lesions. There are no effective treatments or control measures.
FURTHER READING Guy J S 1998 Virus infections of the gastrointestinal tract of poultry. Poult Sci 77: 1166–1175 Guy J S 2003 Turkey viral hepatitis. In: Saif Y M (ed) Diseases of poultry, 11th edn. Iowa State Press, Ames, p 399–403 Macdonald J W, Randall C J, Dagless M D 1982 Picorna-like virus causing hepatitis and pancreatitis in turkeys. Vet Rec 111: 323 McFerran J B 1993 Avian enterovirus infections. In: McFerran J S, McNulty M S (eds) Virus infections of vertebrates, vol 4: Virus infections of birds. Elsevier Science, London, p 497–503 McNeilly F, Connor T J, Calvert V M et al 1994 Studies on a new enterovirus-like virus isolated from chickens. Avian Pathol 23: 313–327 McNulty M S, Guy J S 2003 Avian enterovirus-like viruses. In: Saif Y M (ed) Diseases of poultry, 11th edn. Iowa State Press, Ames, p 326–332 McNulty M S, Connor T J, McNeilly F et al 1990 Biological characterization of avian enteroviruses and enterovirus-like viruses. Avian Pathol 19: 75–87
358