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Microbiology IV

GroupI:dsDNA
Adenoviridae
Many adenoviruses (G. adeno = gland), differentiated from one another by immunological
methods, have been isolated from mammals. A common antigen shared by all mammalian strains
differs from the corresponding antigen of avian strains. However, except canine hepatitis, most
are not serious causes of disease unless animals are immuno-compromised (i.e., immunologically
damaged). Although adenovirus infections of animals are often asymptomatic or subclinical, some
adenoviruses are pathogenic and cause respiratory and/or systemic diseases.
The family Adenoviridae is divided into two genera: Mastadenovirus (G. masto = mammal),
which includes adenoviruses that infect mammals, and Aviadenovirus or avian adenovirus (L. avi =
bird), which includes adenoviruses that infect birds. Adenovirus virions are nonenveloped with
icosahedral symmetry that are medium sized, 70 nm to 90 nm in diameter. The capsid consists of
capsomeres (called hexons) and 12 vertex capsomeres (called pentons). These are the only viruses
with a fiber (the fiber antigen) protruding from each of the 12 pentons. They are composed of 252
capsomers, each with 7nm in diameter. Of these 240 are hexons (hexamers; G. hexa = six) and 12
are pentons (pentamers; G. penta = five). The pentons have been shown to be toxic to cells, and may
represent a "viral toxin". The fiber is the structure of attachment to host cells and is also a type
specific hemagglutinin. The hexon of mammalian adenoviruses contains a
cross-reacting group antigen. The fiber antigen attaches to a specific cell
receptor and initiates replication.
Approximately 40 different proteins are encoded by the adenovirus
genome. Viral DNA replication, mRNA transcription and virion
assembly occur in the nucleus, utilizing both host and virus-encoded
factors. This results in the formation of basophilic and/or acidophilic
intranuclear inclusions. The nucleic acid consists of a single linear molecule of double-stranded
DNA (ds DNA (mol. wt. 20-29106 daltons), which is replicated in the nucleus, where a
characteristic inclusion body is produced. Extended fibers project from the virion surface.
Although suspected, a causal relationship of these viruses to spontaneous neoplasms has not been
established. Adenoviruses are highly species specific.
Many adenoviruses agglutinate red cells of various animal species and some are capable of
malignant transformation in tissue culture cell and oncogenesis when inoculated into laboratory
animals. They are resistant to trypsin and lipid solvents and moderately resistant on premises. The
viruses are usually associated with respiratory and intestinal infections and sometimes with eye
infection.
CAV-1 is most unlikely to be a significant cause of either of these two common diseases of
dogs.Adenoviruses of several serotypes have been recovered from many species which include
cattle, sheep, pigs, horses, dogs, and rats. These virus currently identified by the species of origin
and a number (e.g., adenovirus type 3).
Classification
Dr. Rebanta Kumar Bhattarai, M.V.Sc. (Vety. Microbiology)

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This family originally consisted of only two genera, Mastadenovirus, which infect mammals, and
Aviadenovirus, which infect birds. There are also several as yet unassigned and recently assigned
viruses in the family.
Mastadenovirus
This genus consists of 20 virus species that infect mammals including canine, equine, bovine, ovine
and porcine adenoviruses. All 20 species share a common antigen. Important diseases are infectious
canine hepatitis, canine adenovirus 2 infection, and equine adenovirus A infection.
Aviadenovirus
This genus includes the viruses of inclusion body hepatitis, quail bronchitis, marble spleen disease
and a number of adenoviruses of poultry and birds that are not associated with significant diseases.
Members of the genus share a common antigen.
Table 12. Diseases caused by Adenoviridae
Virus

Disease

Species

Genus: Mast-adenovirus
Canine adenovirus-1
Canine adenovirus-2
Bovine adenoviruses
types 3 and 5

Infectious canine hepatitis


Upper respiratory disease
Bovine adenoviral infections

Dogs
Dogs
Young Calves

Ovine adenoviruses
several different serotypes
Equine adenovirus
Porcine adenovirus
Simian adenovirus
Murine adenovirus

Ovine adenoviral infections

Sheep

Equine adenoviral infection


Porcine adenoviral infection
Simian adenoviral infection
Murine adenoviral infection

Sheep
Pigs
Simian
Mice

Genus: Avi-adenovirus (Avian adenovirus)


Group I (Conventional avian
adenoviruses)

Inclusion-body hepatitis
Chickens
Respiratory disease
Falls in egg production
Viral arthritis/tenosynovitis
Group II (type II avian adenoviruses)
Turkey haemorrhagic enteritis Turkeys
Marble spleen disease
Pheasants
Avian adenosplenomegaly Chickens
Group III (haemagglutinating Adenovirus)
Egg drop syndrome 1976
Chickens
1. Infectious Canine Hepatitis (canine adenovirus 1) Virus

Dr. Rebanta Kumar Bhattarai, M.V.Sc. (Vety. Microbiology)

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Disease
Also known as "hepatitis contagiosa canis", "Rubarth disease", and canine adenovirus
infection", fox encephalitis Infectious canine hepatitis (ICH) is infectious disease of dogs
caused by canine adenovirus-1(CAV-1), which is antigenically related only to canine adenovirus-2.
Although ICH was recognized for years, it was not established as a separate disease until the
classical report of Rubarth in 1947.
Although once an important disease of dogs, ICH is increasingly rare in much of the world, perhaps
as a result of widespread vaccination. The majority of infections are asymptomatic, but the disease
in susceptible dogs is characterized by fever, hepatic necrosis, and widespread hemorrhage as a
consequence of vascular injury. Affected dogs may exhibit increased thirst, anorexia,
tonsillitis, petechial hemorrhages on the mucous membranes, and diarrhea, and be reluctant to
move. During the acute phase of illness, dogs may also develop conjunctivitis and photophobia.
Severe ICH is most likely to occur in pups that are not immune to the disease.
Most dogs that survive acute ICH recover uneventfully, but transient corneal edema may occur in
some convalescent animals after acute signs disappear. The CAV-1 also has been implicated as a
cause of chronic progressive hepatitis and interstitial nephritis, but its role in spontaneous
occurrence of these disorders in dogs is highly conjectural.
Etiologic Agent
Physical, Chemical, and Antigenic Properties
Canine adenovirus Type 1 (CAV-1) is antigenically related but distinct from canine adenovirus 2.
Canine adenovirus 1 is morphologically similar to other adenoviruses, but antigenically distinct.
The virus of infectious canine hepatitis has the typical icosahedral capsid of an adenovirus, with a
particle diameter of about 55-80 nm. The DNA nature of its nucleic acid component was
confirmed by suppressing virus synthesis in dog kidney cell cultures with FUDR (5fluorodeoxyuridine) and by staining infected cell monolayers with acridine-orange which gives the
characteristic yellow-green colour to the intranuclear inclusions. Electron micrographs of thin
sections of infected cells show clusters of virus particles arranged in characteristic crystalline
arrays within the nuclei.
Resistance to Physical and Chemical Agents
CAV-1 is resistant to ether, alcohols, and chloroform since it contains no lipid, its infectivity is not
affected by treatment with lipid solvents including ether and bile salts. It is stable for at least 30
minutes at a wide range of pH (3 to 9), and is also stable in soiled material at room temperature for
several days. Viral infectivity is lost after heating for 10 minutes at 50 to 60C. Steam cleaning and
treatment with iodine, phenol, sodium hydroxide, or lysol are effective means of disinfection.
The virus, which is stable and withstands 0.5% phenol for several days, may be infective for 10 to
14 days on fomites because it is generally more resistant than distemper virus, kennels remain
infected for longer periods. It survives for 10 to 14 weeks at room temperature and for 6 to 9
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months at 40C, but is inactivated by 0.2% formalin in 24 hours.
Haemagglutination
Canine adenoviruses contain a haemagglutinin for human group O red cells, is also capable of
agglutinating guinea-pig erythrocytes (but not infectious canine laryngotracheitis virus).
Interference
Interferons are not produced by cells infected with canine adenoviruses and these viruses are not
affected by the action of interferon. For this reason, there is no interference between the viruses of
infectious canine hepatitis and canine distemper in live attenuated bivalent vaccines and both will
replicate satisfactorily in the inoculated animal. Indeed, fluorescent antibody staining shows that
the viruses can multiply in the nucleus and cytoplasm, respectively, of the same infected cell.
Antigenic properties
Infectious canine hepatitis virus, like all other animal adenoviruses, shares the soluble
complement-fixing antigens of human strains. Hence, they probably all share at least one of the
major antigenic components on the 'hexon' polypeptide, and another on that of the 'penton base'.
Precipitating antigens are often present in the organs of infected dogs and are readily identified
by agar gel diffusion tests. One of the two soluble antigens produced by infectious canine
hepatitis virus, as demonstrated on agar gel, is specific for canine hepatitis whereas the other is
shared with human adenovirus types 5 and 7. Infectious canine hepatitis virus appears to have only
one immunological type but differences in virulence may occur among strains.
It is emphasized that the virus of infectious canine hepatitis is unrelated to the agent of human
infectious hepatitis.
Replication

Assembling and maturation takes place in nucleus although most of their proteins are synthesized in the
cytoplasm.
After adsorption to the host cells, the virus transferred to the interior in membrane bound vesicle and
uncoating is within nucleus by means of preexisting cellular enzymes. Growth phase is slower but latent
phase is longer.
Viral protein is synthesized at 10 hrs and maturation usually takes place between 13-28 hrs after post
infection
Matured progeny virus is released from the nucleus either by disruption of the nuclear membrane or by
formation of large nuclear protrusions filled with virions which detach in cytoplasm
In all adenoviral infection, there is formation of intranuclear inclusion bodies.

Cultivation
The virus is readily propagated in kidney and testis cell cultures of dogs, other canidae (wolves,
foxes, and coyotes) and also in cultures of pig, ferret, racoon and guinea-pig cells. Growth in
rabbit, hamster, calf, mouse, monkey or human cells has not been recorded. Virus replication in cell
cultures is associated with rounding and swelling of infected cells, sometimes to twice their
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original volume, and the formation of intranuclear inclusions. The initially small, acidophilic
inclusions enlarge, frequently turn basophilic, and are connected to the nuclear periphery by
strands of chromatin. Many infected nuclei are seen to contain aggregates of virus particles
arranged in crystalline arrays when examined under the electron microscope.
Although some strains of CAV-1 and CAV-2 are oncogenic in inoculated hamsters, these viruses
have not been associated with neoplastic disease in dogs.
Host-Virus Relationship
Distribution, Reservoir, and Transmission
Infectious canine hepatitis has a worldwide distribution, although clinical disease is increasingly
rare. The infection is spread through the urine of infected dogs. Dogs may retain virus in their
kidneys and shed it in urine for months after infection. Transmission of Infection is by inhalation
and ingestion. Spread is by direct and indirect contact.
Pathogenesis
Following aerosol infection, the virus localizes in the tonsils and spreads to regional lymph nodes
and then to the systemic circulation. Viremia results in rapid dissemination of virus to all body
tissues and secretions, including saliva, urine, and feces. The virus has a particular tropism for
hepatocytes and endothelial cells, which produces the characteristic signs of the disease. Virusinduced injury to endothelial cells leads to consumptive coagulopathy (disseminated intravascular
coagulation) and a generalized bleeding tendency (hemorrhagic diathesis) that is reflected by
abnormal clotting parameters.
Dogs that die during the acute phase generally have edema and hemorrhage of superficial lymph
nodes and cervical subcutaneous tissue. The abdominal cavity often contains fluid, which may
vary in color from clear to bright red. Hemorrhages are present on all serosal surfaces. A fibrinous
exudate may cover the liver, which can be swollen and congested. The gall bladder
characteristically is edematous. Large characteristic intranuclear inclusion bodies may be
present in hepatocytes, vascular endothelium, and macrophages.
The ocular lesions that develop in some dogs that recover from ICH are the result of deposition
of immune complexes within the ciliary body of the eye.
Recently weaned puppies are particularly susceptible and show the highest mortality rates, whereas
relatively few adult dogs become sufficiently ill to show definite clinical symptoms. The usual
incubation period after natural exposure is 2-5 days but periods of 10-14 days may occur,
depending upon the virulence of the strain. In the most severe form, an apparently healthy dog
suddenly collapses with acute abdominal pain, intermittent vomiting and diarrhoea, and dies within
12-14 hours. The presence of blood in the vomit and faeces generally indicates impending death. In
less acute and recovering cases there is high fever accompanied by leucopenia, enlargement of
the tonsils and submaxillary lymph nodes and, sometimes, oedema of the cornea giving rise to
transient corneal opacity ('blue eye'). In older dogs the disease is mild and symptoms may be few
or absent.
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In foxes, the disease is usually characterized by acute encephalitis with convulsions followed
within 24 hours by paralysis, coma and death.
At necropsy, the predominant features are subcutaneous oedema and the presence of an
haemorrhagic exudate in the peritoneal cavity and intestinal tract. The liver is enlarged,
generally pale in colour and often shows a distinct mottled or lobular pattern. The spleen is also
enlarged and haemorrhagic, and the wall of the gall bladder is oedematous and several times its
normal thickness.
Histological examination of the liver shows a massive centrilobular necrosis and numerous local
dilatations of the sinusoids with an accompanying atrophy of the liver cells. The endothelial
cells are often swollen and many of them, like the liver cells, contain large acidophilic or
basophilic intranuclear inclusions. Basophilic inclusion bodies are also present in the lymph
nodes and kidneys. Fluorescent antibody staining reveals viral antigens in the nuclei or perinuclear
area of serosal, hepatic, Kupffer and vascular endothelial cells of the liver, as well as in the
endothelial nuclei of renal vessels.
Laboratory Diagnosis
Clinical specimens: liver, spleen, kidney, blood, urine, nasal swabs and paired serum samples.
The diagnosis of ICH can be confirmed by serologic testing (complement fixation,
hemagglutination inhibition enzyme-linked immunosorbent assay (ELISA) to demonstrate rising
titers of antibody to CAV-1, polymerase reaction (PCR) detection of viral nucleic acid or virus
isolation from affected tissues, or immunohistochemical staining of tissues with CAV-1-specific
antibodies.
The presence of intranuclear inclusion in smears or tissue sections.
Virus can be isolated in primary or secondary cultures of dog kidney cells from blood, tonsils
and, sometimes, the conjunctival sac. The isolate may be identified as an adenovirus by
complement-fixation.
Alternatively, viral antigens may be demonstrable in the blood by the complement-fixation test,
using specific antisera produced in experimentally infected animals.
Among survivors, a diagnosis can be obtained by demonstrating precipitating antibodies in the
sera or by showing a four-fold or greater rise in neutralizing or complement-fixing antibodies
in acute and convalescent paired sera taken at 14-day intervals. The antigen used may be in the
form of purified cell culture virus or an extract of infected liver tissue.
Haemagglutination-inhibition tests using albino rat or human group O red cells can also be used
for the detection of serum antibodies, and the litres are believed to correlate more closely with
neutralizing than with complement-fixing titres.
At necropsy, agar gel diffusion tests are especially useful for the rapid detection of specific
precipitating antigens in infected tissue fragments. Virus can be readily isolated from the affected
tissues by inoculation of cell cultures and its identity confirmed by complement-fixation or virus
neutralization.
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Fluorescent antibody staining can be employed for detecting antigens in smears, tissue sections or
infected cell monolayers,
Treatment and Control
Therapy for dogs that develop ICH involves supportive and symptomatic treatment. Control is by
vaccination and strict sanitation of affected premises with quarantine of exposed dogs. Available
vaccines include both inactivavated (killed) and modified live virus varieties, including vaccines
that induce heterologous protection against CAV-1. Vaccine often in combination with parvovirus and
canine distemper antigens. Modified live vaccines induce a longer lasting immunity, but a small
percentage of vaccinated dogs may develop ocular or renal lesions. Vaccine must be exercised to
ensure that maternal do not interfere with active immunization of pups, because vaccination
success is directly related to the level Neutralizing antibody.
Host immune Response to Infections
Recovery from ICH, regardless of the severity of illness, results in long-lasting immunity that likely
is lifelong. Recovered animals have high titers of neutralizing antibody to CAV-1.
Complement-fixing and precipitating antibodies appear in the blood of infected dogs in about 1421 days and reach their maximum titres within 10-12 weeks. Thereafter, the titres gradually decline
until they are often barely detectable by the 12th month. The immunity resulting from an active
infection with Rubarth's virus is solid and long-lasting, hence the absence of complement-fixing
antibodies is not a reliable indication of susceptibility. Dogs without complement-fixing antibodies
may still be immune to challenge with virulent virus and it is perhaps significant that these animals
mostly show measurable amounts of neutralizing antibodies in their sera.
Passive immunity is transferred to puppies the colostrum of recovered or vaccinated bite and the
protective antibodies have a 'half-life about 8 days.
Avian adenovirus
Adenoviruses infect poultry and other bird species worldwide. Although adenoviruses are often
isolated from apparently normal birds, specific diseases also are associated with adenovirus infections.
These include egg-drop syndrome, a disease of both wild and domestic birds that is characterized by
production of eggs that lack shells or have abnormally soft shells, and hemorrhagic enteritis of
turkeys and marble-spleen disease of pheasants, which are similar diseases characterized by
intestinal hemorrhage and enlargement of the spleen of affected birds.
Avian adenovirus are divided into 3 group.

Group I - Group I isolate share common Antigen

Group II- Turkey Hemorrhagic Enteritis Virus and Marble Spleen Disease Virus.
This virus shares a common group antigen which is different from Group I virus.

Group III- Haemagglutinating virus associated with EDS -76. This virus partially
shares the group I Common Antigen.
Group I
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In marked contrast to the clear association of Group II and Group III adenoviruses with disease, the
role of Group I adenoviruses as pathogens is not well understood.
12 serotype on the basis of serum neutralizing test
2. Inclusion Body Hepatitis /Hydropericardiaum syndrome
Angara disease and leetchi heart disease. Usually seen in meat producing chickens 3-7 wks of age,
characterized by sudden onset of mortality. No separate IBH virus. Virtually every serotype of
adenovirus Group I isolated from naturally occurring disease. All serotype produce hepatitis in young
chickens if inoculated parentererally. They produce eosinophilic and basophilic intranuclear
inclusion bodies in the hepatocytes in most natural cases and experimental infection respectively.
Cause
A number of serotypes of aviadenoviruses have been implicated.
Occurrence
The disease, which most often affects young chickens, occurs infrequently worldwide.
Transmission
Aviadenoviruses are transmitted horizontally and vertically.
Clinical & Pathologic Features

Infected chickens may appear anemic and weak but other characteristic clinical signs are absent.

The mortality is low in the uncomplicated disease, but as high as 30% in birds that are
immunosuppressed as a result of concurrent infection with the chicken anemia virus or the virus
of infectious bursal disease.

The liver of affected chickens is enlarged and pale and frequently with hemorrhages throughout.

Microscopically there is evidence of diffuse hepatitis with intranuclear eosinophilic inclusion


bodies in hepatocytes.

Anemia and icterus involving the musculature and subcutaneous tissues may be noted.

It is not certain that all the tissue changes seen in the disease can be attributed to aviadenovirus
infection alone.

Diagnosis

Clinical specimens: Sick, live birds are preferred.

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This is usually based on macroscopic liver lesions and microscopic examination of characteristic
intranuclear inclusion bodies in hepatocytes.

The virus can be propagated on the chorioallantoic membrane of chicken embryos or in avian
kidney cell cultures and identified by the virus neutralization test, but these procedures are
expensive and seldom performed. Because of the widespread distribution of adenoviruses in
chickens, isolation of virus is not necessarily meaningful.

Group II
3 disease
1. THE- It is acute viral disease of turkey 4 wks of age, and older characterized by
depression, bloody drooping and death. Microscopically, nucleus of infected cells in the
spleen contains characteristic intranuclear inclusion bodies.
2. MSD of pheasants- pheasant of 3-8 months and death due to asphyxia. Produce
intranuclear inclusion and spleenic lesion similar to THE virus
3. Avian adenosplenomegaly(AAS) virus - Spleenomegaly in young and market age
broiler. Splenomegaly with pulmonary ingestion and edema in mature birds. Death due to
asphyxia. Intranuclear inclusion and spleenic lesion are similar to THE Virus.
Group III
1. EDS-76 disease

Major cause of loss of Egg production throughout world. Characterized by otherwise healthy birds
produce and thin shelled or shellless eggs.
EDS Virus differ from Group I serotype I adenovirus.
Although virus share conventional avian adenovirus group Antigen, this can be detected only by
indirect means.
All EDS virus isolates belong to one serotype.
Occurrence
Egg drop syndrome (EDS) is common and worldwide in distribution. It occurs most frequently in
broiler chicken breeder flocks 5 - 6 weeks of age. Infact, EDS virus is naturally occurring adenovirus
of duck and geese which has infected fowl.
Transmission
It is mainly transmitted vertically through the egg. The virus is shed in the feces and spread can be by
contaminated water and fomites. Sporadic outbreaks have been attributed to wild birds
contaminating water.
Morphology
Replication

Same as previous

Dr. Rebanta Kumar Bhattarai, M.V.Sc. (Vety. Microbiology)

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Cultivation :
Propagated readily in chicken embryo causing death and stuning of chick embryo.
Cell culture:
It grow best in duck and geese cell culture, but also well in chicken embryo liver and chicken kidney
support the growth of viruses and produces CPE.
Pathogenesis :

Incubation period is 7 days


After horizonatal transmission, virus grows low titre in the nasal mucosa, viremia and virus
replicates in lymph nodes.

8 days of post-infections, virus massively grows in pouchshell gland of egg region of oviduct.

Results in egg shell changes

Both exterior and interior of the eggs produced between 8-18 days after infection contains virus.
Copious exudate of oviduct is rich with virus and contaminates drooping.

Clinical & Pathologic Features

Infected birds appear healthy. Egg production is variably depressed and abnormal eggs are
produced.

Shells of the latter may be absent, thin, under-pigmented and rough surfaced. Outbreaks last 4 to
10 weeks.

Among the effects noted are inactive ovaries, atrophied oviducts, edema of the uterus, exudates
in the cell gland and intranuclear inclusions in tissue of the cell gland.

Diagnosis

Tentative diagnosis through history and clinical symptoms. Loss of egg production with
abnormal eggshells suggest EDS.

Clinical specimens: eggs and reproductive tissues including the shell gland.

By isolation of virus in chicken embryo and different cell cultures.

Virus can be cultivated in duck and goose embryonated eggs (preferred) and also in duck kidney
or fibroblast cell lines.

The presence of virus is indicated by hemagglutination of avian red cells.

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In cell cultures, these viruses produce cytopathic effect. The nucleus of infected cells enlarged
and filled with basophilic granules and later: intranuclear inclusion bodies can be seen.

Diagnosis is made with the hemagglutination inhibition test. It is used to screen flocks but
negative tests do not indicate that birds are necessarily free of infection.

Serological tests- HI test, ELISA, Serum neutralization is used for conformation.

Prevention

Replacement birds should be from uninfected flocks.

An oil adjuvant inactivated vaccine provides immunity for a year. It reduces but not prevent
virus shedding. Mostly given intramuscularly during growing phase usually at 14-18 weeks old

Combined (EDS+ND) vaccine is also applicable

Good hygiene to prevent lateral spread particularly from infected egg contamination.
1. Sheep pulmonary Adenomatosis

A very slowly progressive shronic lung infection of sheep characterized by gradually increasing
dyspnea, ematiation and eventually death.
Morphology :

Particles vary in size from 80-100nm in diameter.

Capsids are hexagonal with hollow elongated peripheral capsomeres

Enveloped, ds DNA

Infectivity is abolished by a temperature of 56oC for 60minutes or by exposures of chloroform.

Partially acid labile.

Cultivation:
1. Cannot be propagated in embryonated hens egg and does not produce conjunctivitis in rabbits by
corneal scarification or deaths in day old mice injected intracerebrally.
2. Cell cultures
In cultures of foetal human fibroblasts and foetal sheep lung, produce CPE.

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Pathogenesis:

Transmitted naturally by herding healthy and diseased sheep together.

The signs of disease seldom occur in sheep under 2-3 years of age and most common in animals
4 years old or more.

The IP is very variable but is probably longer than 6-9 months.

The clinical illness usually lasts 2-8 months


Condition is seen as a febrile, chronic progressive respiratory condition characterized by
increasing dyspnea, coughing, abundant nasal discharge, inappatance, and ematiation, death.

Mortality rate is about 1-3%

Recovery from sheep pulmonary adenomatosis(SPA) has not been reported.

Necropsy Findings:

Small grey multifocal nodules in the apical, cardiac and lower part of diaphgramatic lobes of
lung.

In advanced case, lesions may be consolidated due to secondary bacterial infection.

On squeeze, a thin frothy fluid usually exudes from the cut surface.

Peribronchial lymphnodes are hyperblastic and markedely enlarged.

Peribronchial lymphnodes are hyperplastic and markedly enlarged.

Histologicallyo Accumulation of large mononuclear cells.


o Proliferation and hyperplasia of the cells lining the bronchioles and alveolar ducts.
o Papilliform epithelial projections may extend from alveolar septa.

Diagnosis:
1. Clinical symptoms are very similar to other chronic lung disease and hence accurate diagnosis is
possibly cy histopathological examination.

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2. Animal inoculation.
In susceptible sheep, when we inoculate extracts of or filtrates from lesions of diseased lung, by
intranasal or intratracheal inoculation- similar lungs lesions will develop that can be observed
after slaughter.
3. Serological test- no serological tests for the detection of specific antibodies.
4. Cell cultures- human foetal fibroblast and sheep foetal lung cells----------this virus produce CPE.
Control

No active method immunizatoion is yet available.

Eradication is only possible after slaughtering.


Papovaviridae

Grouping of three different viruses leading to the acronym (a word formed from the first letter of a
group of words).
Papilloma virus
Papova
Polyoma virus
Vacuolating agent
Papovavirus are small (45-55 nm in diameter)
Non-enveloped
Icosahedral virions
The genome consist of one molecules of double stranded DNA(dsDNA), codes 8-10 protein, 2
are structural and remaining are non structural protein required for virus replication.
The family contains two genera
Papilloma virus
Polyoma virus, which are immunologically distinct.
Only papilloma viruses are veterinary importance and widespread among mammals (cattle,
sheep, goat, horses, dogs, pigs and birds).
The genus papillomavirus contains virus responsible for benign papillomatosis (e.g. wart,
verrucae vulgaris) in human and other animals. Virus induced papillomas (wart) are benign,
hyperplastic epithelial proliferation of the skin or mucous membrane that may undergoes
malignant transformation in certain circumstances.
Virus induced papillomas (wart) are common in horses and cattle and infrequent in dogs, sheep
and goats.
The viruses are highly species specific, with exception of bovine papilloma virus and are
contagious only to the animal species in which they naturally occur.
As with oncogenic DNA virus, viral replication doesnt occur in the transformed, rapidly
dividing cells.
Therefore, viral particles and viral antigens are not demonstratable in the cells of the germinal
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layer.
However, the cells of the upper layers i.e. the epidermis are permissive for viral replication and
viral particles can be localized here.
Basophilic intranuclear inclusion bodies may also be seen in these cells.
Only human and bovine papilloma virus has been characterized in detail.

Papilloma types

Papilloma viruses are highly restricted in both their host. Specifically, tissue and cell tropism and
sequence relatedness.
They are distinguished on the basis of lesion thy induce, including skin papillomas (warts),
proliferation of non-stratified squamous epithelium (polyps) and subcutaneous fibromas with or
without associated cutaneous papillomas (fibropapillomas)

Bovine paplloma virus

At lest 6 serotype of bovine papilloma virus are distinguishing on the basis of their antigenic and
nucleotide sequence homologies.
They can be further distinguished on the basis of the nature of the lesions that they cause in
cattle.
Cutaneous fibropapillomas (warts)
1.

Papillomatosis

It is benign tumors (warts) of the skin and mucous membrane of animals caused by papilloma virus.
Morphology:

Non-enveloped, icosahedral virions about 45-55nm in diameter.


72 capsomeres (each 8 nm across) in a skew arrangement but hollow and tubular or filamentous
forms may also be present.
ds DNA which is often circular or supercoiled (MW-3-5106 daltons)

Cellular transformation:
Two types of virus-cell interactions can occur following infection. Either there is productive or lytic
cycle resulting in synthesis of progeny virus followed by death of the infected cells or infection is
abortive, whereby little or no infectious virus is released and infected cell survives although it is
permanently attend or transformed.
Transformed cells produce, new proteins type of viral antigens distinct from the structural proteins of
the virionsi.
T-antigen (t for Tumor)= occur mainly in nucleus.
ii.
Transplantation antigen=occur in plasma membrane of transformed cells
The continued synthesis of these new proteins is due to permanent retention of viral genetic
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material within the transformed cells, which is incapable of releasing mature infectious virus.
Production of infectious virus can be induced following exposure to ultraviolet irradiation or
experimentally with co-cultivation, whereby transformed are fused artificially with permissive cells
by means of inactivated sendai virus.
Cultivation:

Growth on chorioallantoic membrane (CAM) of embryonated hens egg has not been reported.
Papillomavirus cannot be propagated in cell culture. The virus species induce papillomas in the
host of origin but rarely cause cell transformation or cytopathic effects (CPEs) in cell culture.
In experimental house, subcutaneous (S/C) inoculation of the virus gives rise to connective tissue
tumors.
Intracerebrally injected calves may develop meningeal fibromoda.
Unweaned hamsters and certain strains of mice (C3H/eB) on subcutaneous inoculation develop
tumor at the inoculation site within 100 days. The tumors are firm, multiple or singe, measuring
about 8-15 nm in diameter and persist for 4-6 months without evidence of metastasis.

Pathogenesis:

All ages of cattle are affected but the incidence is higher among calves and yearlings than adults.
The lesions (which are firm nodules to large cauliflower like growth) appear more frequently
when the animals are housed than when on pasture. In dairy cattle, lesions re mostly found in teat
and udder.
In beef cattle, wart occurs on the skin around the eyes, mouth, ears, neck, shoulders.
Occasionally, lesions are found in prepuce of bulls, vulva and vagina of heifers, the urinary
bladders of calf and oesophagus or omasum of older cattle but the curve of these is due to
papilloma virus is still not known.
The mechanism of tumor induction by papilloma virus is largely unknown.
The virus probably gains entry
o Directly via abrasion in the skin by direct contact with infected animals.
o Indirectly by means contaminated inanimate objects like
Halters, syringe and needle, tattooing machine or other equipments used for managemental
practices.
Papillomavirus affect cutaneous and mucosal squamous epithelium and induce benign
circumscribed tumor, which tends to regress spontaneously after a period of time.

Diagnosis:

A viral wart is -byand there is no need for confirmatory diagnosis.


Confirmed by demonstrating the presence of viral particle in the negatively stained specimen of
wart tissue by electron microscope.

Immunity and control:


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Formalinized wart tissues have been used as autogenous vaccine either for propylaxis or cure.
Dose 10ml S/C repeat after 10-14 days.

Polyoma Virus

The genu (G. Poly= many, oma= tumor) is made up of several virus of scientific interest because
of their oncogenic and transforming effects on mammalian cell systems.
Poxviridae

Pock means a postule or ulcer


The double-stranded DNA virions of this family are the largest and most complex of known animal
viruses. They infect many vertebrate and insect species. Unlike other viruses, some poxviruses are
large enough to be seen with a light microscope.

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Microbiology IV

Poxviruses are largest (170300nm), enveloped (some virions contain double envelope), and
most complex ds DNA viruses that replicate wholly within cytoplasm.

Fig. Poxviridae (220 - 450 x 140 - 260 nm). Indicated are the surface tubules, the envelope
(present only when virions have budded), the biconcave core that surrounds the nucleoprotein,
and the lateral bodies (function unknown). To view click on figure

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Microbiology IV

The capsid / nucleocapsid is brick-shaped to ovoid or wool ball shaped containing the genome
and lateral bodies (function unknown).and outer envelops.
The capsid symmetry is complex.

The large complex genome consists of a single, linear molecule of double stranded DNA that
codes for approximately 200 proteins. The ends are ligated to each other so the DNA molecule is
continuous, without free ends.

Although the nucleocapsid is enveloped, most members are resistant to ether but few e.g.
neethling strain of lumpy skin disease are partly or highly ether sensitive.
The mol. Wt. of poxvirus DNA is 150-160106 daltons.
These viruses are acid labile.
Poxviruses infect epithelial cells where they produce papules, vesicles and pustules (pock
formation)
In the cytoplasm, the dsDNA is used as a template for both mRNA production (for translation of
proteins) and copies of the genome for progeny virions; viral enzymes largely mediate both
processes. As the virions are large and complex, the mechanism associated with virion assembly
is largely unknown. Virions are released from the cell by budding.

Viruses of this family possess at least 10 major antigens with a common nucleoprotein antigen,
which accounts for cross-reactivity among species.

There are at least 10 viral enzymes contained within the virus particle, many of which function in
nucleic acid metabolism and genome replication.

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Poxviruses remain viable in scabs for long periods.

Some (mainly orthopoxviruses) produce hemagglutinins that agglutinate red blood cells.

Eosinophilic inclusions called Guarnieri bodies may be produced in infected cells/tissues.


Classification

The Poxviridae consists of two subfamilies, Chordopoxvirinae (poxviruses of vertebrates) and


Entomopoxvirinae (poxviruses of insects).
There are eight genera in the subfamily Chordopoxvirinae. They are, with significant diseases, as
follows:

Genus
1. Orthopox virus
2.
3.
4.
5.
6.
7.
8.
9.

sps:

Vaccinia virus, monkey/camel/cow pox virus,


Variola. Horsepox, Buffalopox
Capripox virus
sps: Sheep pox, goat pox, Lumpy Skin Disease
Aviapox virus
sps : Fowl pox, pigeons pox
Parapox virus
sps : Contagious Ecthyma / orf virus, bovine popular stomatitis virus,
pseudocowpox virus/ milkers nodules
Leporipox virus sps: Myxomavirus of rabbit
Fibromavirus of rabbit
Suipox virus
sps: Swine pox virus
Molluscipoxvirus sps: Lumpy skin disease(neethling)
Mollusccum contagiosum: A common disease of children
Suipoxvirus:
sps: Swinepox
Yatapoxvirus:
sps: Yaba monkey tumor virus and related viruses

Pox viruses
Poxviruses infect the epidermis and produce focal lesions that frequently become proliferative
and later necrotic.
Rare generalized infections can be fatal.
Poxviruses occur naturally in most veterinary species, except the dog.
Many poxviruses produce an infection resulting in changes conveniently summarized in order of
development as papule, vesicle, pustule, and finally scabs or crusts. Secondary bacterial
infections are not uncommon.
Recovery from poxvirus infection usually is followed by long-term immunity.
Morphology:

Brick or wool ball or round shaped virions, 250-350 nm in length 200-250 nm in width.
Complex structure with core, lateral bodies and outer membrane (envelop).
The central core or nucleoid contains genome which is a single molecule ds DNA (Mol.wt. 150160106 daltons)

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Replication:

Little is known about poxvirus replication. The viruses have more than 100 proteins and several
enzymes (including DNA-dependent DNA polymerase and a DNA dependent RNA polymerase).
Replication poxviruses occurs in the cytoplasm and can be demonstrated in enucleated cells.
However, a function associated with the nucleus appears to be required for efficient replication.
The cellular RNA polymerase-II is associated with the viral RNA polymerase during replication.
After fusion of the virion with the plasma membrane or entry via endocytiosis, the viral core is
released into the cytoplasm. Initial transcription occurs within the viral core, leading to synthesis
of early proteins. Some of which lead to uncoating of the viral DNA, allowing further
transcription and DNA replication.
Virion formation occurs in the circumbriscribed areas of the cytoplasm (viral factories). Some
of these mature particles move to the vicinity of the golgi complex, acquire an envelop, and are
released from cell virions are released by budding (enveloped virions) or by cell destruction
(non-enveloped virions)

Cultivation:

Most but not all grow readily in embryonated hens egg and produce pocks or nodular focal
lesion on the chorioallontoic membrane. The pocks vary in size, shape and color according to the
nature of infecting viruses, and their appearance can be useful for the rapid identification of
viruses in clinical specimens. Stained sections of infected membrane show ballooning
degeneration of the affected cells and round or oval acidophilic cytoplsamic inclusions.

It can also be propagated in cultured cells of many different types- bovine kidney, rabbit kidney,
HeLa cells and chicken embryo fibroblasts. Cytopathic effects are not always obvious, but
infected cells may haemadsorbed and stained monolayer preparations usually contain
cytoplasmic and nuclear inclusion.
Because of their large size, poxviruses can be seen with light microscopy in stained smears. Virus
elementary bodies stained by various procedures, including Gutstein's and Giemsa, can be readily
seen either as aggregates (acidophilic cytoplasmic inclusions) or singly.
Poxviruses may survive for years in dust.
Some mammalian poxviruses are considered oncogenic and have been associated with epidermal
and fibromatous hyperplasia.
Pathogenesis of infection with a generalized rash:
Pathogenesis of all pox viruses are infections are almost similar. Poxviruses are pathogenic for
man and many species of animals including birds. The mode of infection may be via minute
abrasions on the skin or, perhaps, in animal to animal infections by respiratory routes. During the
incubation, period of 12-14 days, the virus probably replicate within cells of the
reticuloendothelial system, notably in lymph node, which may be enlarged and tender several
days before the rash appears
There is then viremia (cell associate), and the epidermal cells in various parts of the body are
infected, to produce a rash, which is most extensive on the face, extremities and udder region.
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The rash goes through stages of macule, papule, vesicle, postule and scab over a period of about
2 weeks.
The extent of general signs (fever, prostration, toxemia) is roughly proportional to the extent of
rash.
Pathogenesis of localized infections:
Cowpox and vaccinia viruses to produce localized infections to the site of entry of virus through
the skin. In infections with these viruses, the local lymph node usually became tender and
swollen a few days after infection occurs, and there may be transient viremia.
Transmission:

Spread of infection is commonly by the respiratory route.


Through skin abrasions
Swine pox viruses are transmitted mechanically by arthropods.

Diagnosis:
Can be made by1. Direct demonstration of virus/particles in scrapings from maculopapules, vesicle fluid or
crusts when viewed under electron microscope.
2. The common pox group antigen is demonstratable when the patients lesion are used as
antigen and tested for complement fixation with suitably prepared antisera or agar gel
diffusion.
3. The isolation of causative viruses can be made on chorioallantoic membrane of chick
embryos and observed for the development of pocks
4. The presence of cytopathic inclusions or other cellular changes can be examined on
susceptible cell culture systems.
5. Serological tests Complelment fixation
Neutralization
Haemagglutination inhibition
Immunodiffusion
These test use for detection of serum antibody, where applicable.
Control:

Both lice unmodified and live attenuated virus vaccine are useful for the control of many pox
viruses infections.
Only method of prevention is by avoiding contact
1. Cow Pox Virus:

A benign disease of cattle affecting principally the skin of udder and teats. This disease occurs
naturally in milking cows. It is occupational diseases of man.
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Cause
Cowpox virus (orthopoxvirus).
Occurrence
Additional hosts are human beings and various animals, including large zoo cats, domestic cats,
anteaters, and rodents. The latter are considered the natural reservoir hosts.

Transmission

Milkers and milking machines are the main means of spread. Insects may also serve as mechanical
vectors for the virus.
Morphology:
Cultivation:
Virus grows well on CAM of 7-13 days old embryonated eggs. The pocks are characterized by an
intense hemorrhagic reaction.
Pathogenesis:
After and incubation period of 3-7 days, affected cattle show slight fever and anorexia followed by
appearance of lesions on teats and udder which pass through all stages of pox infection. The cowpox
lesions are 1-2 cm in diameter and are thick, tenacious and yellow brown to red in color. Mostly
these lesions are confined to teat and udder but in severe cases, the lesions may spread to inside of
the thighs and rarely to perineum, vulva and mouth. In bulls, the lesions usually appear on the
scrotum. Recovery results in an active immunity and calves born from immune dam acquire
protection through colostrums. After, a few days, the vesicles show a central depression with a raised
firm edges (umblication) unless rupture earlier as a result of trauma. It is quickly followed by
suppuration and the formation of dry crusts. The swelling and induration slowly subside and the
crusts fall off after about a fortnight, leaving white depressed cicatrices in the skin.
Clinical & Pathologic Features
Cowpox virus produces what is usually a benign infection of the udder and teats. Papules are first
seen, followed by vesicles, which rupture leading to scab formation. Scabs drop off in about two
weeks. Losses in milk production result from the soreness of affected teats and also from secondary
bacterial infection, which may complicate the disease and contribute to development of mastitis.
Immunity:

Recovery from the cow pox disease usually occurs in an active immunity.
The duration of the immunity against it may remain up to 2-3 attacks,

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Generally, the herd in which an outbreak has occurred is not likely to suffer again cowpox for
several years.
Calves born of immune dams acquire passive protection through the ingestion of antibodies in
the colostrums, but duration of protection is unknown.

Diagnosis:

Clinical specimens: Vesicular fluid, scabs, and scrapings from lesions.

Appearance of firm, dark red circular or oval lesions with raised edges and a depressed centre on
the skin of the teats or udder
It is difficult to clinically distinguish cowpox from pseudocowpox and other infections of the
teats.
Confirmatory diagnosis can be made by isolation of the virus from the lesions and by serological
methods for its identification.
Confirmatory diagnosis and be made by isolation of the virus from the lesions and by serological
methods for its identification.
There is a bright red, haemorrhagic pock on the CAM of embryonated hens egg.
Diagnosis is most easily confirmed by the examination of distilled water lysates of lesion
material by electron microscopy. Orthopoxviruses are "brick-shaped" as opposed to the virions of
pseudocowpox (a parapoxvirus), which are ovoid in appearance.

Cowpox virus can be cultivated in cell cultures of bovine and human origin, and on the
chorioallantoic (CA) membrane of chicken embryos. The latter method of cultivation also
provides a means to differentiate cowpox virus from pseudocowpox virus, which does not grow
on the CA membrane. Vaccinia virus produces smaller pocks on the CA membrane than does cow
pox.

Prevention

Vaccination is not practiced.

Prevention is best accomplished by sound milking practices. Milkers and milking machines
can spread the virus.

Public Health Significance


Milkers may contract the infection from cows. The human infection usually involves a single, benign
lesion on the hand or face. Serious systemic disease has been reported in immunosuppressed
individuals.
2. Sheep pox virus:
It is the most severe diseases of the domestic animals, sheep pox (SP) is highly contagious, febrile
and often fetal illness characterized by epithelial hyperplasia and micro-vesicle formation in the
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epidermis which is particularly seen on the exposed part of the body.
Morphology:

Smaller and more elongated than other pox viruses


Measures approx.115nm

Replication: same as general


Cultivation:

Replicate in sheep and goat kidney cell cultures


Produces specific cytopathic changes within 4-6 days but the infection tends to self limiting and
rarely involves more than half of the monolayer.
Reports of cultivation in chick embryo are conflicting.
The viruses produce intracytoplasmic inclusion bodies in the infected cells.

Pathogenesis:

The incubation period varies fro 4-8 days.


On the first day, there is rise in temperature, increased respiration rate, swollen eyelid and a
mucus discharge from the nose.
On the 2nd day of clinical illnesso Intense inflammatory reaction in the dermis, dermal oedema becomes marked, skin
proliferates until it is 12-15 cells thick and there is raised circular thickened plaques with
congested borders can be seen on the skin.
This cutaneous reaction passes through the various stagesi.e. papules-----micro vesicles-----pustular pocks-----scab
The eruptions may be widely distributes but are most prevalent on the cheeks, lips, ears and wool
free skin. In more severe cases, lesions in buccal, digestive and respiratory mucosa also develop.
The mortality rate may reach upto 80%
At the time subside of clinical symptoms, temperature falls and during the resolution of the
pocks, there is itching, shedding of the wool and exfoliation of the epidermis.
The courses of the disease are 3-4 weeks.
Postmortem examination reveals hemorrhagic inflammation and ulcers of the mucous membrane
of the respiratory and intestinal tracts as well as swelling of the lymph nodes, petechiation of the
serous membrane and numerous small spherical nodules in the lungs and kidneys.
There is gelatinous oedema of subcutaneous and intramuscular tissues

Diagnosis:

Clinical diagnosis can be made on the basis of history, course, and symptoms of the disease,
together with postmortem findings.
The confirmatory diagnosis is achieved by the isolation of virus in the susceptible sheep or cell
cultures.

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The antibodies can be detected in serum of recovered animals by using neutralization,


complement fixation, gel diffusion and fluorescent anibody tests.

Immunity:

Lamb born of immuned ewe may acquire passive protection through the ingestion of antibodies
in the colostrums.
Immune serum can be used for passive immunity
The protection gained by passive immunity is immediate but short-lived .
Animals recovered from nodural infection develop solid immunity.
A Chinese strains of sheep pox adapted to CAM of chicken embryo is reported to be avirulent
and induces a high level of protective immunity.
3. Goat pox virus:

It is an epizootic disease of the goat and is characterized by fever an mucopurulent nasal discharge
and the development of a generalized cutaneous eruption.
Morphology and replication: same as general
Epidemiology:

Virus is host specific but some strains affects sheep also.


The disease spreads by contacts
Mechanically spreads by insects.

Cultivation:

Grows readily in cell cultures of


Lamb kidney
Kid kidney
Kid testes
Lamb testes
Produces cytopathic effects (CPE) as like of sheep pox and there is complete degeneration for the
infected monolayers within 5 days.
Its culture on CAM is conflicting but few strains can grow

Pathogenesis:

Incubation period varies between 5-14 days. The papules develop on the hairless part of the body
like around eyes, nose and mouth, udder, teats or scrotum, inner aspect of thighs.
It passes all the stages and subsequently heal within 3-4 weeks
More common in young and lactating animals
Mortality rate o-50%

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Diagnosis:

By isolation of virus in cell culture


Gel diffusion is preferred for detection of specific precepitnogens in affected tissues
Clinical specimens: Lesion material (scabs, liquid).

A highly presumptive diagnosis is based on clinical signs and lesions. Confirmation is most
easily and rapidly obtained by the electron microscopic demonstration of poxvirus in distilled
water lysates of lesion material. The virions are morphologically similar to the orthopoxviruses,
being "brick" shaped as opposed to the virions of contagious ecthyma (a parapoxvirus), which
are ovoid in appearance.

Eosinophylic intracytoplasmic inclusions are seen in skin cells.

The virus can be propagated in cell cultures derived from goats, sheep, and cattle.

An antigen-trapping ELISA can also be used in detection of the virus.

Other serological procedures including indirect immunofluorescence assay and virus


neutralization are used to detect antibodies but are not ordinarily used for diagnosis.

Immunity:

Passive immunity in kid through colostrums


Recovered animals develop a solid, life long immunity.
4. Fowl pox virus

Fowl pox is an infectious viral disease of poultry and other birds. It occurs in two forms1. A diptheritic form (avian diphtheria), which causes whitish caseous (cheesy) membranes in
the mouth and trachea.
2. Cutaneous form: there is grey spots on the comb, wattles, eyelids and beak, feet and legs of
chicken that enlarge into brownish wart like lesions and spread over face.
Diptheritic form is mostly responsible for mortality associated with the diseases.
Cause
Antigenically related fowlpox viruses of the genus Avipoxviruses.
Transmission
Mainly by direct contact with infected birds and fomites, particularly contaminated litter.
The virus enters the skin (cutaneous form) through minor abrasions or by the bite of mosquitoes; or,
entry may be via the oral or nasal mucous membranes via aerosol droplets leading to the diphtheric
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form of fowlpox.
Host affected:

The disease is highly contagious and occurs commonly worldwide.


The disease mostly occur in chicken but also affects turkey, guinea fowls, peacocks, pigeon,
waterfowl, pheasants, canarie and sparrows may be affected.
There are 4 strains of virus Fowl pox virus
Turkey pox virus
Pigeon pox
Canary pox

History and distribution:

Occurs in all countries of the world where poultry are kept in large scale
Appears most frequently in early spring and late autumn.

Morphology:

The elementary or Borrel bodies are morphologically similar to other pox virus.
Measures about 3330280nm in size.
Under the electron microscope, there is a centrally placed electron densed pepsin resistant core or
nucleoid which contains ds DNA (MW 200-240106 daltons)
The virus particles stained with uranyl acetate and shadowed with platinum appear to have a
knobbly surface and resemble a mulberry.

Replication: as described in general


Cultivation:

Grows well on the chorioallantoic membrane (CAM) of chickens embryos, produces large,
raised, whitish pocks within 3 days and latter there is necrotic foci.
Marked cytopathic effects (CPE) on cell cultures (chicken embryo fibroblast) and large
intracytoplsamic inclusions (Bollinger bodies) can be seen in infected cells.

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Pat
hogenesis:

All ages of birds are affected but most prevalent in 5-12 months chicken.
Incubation period (IP) is 4-8 days; the course of disease is 3 or 4 wks.
After entering the skin, multiplication of virus results in the proliferation of epithelial cells giving
rise to typical pox-like eruption, scab or nodules formation on the skin, comb, wattles, oral
commisures, feet and sometimes in vent region. Involvement of the eyes may lead to
conjunctivitis and temporary blindness, and caseous material may accumulate in the intra-orbital
sinuses.
In diptheritic forms small caseous white nodules appear at the side of the tongue, on the roof of
the mouth and around the epiglottis. These may coalesce to form an extensive yellow to white
necrotic membrane resulted finally in death from suffocation by occlusion of the larynx. When
the lesion involve the larynx and trachea. Affected birds show severe respiratory symptoms. The
exudates of these birds contain large amount of viruses

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Mortality is usually low, but may be as high as 50% with the diphtheric form.
Lesions that resemble those of other pox diseases are normally present on the comb, wattles,
around eyelids, and on other featherless areas. Birds usually recover within 1 month.
The diphtheric form is more severe and is often complicated by secondary bacterial
contamination. Lesions involve the mouth, pharynx, trachea, orbit, and sinuses. This form of the
disease may be confused with infectious laryngotracheitis as typical cutaneous pox lesions may
not be seen.

Diagnosis:

Is usually based on typical clinical and pathologic findings. Typical pox lesions on the comb,
wattles, legs etc are indicator of infection. On diptheritic form, there is severe respiratory
depression

Confirmatory diagnosis can be made by isolating virus in chicken embryo. The virus can be
readily cultivated on the chorioallantoic membrane of chicken embryos, where it produces focal
or diffuse pock lesions.

The specific antibodies can be detected by CFT (Complement fixation test) and immunodiffusion
test.
Demonstration of acidophilic cytoplasmic inclusions called Bollinger (aggregates) and Borrel
(single) bodies in tissue scrapings and sections is diagnostically significant.

The electron microscopic examination of distilled water lysates of lesions is a rapid means of
diagnosis.

Immunity/Prevention:

The recovered birds develop solid, lifelong immunity.


By vaccination:
Two types of live vaccines are used-fowl pox and pigeon pox. These vaccines are
prepared from infected CAM of chicken embryos. Fowl pox vaccine prepared in tissues
culture is also in use.
Vaccine prepared by HP1 (German) strain can be given orally at 5 days old.
The age of birds at vaccination by cutaneous method is 4-8 wks.
Vaccination is widely practiced in high-risk flocks. The most widely used and safest product for
chickens is the pigeon poxvirus, which is highly immunogenic, but is of low pathogenicity for
chickens. It is propagated in embryonated eggs and administered by the wing-web stick method
or by brushing defeathered follicles.
Vaccination during the first few weeks of life, with revaccination at 8 to 12 weeks is
recommended.
Turkeys are usually vaccinated at 2 to 3 months of age with a fowlpox vaccine by the thigh stick
method.

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Microbiology IV

Herpesviridae

Gr. Herpes=to creep


Enveloped, ds DNA virus with molecular weight 50-100106 daltons.
Virion measure about 150-200nm in diameter (larger virions are enveloped but smaller (100110nm) naked virions are also frequently present).
The capsid has cubic symmetry having 162 prismatic hollow
capsomeres (150 are hexagonal and 12 are pentagonal),
surrounde by a granular zone composed of globular
protein(integument) and encompassed by a lipid envelop.
All viruses are ether sensitive and acid labile.
Relatively stable at room temperature, heat labile being
inactivated in 30 min at 50oC.
These virus can be preserved in the frozen state at -50oC or at
4oC
The family heprpesviridae consist of three major
Equine "slow herpesvirus"
intranuclear inclusions
subfamilyes, alpha-, beta-, and gammaherpesvirinae,
which are initially distinguished by host range, duration of
reproductive cycle, cytopathology, and latent infectrion characteristic.
Genus:
1. Alphaherpes virus
Variably-resticted host range, are generally highly cytopathic in cell culture, have a relatively short
replicative cycle (24 Hrs), and frequently cause latent infection in sensory ganglia.
Species: Pseudorabies virus
Feline rhinotracheitis
Avian infection laryngotracheitis (ILT)
Infectious bovine rhinotracheitis
Bovine ulcerative mammilitis
Fish herpes virus
Herpes simplex virus
2. Betaherpes virus
Variable host range and long replicative cycle; infected cells often become
enlarged(cytomeglia, so as
Fig 1. Herpesviridae (100 - 200 nm).
cytomegaloviruss).latency in numerous tissue
Between the capsid and the envelope
Species:
cytomegalo virus of
there is a protein-filled region known as
different animal species
the tegument.
3. Gammaherpes virus
With some exception, tends to be tropic for B or T lymphocytes (lymphotrophic), replicate in
lymphoblastoid cells, and may cause lytic infections in certain types of epithelial and fibroblastic
cells.
Species:
fowl herpes virus (Mareks disease virus)
1.

Mareks disease virus (MDV)

Mareks disease is a lymphoproliferative disease of chicken that may involve numerous tissue. Most
frequently peripheral nerves are affected. It is a transmissible viral disease of domestic chicken
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flocks world wide distribution and is considered economically important, affecting mainly poultry
and rarely seen in turkeys, pheasants, red jungle fowls and Japanese quails. The virus can persist in
excreta, litter, and poultry house dust, and horizontal infection via aerosols of chickens appears to be
main method of transmission. Egg transmissionis doubtful. This disease is characterized by
mononuclear infiltration of peripheral nerves and to lesser extent in skin, muscles, gonads, iris and
various internal organs.
MDV matures to its enveloped infectious form only in the feather follicles and can then be spread
to the environment via desquamated cells.
Whole live cells form blood or tumor material, or infected whole cell cultures, are infectious
experimentally.
Cell-free fluid does not appear to be infectious. Certain chickenlines are genetically resistant to
MDV, and resistance is associated with the development of serum-neutralizing antibody.
Morphology:

Electron microscopy of ultra thin section of infected cell cultures reveals hexagonal, naked viral
particles about 85-100 nm in diameter, sometimes larger enveloped virions of 150-170 nm in
size can be observed.
The enveloped virions are of two types:
1. Infectious:
predominant in the cytoplasm and epithelial cells of feather follicles.
Measure about 250-280 nm in diameter.
2. Non-infectious: found in the nucleus of infected cells and is about 150-170 nm in diameter.
The symmetry of capsid is cube (icosahedral)
Consists of a central core of ds DNA (MW=1106 daltons), surrounded by a protein capsid.

Resistance to physical and chemical agents

Cell free virus is readily inactivated at temperatures greater than 37 oC and is only relatively
stable at 25oC (4 days) and 4oC (2wks).
MDV can be maintained for long periods at 27oC.
Virus is inactivated by pH 3 and pH 11.
Infectivity of dried MDV infected feathers is destroyed by chlorine, or organic iodine, a
quaternary ammonium compound, salicylic acid, synthetic phenol, and sodium hydroxide.

Replication

Mareks disease virus are adsorbed and transformed to cell in a membrane bound vesicle. Most
virions lying within the vesicles are enveloped and engulfed more efficiently. The phagocytic
cytoplasmic virus detached wholly or partially form their nucleocapsid whereas, several nonenveloped virions may be found lying freely in cytoplasm near the nucleus. Viral nucleic acid
enters the nucleus.
Viral mRNA are synthesized by host cell RNA polymerase on the viral DNA template.
Messengers are exported form the nucleus and initiate, synthesis of viral proteins.
These proteins enter the nucleus, synthesize additional viral mRNAs,

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Viral DNA polymerase enters the nucleus and initiates DNA replication.
DNA is cut into unit length and packaged into nucliods with an icosahedrals.
Budding of the icosahedral shell takes place through the nuclear membrane and acquired a
lipoprotein enveloped containing viral glycoproteins.

These matured viruses are transported in vesicle to the plasma membrane and fusion between vesicle
and plasma membrane results in the release of virus into the extracellular space.
Cultivation
Virus can be cultivated in susceptible chickens, chicken embryos and cell cultures
1. Susceptible chickens:
The suscpected materials are inoculated intraperitoneally into the susceptible day old chicks where
they produce lesiono Which can be detected histologically in ganglia, nerves and certain viscera after 2-4
weeks?
o Whereas, gross lesion are apparent after 3-6wks.
Fluorescent-antibody technique show many tissues with virus. Specific viral antigen can be detected
apparently in epithelium of healthy kidney tubules or in epithelium of feather follicles.
2. Embryonated eggs of hen
a. Chrioallantoic membrane(CAM): Produce discrete while pocks on the surface of CAM
b. Yolk sac: Most strains possess the affinity to grow well in the yolk sac of genetically
susceptible chick embryos.
3. Cell culture:
Grows in chicken kidney and duck embryo fibroblast.
Cytopathic effect is produced within 6-14 days, which is characterized by the development of
discrete focal lesions consisting of clusters of rounded, highly retractile degenerating cells about
7 days after inoculation. During the preceding 7 days, these clusters of refractile cells enlarged
and detached from the glass giving rise to microscopic plaques.
The stained monolayer preparation contains intranuclear inclusion body.
Pathogenicity:

The severity of infection depends on the viral strains, strain of birds, route of infection, dose, age
sex, immune status and genetic susceptibility of chickens.
It usually occurs in chickens between 2-5 moths old chicken and is not seen in birds older than
22weeks.
The virus primarily affects the nervous system although visceral organs and other tissues may
also involved.
Lesions are present in the nervous system and involve peripheral nerves and spinal roots.
The principal nerve trunk involved shows gross lesions consisting of a grayish-white swelling,
which histologically are characterized by extensive lymphocytic infiltrations (in optic nerve
also).

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Oedema and myelin degeneration of nerve sheaths may be apparent.


Ocular lymphomatosis is another possible outcome of MDV infection, with blindness resulting
due to iris involvement.
In the visceral form, lymphoid tumors of varying degree of severity infiltrate the gonads, liver,
lungs and skin.
Affected chickens have enlarged visceral organs with white nodular or military foci.

Mode of infection
The virus has the ability to survive in poultry house, litter and dust for about 4-8 months in the
shaded areas. Commonly, the entry is via inhalation of infective particles. Infection may start
even in first week after hatching but the presence of maternal antibodies delay the infection until
3-4 wks of age, by which time antibodies get depleted. It has not been found to be transmitted
vertically (i.e. through eggs)
Once the infection enters to susceptible, unvaccinated chickens, all the birds may show viraemia
by about 8 weeks of post infection, where there is maximum shedding at 5 wks of age and may
continue indefinitely but in lesser extent.
The incubation period is 3-4 wks and outbreaks occur between the age of 8-16 weeks. Recovery
from overt disease is unusual and mortality in an affected flock may vary: 25to 60% in acute Mareks disease(AMD)
10 to 30 % in classical Mareks disease(CMD)
Sometimes, both forms can be seen in the same flock
It is a lympho-proliferative condition affecting peripheral nerve and sometime, the visceral organ,
., there is slight paresis to spastic and rarely flaccid paralysis.
The infection possesses 3 forms
a. Neural form: Characterized by- infiltration of peripheral nerve by lymphoid cells.
1.
Causing paralysis of brachial nerves of wing, sciatic nerves of legs, vagus nerve along the
neck or mesenteric nerves of the abdomen.
b. Visceral form- lymphomas develop in the live, spleen, gonads and other tissues
c. Ocular form- lymphocytic infiltration of iris.

Normal black colored iris ha the appearance of grey or pearly.

Pupils get contracted.


The acute type of disease is generally occur in younger birds characterized by severe depression,
ataxia, diarrhea, emaciation and high mortality.
Diagnosis
1.
2.
3.
4.

On necropsy, gross lesions are common in peripheral nerves, root ganglia, and the spinal
roots.
lymphomatous lesions are characteristically composed of small lymphocytes,
lymphoblasts, and reticulum cells.
Arterial lesions of atherosclerosis are often present.
A tentative diagnosis id made on the basis of symptoms and lesions:

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Figure 1 Differential features of Mareks Disease (MD) and Lymphoid Leukosis (LL).

5.
6.

7.

Classical Mareks disease


Unilateral paralysis of legs and wings.
Curling of the toes
Torticolis
Dilations of the of the crop
Diffuse grey opacity o
f the iris
Granular .
Slightly thickened nerves or as much as 3 times
Flattened and striated appearance of the nerves becomes rounded.
Visceral gross lymphomatous tumor like lesions are seen in ovary but sometimes also in
spleen, liver, heart, lungs, kidneys, proventriculus and other tissues.
Acute mareks Disease
Rapid spread
Severe depression
Ematiation
Death
Confirmatory diagnosis is made by viral isolation or by antigen detection using
fluorescent antibody, immunoperoxidase, or ELISA on feather follicle cells.
Isolation of MDV can be achieved by inoculation to susceptible chickens, embryonated
chickens egg and cell cultures. The suspension materials of choice are intact viable cells
from buffy coat, spleen, tumor tissues.
The antibodies can be demonstrated in the sera of infected or recovered birds by
serological testsAgar gel precipitation test(AGPT)
Agar gel immunodiffusion test
Indirect fluorescent antibody(IFT)
Passive haemagglutination test(PHT)
Virus neutralization

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Control and immunity

1.

2.

Immune response to MDV is complex, in that both humoral and cell-mediated immunity (CMI)
develop in normal birds.
MDV infection is immunosuppressive.
In addition, the immune response may be involved in tumor formation.
Bursectomized birds survive experimental infection, suggesting that CMI is important.
In chicks, passively acquired antibody is thought to limit the extent of infection rather than
prevent it or clear the virus.
Viral specific antibodies appear within 1-3 wks following infection and neutralizing antibodies
persist for the life of the bird.
Following infection, transient CMI suppression is common; it may persit in birds that develop
neoplasm.
Both B and T lymphocytes have been identified in tumors
Experimentally, MDV-free flocks maintained by strict isolation, constant surveillance, and
frequent monitoring for virus and antibody, but these technique hve been of limited commercial
use.
Commercial vaccine is available and has been effective in reducing the incidence of MD.
Through vaccination
Day old chicks: HVT(Herpes virus of turkey)
Layers=0.2ml S/C
Broilers=0.1ml S/C
Booster dose about 18-21 days of age.
There are three serotypes line available live vaccinea.
Serotypes-1 lesions herpes virus of turkey(CV 1988)
b.
Serotype 2 apathogenic strains of MDV- SB1
c.
Serotype 3 cell cultures attenuated MDV- HVT or FC 126
HVT vaccine is the most economical to produce and is most effective when exposure is not
heavy.
Vaccination does not prevent infection or shedding or virulent MDV, but it does prevent tumor
formation.
Bivalent and polyvalent vaccines used successfully where monovalent vaccines have been
ineffective.
Embryo vaccination is currently used in at lest 55% of broiler embryos.
Genetically resistant lines of chicken have been maintained experimentally.

All these vaccines does not prevent super infection, replication or shedding of virulent MDV but they
do protect against overt disease. This resistance is of life long and mechanism of protection is due to
cell mediated immunity response.
2. Malignant catarrhal fever (MCF)
MCF is an acute, sporadic, infectious and fatal disease of cattle and some other bovidae and cervidae
characterized by high fever, catarrhal mucopurulent inflammation of respiratory and alimentary
epithelium, extensive lymphoid hyperplasia, keratoconjunctivitis, encephalitis and rapid loss of
conditions. The sheep and wildbeest may act as symptomless carrier. Morbidity is low but mortality
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is extremely high.
Morphology
Ds DNA, surrounded by proteins capsid, enveloped, cubic symmetry
Replication same as MDV
Cultivation:
1. Cell cultures:
The virus produces a marked cytopathic effect with syncytia and cowdry type-A intranuclear
inclusion when cultivated on bovine thyroid, bovine adrenal or calf kidney cell cultures.
The richest samples used for cultivation are from lymphnodes, thymus, spleen, bone marrow and
buffy coat from peripheral blood.
Pathogenesis:

Occur sporadically throughout the year. Morbidity is low but mortality rate is high and reached
upto 60-90 %
Under natural condition, incubation period (IP) is about 4-20 weeks or longer but under
experimental condition; it is usually 10-30 days.
The portal of entry of MCF virus is probably upper respiratory tract, nasal mucosa and
sometimes tonsil. It can also transmitted through aerosol or intratracheal routes. congential
transmission is evident in reservoir host and also demonstrated only once in cattle.
The first sign of infection are high fever, which persists throughout the course of disease,
accompanied by slight ocular and nasal discharges.
Within 24-72 hours, oral and nasal mucous membrane get inflamed followed by congestion,
necrosis and erosion of oral mucosa.
The disease is characterized by marked, swelling of the joints, hyperplasia of the lymph nodes
and progressive severe depression n. nervous sign may develop and the animal becomes excited
and aggressive. There is photophobia and marked corneal opacity leading to complete blindness.
Cystitis is sometimes present. Death may occur generally within 24 hrs but sometimes disease
may last upto fortnight (15 days) or more.

Necropsy findings

Epithelial necrosis of gastro-intestinal, respiratory or urinary tract associated with mucosal or


dermal lymphoid inflammation, lymphoproliferation interstitial infiltration of non-myeloid tissue
and vasculitis.
Most lymph nodes are hyperplastic.
Interstitial infiltration of lymphocyte is seen in organs e.g. heart, liver, adrenal gland, meninges,
CNS vascular spaces and kidneys. In kidney, there is a white raised focus under the capsules.

Diagnosis
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1. On the basis of histopathological findings
2. A confirmatory diagnosis can be obtained by isolation and identification of causal virus. In calf
thyroid tissue culture, suspensions from affected animals are leucocytes or lymphnode produce
cytopathic effect after 6-9 days of incubation. CPE is characterized by syncytia formation and
there is intranuclear inclusion body
3. Serological test
Virus neutralization (VNT)
Indirect immunofourescence
ELISA
Immunity and control

Recovered animals develop solid immunity against homologous strain for 2-3 years.
Virus can be neutralized by sera of recovered animal, but the immunity is often poor.
Separation of seep from cattle to check transmission.
Contact with wildebeest and wild ruminant should be checked.
3.

Infectious laryngotracheitis

Infectious laryngotracheitis is and acute, highly contagious respiratory disease of poultry caused by
herpes (ILTV) and is characterized by moist rales, sneezing, coughing and marked depression. The
virus produces sign of respiratory distress and coughing that often produce a bloody discharge. All
breeds and ages of fowls are affected. Pheasants may also be susceptible and occasional infection of
ducks, pigeon and turkey is also reported
Morphology same as general of herpes virus
Replication same as MDV
Physical, chemical agents
ILTV is inactivated by 3 % cresol, 1% sodium hydroxide, and 1% lye, 24hrs exposure to ether, and
10 to 15 minutes at 55oC. The virus can be stored for lengthy periods by lyophilization and freezing.
Cultivation
1. Chorioallontoic membrane (CAM): The virus can grow in CAM of 10-days old chicken embryos
producing isolated Pocks with an opaque raised edges and depressed central area of necrosis. It
can also propagate in CAM of embryonated turkey egg.
2. Yolk sac: Can be grown satisfactorily in the yolk sac of developing hens egg.
3. Amniotic inoculation: Amniotic inoculation causes lesions in the trachea and bronchi of the
infected chick embryo.
4. ILT virus can grow on chick embryo, respiratory epithelium, chick embryo lungs, chick embryo
kidney monolayer cell culture. The cytopathic effects are only confined to epithelial cell cultures.
The CPE are produced within 3-5 days characterized by large syncytia formation many of which
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contain intranuclear inclusions. Growth can also taken place on chicken fibroblast and HeLa cells
without CPE.
Pathogenicity:

All ages of birds are susceptible but most common in birds of 4-18 moths. This virus is usually
introduced into a flock by carrier bird and is transmitted by aerosol and inhalation, less
commonly by ingestion. Never transmitted vertically. Recovered birds act as carrier upto 2 hr or
more because it harbors the ILT virus.
ILTV in natural conditions enters through the upper respiratory tract and ocular tract.in the
natural disease, the greatest concentration of ILTV is found in the trachea, since the virus
replicates only in the nasal cavity, trachea, and lower respiratory tract.
Latent virus has been demonstrated in the trigeminal ganglion.
After incubation period of 2-8 days, there is mild coughing and depression.
There is hemorrhagic tracheitis which results in spasm of gasping, coughing and sneezing, birds
frequently shake its head and an attempt is made to dislodge blood stained or caseous exudates
obstructing the trachea or larynx.
The mortality rate comes according with viral strains, may vary between 10-60%.
The main pathological changes includes
o Hemorrhagic inflammation and edema of larynx, trachea and bronchi
o Presence of a diptheritic membrane in the trachea or caseous exudates in the air passage
and air sacs.
o Small area of lungs is sometimes congested.

Diagnosis

The acute form is readily recognized by its sudden onset, characteristic symptoms of rales
accompanied by blood, mucous and caseous exudates in the trachea, rapid spread and high
mortality.
By inoculating a suspension of suspected material into 10-12 days embryonated hens egg and
examining the CAM for pocks lesion after 5 days of incubation.
Virus can be isolated from tracheal and lung tissue in embryonated chicken eggs, cell culture, and
ILTV DNA can be demonstrated by PCR.
Immunoflourescent staining of trachea can demonstrated presence of viral antigen upto 14 days
postinfection.
By demonstration of inclusion bodies in the lesions, which is intranuclear cowdry A type.
Serologically by neutralization test; agar gel diffusion and fluorescent antibody test.

Immunity

The first signs of ILTV infection usually appear 6-12 days following natural exposure.
Resistance to disease following the infection or vaccination usually persists for approximately 1
year.

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Infected bird develop precipitating and serum neutralizing antibodies; however, the cell-mediated
response appears too important in resistance.

Control

Use of live ILTV vaccine results in carrriers, which can shed to nonvaccinated susceptible birds.
Since the virus can survive for 10 days at temperatures or 13-23oC, the cleaning of infected
premises is very important.
Complete depopulation and disinfection of premises has been used to control the disease.
Vaccination has been successful via the cloaca, infraobital sineuse, intranasal instillation, feather
follicles, and drinking water.
Birds younger than 2 weeks of age donto respond as well as older birds.
Water administration of ILTV vaciine doenot give as complete or long lasting immunity s other
methods.
For water vaccine to be effective, the has to penetrate into the nasal cavity or trachea.
Cloacal administration of the vaccine leads to rapid absorption of the virus into the bursa of
fabricius and results in and immune response.
Recovered birds produce immunity for 1 year.
In enzootic areas, living virus vaccine should be used.
Attenuated virus prepared in duck embryo tissue cultures can be used.
Eradication of ILT is achieved by complete depopulation and disinfection of infected premises,
followed by restarting at least after one month.
3.1. Pseudorabies virus

Synonyms: Aujeszkys disease, Mad itch, Herpes virus suis, infectious bulbar paralysis.
This disease was firs of all described by Aujeszky in Hungery in 1902 and its viral aetiology was
confirmed in 1910.
Occurs naturally in a wide range of animalsSwine, cattle, sheep, dog, mink, ferrets, foxes, cats and rats
Among these, the most common primary hosts are swine however , the adult pigs are probably the
main reservoir of the virus.
Morphology:

Measures about 110-150 nm in diameter (but some particles upto 230 nm)

More frequently oval, sometimes appears round in thin sections.

The dense central body or nucleoid contains ds DNA with molecular weight of 68-70106
daltons.

The capsid is composed of 150 hexagonal and 12 pentagonal hollow elongated capsomeres s
(12nm9nm, 4nm in diameter).

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Viral symmetry is icosahedral

Enveloped, some virus may have peplomers.

Physicochemical properties:

Retains infectivity in glycerol saline or skimmed milk at refrigeration temperature.

Preserved in best way in saline buffered with Tris at pH 7.5 containing 1% serum albumin at
-70oC.

Inactivation in an exceptional manner by X-rays or U-V light irradiation.

Highly susceptible to lipid solvents e.g. ethyl ether, sodium desoxycholate and chloroform.

Trypsin and certain other enzymes inactivate the virus without destroying the viral capsid.

Can withstand 3% phenol but destroyed by 5% phenol

Immediate inactivation can be achieved by 0.5-1% NaOH.

In natural condition. It persists viability on hay for a month or even longer.

Cultivation
A. CAM: This is being typical herpes virus grows readily on the CAM of embryonated hens egg.

Where, it produces moderately large, raised, white pocks after 3-4 days of incubation.

Apart form pock, lesion, many strains are lethal to the embryo and cause haemorrhageic
destructuion of nervous system.

B. yolk sac:

Virulence can be increased by serially passaging this virus into yolk sac.
5. Iridiviridae

Irido means shining/ iridescent)


Enveloped viruses that have Icosahedral symmetry measuring about 120-200 nm in
diameter(occasionally 300nm)
Most members are naked but some have lipid containing envelop e.g. African swine fever(ASF)
virus
The genome consists of single molecule of ds DNA having mol. Wt. 130106 daltons.

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The capsid shell consist of about 812 protein subunit and as many as 1500 capsomeres
Genus:
o Iridovirus sps: African swine fever virus.
4.

African swine fever (ASF)

A highly contagious often peracute viral disease of domestic swine characterized by high fever,
leucopenia, often followed by the appearance of erythema (red areas on the skin), weakness,
accelerated respiration and pulse, vomiting, bloody diarrhea, nasal and conjunctival discharges.
Mortality rate (MR) may reach upto 100%. ASF virus infects domestic and wild pigs (not a African
wild boars). Soft ticks of the genus Ornithodoros transmit the virus as biological vectors.
Morphology:

Enveloped DNA with nucleoprotein core or 70-100 nm that is surrounded by lipid layers and a
hexagonal icosahedral nucleocapsid of 170-190 nm in diameter.
Capsomeres are 812-1500 in number.
The genome consists of single molecules of ds DNA having molecular weight 130106 daltons
The virus has more than 20 structural proteins and several virion associated enzymes (at least 50
proteins).
In 1921 it was kept under iridoviridae.
Ether sensitive. Retain viability around 5oC.
Spleen -70oC viability 2years.
Virus along with 25 % suspension of serum virus can be kept for 24hrs under body temperature.

Resistance to physical and chemical agents


ASF virus is stable in tissues and excretions. The virus can withstand a considerable range in
pH(pH 4-13).
The virus is inactivated by heating to 60oC for 20 minutes and by lipid solvents and some
disinfectants (paraphenylphenolic disinfectant are very effective against the virus)
Replication:
ASF virus replicates in pig macrophages.
Replication occurs in cytoplasm(although the nucleus is needed for viral DNA synthesis)
Virions are released by budding.
Cultivation
Growth occurs in the yolk sac of embryonated hens egg.
Some strains are capable of killing the embryos in 6 or 7 days provided the virus has been
previously passaged through a series of alternating pig/rabbit passages.
Most strains replicate in the buffy coat cells of swine bone marrow.
Propagated in vitro on culture of swine bone marrow cells, monocytes, and alveolar
macrophages. Growths can also occur in pig, bovine and chicken kidney cell lines. Virus that
adapted to grow in pig kidney cell lines may be grown in bovine or chicken kidney cell cultures
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as well as in variety of established lines such as BHK-21 and VERO cells with or without
production of nuclear or cytoplasmic inclusion.
Pathogenicity:
Following oral or nasal exposure of domestic pigs to ASF virus, virus replicate initially in the
URT with subsequent dissemination to adjacent lymphnode and then systemic spread via
leukocytes, erythrocytes, or both in the lymph and blood; this occurs within 3 days postinfection
and corresponds closely with the onset of pyrexia.
The virus replicate in macrophages; thus highest titre of virus occur in those tissues in which
macrophages are most abundant.
There is sudden onset of clinical symptoms. The incubation period (IP) varies from 5-15 days
and mortality may approach 100%. In case of peracute infection, the death is the first indication
of the disease.
In acute infection, there is early symptoms like
1. Rapid temperature rise upto 40.5oC or higher.
2. Marked leucopenia.
After 3-4 days of fever, affected animals stop eating, become weak, incoordinate and cyanotic. Some
pigs show respiratory distress, coughing, vomiting and blood stained diarrhea.
Death usually occurs about 5-7 days after the onset of fever, Mortality 90%
After entering the virus mainly by ingestion or inhalation, virus gets adsorbed onto RBCs and
undergoes haematogenous dissemination. These viruses have a marked affinity for the vasculoendothelial cells where it causes generalized damage to the walls of small blood vessels usually
gives rise to oedema, congestion, hemorrhages, infraction and thrombosis.
On PM examination, following gross lesions can be observed Profuse hemorrhages may be seen throughout the entire carcass, including the alimentary tract.
Marked petechiation of the larynx, urinary bladder, renal cortex, heart, lungs and visceral
surfaces of organs.
The lymph nodes are enlarged and severely hemorrhagic looking like as hematoma.
Excessive fluid can be observed in pleural, pericardial and peritoneal cavity.
The spleen is enlarged upto 3 times than its normal size.
Perivascular haemorrhages and oedema may be widespread in the CNS in early stages of disease.
Pin point hemorrhages in lungs, liver.
Diagnosis:
Laboratory test are required to distinguished between hog cholera and ASF , because the tow
disease cause very similar signs and lesions in susceptible pigs, including fever, high mortality,
and hemorrhages within internal organs.
Tissue submitted should include spleen, liver, lymph nodes, and blood.
When there is peracute or acute, contagious hemorrhagic syndromes accompanied by high
mortality (some times 100%), the ASF can be suspected.
Provisional diagnosis can be made on the basis of clinical symptoms and PM lesions.
The confirmatory diagnosis can be achieved by inoculation of suspected material from both on
susceptible and immuned pigs under strict isolation.
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No intracytoplasmic inclusion bodies.


CFT, immunofluorescence and gel diffusion tests can be used to demonstrate the presence of
viral, antigen in tissue suspension of reacting animals in the antiserum of ASF.
CFT and gel diffusion tests are applied for detection of African swine fever virus antibodies.
For rapid diagnosis, haemadsorption test can be considered and this test is proved to be great
practical value.
PCR
Histopatholgical examination of spleen, lymph nodes and brain may aid in diagnosis. The most
important distinguishing features being severe necrosis and karyorrhexix of lymphocytes in the
lymphoid tissue and marked leucopenia.

Immunity:
The ASF viruses are thought to be unusual because most strains are of high virulence but low
immunogenicity, although complement fixing, precipitating, haemadsorption inhibiting and other
antibodies are usually produced within 7-21 days. The protective antibodies thus produced are of
low potency and short duration, whereas viraemia invariably persist for many months or even
years. Pigs recovered from the disease are immune to homologous strain but not heterologous
strain. No any cross immunity.
Control:
Vaccination with killed as well as live virus vaccine has not been effective.
Prevention can be made by preventing the susceptible pigs accessed to the infected pigs, carcass
and food products including uncooked s.
The best method of eradication is slaughter and strict import regulations for meat products as
well as live animals.
`not only saughter all pigs but also must be treated with insecticide and disinfectant containing ophenylphenol with surfactant and must remain free of livestock for at least a month.
If this virus is passaged in live rabbit it does not cause infection but only causes hemorrhage
(slight)
All species of pigs are highly susceptible.
Feeds should be given boiled.

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GroupII:ssDNA
6. Parvoviridae(picodna viridae)
Pico/parvo menas very small: dna=DNA)
Very small, naked, cubic ssDNA with mol.wt 1.2-1.8*106 daltons
The capsid of parvovirus is small (18-24nm)
There are capsomeres each having 2-4 nm diameter
All viruses are resistant to ether and chloroform.
Multiplication takes place within the nucleus of the host cells.
Resistant to high temperature(700C for 60 minutes) and are stable between pH 3 and 9.
Viruss remain infective for long periods of time at low temperature and in 50% glycerol
Remain viable in fomites for may years.
Genus:
Parvovirus sps: human parvovirus
Bovine
Caprine
Equine
Chicken
Dependovirus sps: bovine/canine/equine/ovine/avian adeno associate(adenoassociated viruss)

GroupIII:dsRNA
7. Birnaaviridae : Bi=two and RNA= Ribonucleic acid)
Naked , icosahedral symmetry and measure about 60nm diameter.
The genome is ds RNA which is linear and remain in two segments.
Genus : birnavirus sps : IBD virus

5.

IBD/Infectious bursal disease virus/


Gumboro disease/Avian nephrosis virus
Introduction: Gumboro Disease or Infectious bursal disease virus (IBDV) is a chicken disease
targeting the Bursa of Fabricius, an important organ in the young chicken's (3-6 weeks most
susceptible) developing immune system. The causative agent, a Birna virus, destroys immature Blymphocytes in the Bursa of Fabricius resulting in immunosuppression. Very virulent strains of
IBDV can result in mortality of up to 40%. Control is best achieved by improved biosecurity and
vaccination
Acute, contagious of young chicks
BF primary site of infection
First described in Gomboro, Delaware, in 1962
Now, reported from most major poultry producing area of world
Agents similar to IBDV have also been reported in turkeys and ducks
Previously reported as reovirus but now in birnaviridae
Morphology: Family: Birnaviridae.
The Virus small, non-enveloped double stranded RNA virus Icosahedral having size of
60nm diameter
Virions relatively heat stable(5 hrs at 560C) and resistant to ether, chloroform and pH 3
Replicates in the cytoplasm without depressing cellular RNA or protein synthesis.
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IBDV replicates in both chicken & mammalian cells and produce CPE in 3-4 days
Also replicates in CEBF cells and RK-13 cells and has been adapted to CE kidney and
Vero cells
Physical and chemical properties
Very stable hardy virus.
Able to withstand a wide pH range (pH 2-12).
Heat stable (still viable after 30 minutes at 60C).
High level of resistance to most commonly used disinfectants.
Survives in the poultry house environment for extended periods of time.
Inactivated by formalin and iodophores.
Replication: Same as general, replication in cytoplasm and release via cell destruction
Cultivation:

8. Reoviridae:
The family name represents- respiratory enteric orphan virus(REO)
Members are found in both respiratory and enteric tract of man and most of animals.
Naked, icosahedral symmetry with a diameter and enteric tract of man and most of animals.
The virion is cubical, unenveloped, roughly hexagonal in shape and posses an inner and outer
capsid which renders it very stable.
The virion contains ds RNA in 10-12 pieces with a mol. Wt of 10-16*106 daltons
Virus multiply in the cytoplasm
Reoviruss are resistant to the action of dietyl ether and chloroform because it lackes essential
lipids in the virus particles.
Genuc :
Reovirus sps: mammalian and Avian reovirus
Orbivirus sps : blue tongue virus , African horse sickness virus
(orbis=ring) all orbiviruss are arthropod transmitted
Rotavirus sps : human/bovine/porcine/avian rotaviruss.
Very resistant to heart and viral infectivity survives heating to 56 0C for 2hrs or 600C for 30
minutes but is rapidly inactivated thereafter and is completely destroyed in 2-3 hrs.
Stable at wide range of Ph i.e. between 2.2 to 8.0 and survives at least 1hr at room
temperature in 1% H2O2, 1% phenol, 3% formaldehyde or 20% Lysol.
Heating at temperature of 50-55 0C for 5-15 minutes in the presence of high concentration of
magnesium chloride resultsin an increase in infectivity but at -20 to 40oC , infectivity are
completely destroyed.

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GroupIII:ssRNA

+vesensessRNA
9. Picornaviridae:
Pico= very small, rna=ribonucleisc acid)
Very small, naked spheres about 20-40 nm in diameter.
The genome is one piece linear, +ve sense, SS RNA with molecular weight about 2.5*106
daltons.
The virions are resistant to the action of ether, chloroform and bile salts (infact remarkabley
stable to inactivation by most agents)
Most picornavirus grow well in cell cultures producing a marked cytopathic effect(CPE)
which is usually sharacterized by granularity and roundingup of the affected cells and
complete destruction within 48-72 hours.
Virus synthesis and maturation occur in the cytoplasm with occasional formation of
crystalline arrays of viral particles.
Cubic symmetry
For practical purposes, picornaviruss are mostly cultivated in cells derived from kidneys of
the animal from which the virus was isolated.
Poliomyelitis virus having marrow host range require primates cells for propagation whereas
FMD virus have wide host range and grow well in a variey of cell types including primary
and secondary pig or Ox kidney cultures and the BHK- 21 lines of baby hamster kidney cells.
Most picornaviruss inabit the intestinal tract and commonly cause no clinical illness.
Some viruss may spread from the gut and cause destructive lesion in CNS (Poliomyelitis
virus) while other parasitize the upper respiratory tract (rhinovirus).
Genus
Enterovirus sps: poliomyelitis, bovine enterovirus, porcine enterovirus, duck hepatitis, avian
encephalomyelitis virus.
Rhinovirus sps human /equine/bovine rhinovirus
Cardiovirus sps : encephalomyocarditis virus
Apthovirus sps : FMD virus(aptha= vesicle in mouth)
10. Togaviridae(L Toga= cloak)
More than 260 members of this family have been recognized to date and all contain ssRNA with
mol. Wt of 3-4*106 daltons.
The virions so far studied by electron microscopy are small, spherical particles of diameter 4070nm surrounded by a non-rigid and delicate lipoprotein (envelop).
Ether sensitive, acid labile and have haemagglutinin with agglutinate the erythrocytes of nearly
hatched chicks and geese.
Unstable at room temperature but survives for a few montshs at -20 0C.
Wide range of animal is infected and different species of laboratory animals including unweaned
mice and hamsters highly susceptible.
Propagated in embryonated hens eggs by chorioallontoic membrane or yolk sac routes.
Cubic symmetry.
Produces marked cytopathic effects in primary and secondary cultures of chickes embryo or
mouse embryo cells as well as variety of stable cell line e.g. HeLa.

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The virus multiply in the cytoplasm and mature by buddind from cell membranes.
The arthropods borne viruses multiply in arthropods as well as in mans.
Genus:
Alphavirus sps : equine encephalomyelitis virus.
Pestivirus sps i. swine fever
Bovine viral diarrhesa
Mucosal disease
Louping ill illness.

11. Coronaviridae :

(Gr. Corona=crown)
The virions are enveloped, somewhat pleomrphic medium sized i.e. 80-160 nm in diameter.
The central core of the virions is composed of +ve sense, SS RNA ithh molecular weight 5.56.1*106 daltons.
The symmetry of coronavirus has been determined but may be helical.
There are widely spaced, pear shaped peplomers in the lipoprotein envelop.
Sensitive to ether, chloroform and other fat solvents.
Growth of coronavirus takes plce in the cytoplasm and maturation is by budding into
cytoplasmic vesicles.
Genus
Coronavirus sps avian infectious bronchitis
Transmissible gastroenteritis (TGE) of swine
Haemagglutinationg encephalomyelitis (HEV) of pigs
Feline infectious peritonitis virus.
Neonatal calf diarrhesa virus
Turkey blue comb birus
Mouse hepatitis

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Microbiology IV

12. Orthomyxoviridae : (Gr. ortho-free, myxo-slime/mucus)


Orthomyxoviruses derive their name from the affinity they possess for the mucous and other
mucoprotein substances present in the respiratory tract and elsewhere.
Enveloped, pleomorphic particles
with 80-120 nm in diameter.
Medium sixed, etther seisitive,
acid labiles, -ve sense SS RNA
viruses with molecular weight 2.5
106 daltons.
The nucleocpasid has a helical
symmetry.
Genus: influenza virus- human,
porcine, equine, avian(fowl
plague)

The Orthomyxoviridae is a family of


RNA viruses that includes viruses which
cause influenza in vertebrates. There are 3
genera of influenza virus, identified by
antigenic differences in their
nucleoprotein and matrix protein.
Morphology:
It has a different gene on each segment. The 8 segments (A &B) are held together by the
helical capsid comprised of nucleoprotein.
Influenza virus particles are highly pleiomorphic, mostly spherical/ovoid, 80-120nm
diameter, but many forms occur, the outer surface of the particle consists of a lipid envelope
from which project prominent glycoprotein spikes of two types, the haemagglutinin (HA),
and neuraminidase (NA), nucleoprotein, 3 viral
polymerases and a large non-structural protein.
The inner side of the envelope is lined by the
matrix protein.
Lipoprotein
membranes
enclose
the
nucleocapsids.
They have a helical symmetry.
The genome consist of 7(influenza virusC) or
8(influenza virus-A & B) segments of linear, ss
RNA with molecular weight 2 106 Dalton.
Antigenic Classification: Based on the differences of the antigenic structure of the nucleoprotein
(NP) and matrix of the viral envelope (M), influenza virus are classified in to 3 types. The division of
types in subtypes is due to HA & NA antigen.
1. Influenza A viruses infects a wide variety of mammals, including man, horses, pigs, ferrets and
birds. Pigs and birds are believed to be particularly important reservoirs. Is the cause of all flu
Dr. Rebanta Kumar Bhattarai, M.V.Sc. (Vety. Microbiology)

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pandemics and are known to infect humans, other mammals and birds. Influenza affects 5-15%
of the world population, and 250,000 to 500,000 deaths worldwide
2. Influenza B viruses infect much man and birds; cause human disease but generally not a severe as
A types.
3. Influenza C viruses infect man alone and pigs.
Antigenic Variation: Influenza viruses show remarkable ability to undergo antigenic variation due
to frequent changes in the MA & NA. The variation is more in type A, less in type B and none in
type C. There are 16 different hemagglutinin subtypes and 9 different neuraminidase subtypes, all of
which have been found among influenza A viruses in wild birds. Only 3 subtypes of HA (H1, H2 and
H3) and two subtypes of NA (N1 and N2) are known to have circulated widely in humans.
Replication:
The viruses bind to a cell through interactions between its hemagglutinin glycoprotein and sialic acid
sugars on the surfaces of epithelial cells in the lung and throat. The cell imports the virus by
endocytosis. In the acidic endosome , part of the haemagglutinin protein fuses the viral envelope
with the vacuole's membrane, releasing the viral RNA (vRNA) molecules, accessory proteins and
RNA-dependent RNA transcriptase into the cytoplasm. These proteins and vRNA form a complex
that is transported into the cell nucleus, where the RNA-dependent RNA transcriptase begins
transcribing complementary positive-sense vRNA. The vRNA is either exported into the cytoplasm
or translated or remains in the nucleus. Assembly occurs via plasma membrane and release by
budding.

Cultivation and cytopathic effect: Influenza viruses are routinely grown to high titre in the
allantoic cavity of 10-day-old fertile hens eggs or more rarely in kidney cells for vaccines and
diagnosis. Virus is detected by its ability to haemmagglutinate red blood cells. A, contains the
veterinary isolates. B and C are human. The A antigens are on the nucleoprotein and can be identified
by ELISA in certain diagnostic tests eg on nasal swabs.
Pathogenesis:
Transmitted by droplet infection (Horizontal Transmitted)

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Primary infection involves the cilliated epithelial cells of the U.R.T. (Specific receptor on cell
surface) i.e; sialic acid receptor.
Necrosis of these cells results in the usual symptoms of the acute respiratory infection.
Death from primary Influenza infection is determined by host factors and 'virulence' of virus.
Damage to respiratory epithelium predisposes to secondary bacterial infections which accounts for
most deaths in animals.
Equine Influenza: It causes an acute respiratory illness of horse.
Morphology: All the structure is similar as in general, the mean diameter of the virion is 100nm.
The equine influenza A virus agglutinates RBC of horse, calf, pigs, guinea pig and human.
Replication: Same as general
Cultivation:
Grow well in embryonated eggs, viruses are routinely grown to high titre in the allantoic
cavity of 10-day-old fertile hens eggs or more rarely in kidney cells for vaccines and
diagnosis. Virus is detected by its ability to haemmagglutinate red blood cells.

Equine influenza virus can be grown in chick embryo kidney, bovine kidney, rhesus monkey
kidney, & human embryo kidney cells. The CPE effect is characterized by an early
development of syncytia & multiple eosinophilic intracytoplasmic inclusions.

It can be adopted to infect mice, introduced intranasally.

Pathogenesis: Aerosol virus infects the ciliated epithelium of the nasal mucosa and then may extend
to the bronchioles with resulting epithelial cell necrosis, which manifests as bronchiolitis and serous
exudation.
Clinical signs: A harsh dry cough follows an incubation period of 1-3 days when the horse also
develops pyrexia (103O-106OF), depression, loss of appetite, and enlarged submandibular lymph
nodes. Secondary bacterial infection follows defective muco-ciliary transport. Streptococcus
zooepidemicus can cause a purulent bilateral nasal discharge. A fatal bronchopneumonia is more
likely if horses continue to be exercised.
Diagnosis: A definitive diagnosis of avian influenza is established by
1) Characteristics symptoms & high contagiousness, sudden onset and rapid spread.
2) Isolation of virus: direct detection of AI viral proteins or genes in specimens such as tissues,
swabs, cell cultures, or embrocating eggs; or Isolation and identification of AI virus. A
presumptive diagnosis can be made by detecting antibodies to AI virus.
3) Immunofluorescence: Viral antigen in the clinical specimens can be detected by
immunoflurescent technique, like ELISA and RIA.
4) Serology: HI is the widely used technique in detecting the virus.
Control:
After recovery the immunity of homologus strains persist up to 2 years.
By vaccination- Inactivated viral vaccine containing both serotypes mixed with oil adjuvents.
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Microbiology IV
1. Primary at 3-4 months of age.
2. Secondary at 2-6 weeks of first &
3. Booster at one year of age.
12.1.

Avian Influenza (fowl plague) virus

It is an acute, highly infectious and generally fatal disease of poultry, tourkey and occasionally duck
and geese. A wide of birds are also affected. Influenza viruses that infect birds are called avian
influenza viruses. Birds are an especially important species because all known subtypes of influenza
A viruses circulate among wild birds, which are the primary natural reservoir for all subtypes of
influenza A viruses. Phylogenetic studies suggest that aquatic birds could be the source of influenza
A viruses in all other animals.
Morphology:
The structure of virion is mostly spherical/ovoid, 80-100nm diameter
The genome has 8 segments and is held together by the helical capsid comprised of
nucleoprotein.
The linear genome is ss RNA with molecular weight 2 106 Dalton.
The outer surface of the particle consists of a lipid envelope

Envelope from which project prominent glycoprotein spikes of two types, the
haemagglutinin (HA), and neuraminidase (NA), nucleoprotein and a large non-structural
protein.
They have a helical symmetry.
The inner side of the envelope is lined by the matrix protein.
Lipoprotein membranes enclose the nucleocapsids.
Resistance characterstics: Inactivated by sunlight within 2 hrs & rapidly inactivated by u.v. light.
Virus may survive at 56c for 50-60 min. or at 60c for 30 min. It is inactivated by lipid solvent or
disinfectants i.e. phenol or strong alkaline compounds. It is killed by formalin & chloroform. The
virus apparently does not survive long in nature after it leaves host. It is quickly destroyed by
putrifaction.
Replication: Same as general
Cultivation: Same as equine influenza.
Antigenic Variation: the Important of haemagglutinin

Attachment of the virus to red blood cells is an important property used in the laboratory to
detect the presence of the virus and to detect antibodies to the virus.
Main determinant for protection. (Provide primary protection)
The extent of infection into host organism is determined by HA
Usually pneumotropic, the reason is that HA is cleaved by tryptase clara which is restricted to
lungs.
The Important of Neuraminidase (Receptor destroying enzyme)/RDE these are the glycoprotein
present on the surface of the virus. It is measured by enzymatic action.
Allowing the virus to be released from the cell surface.
Provide primary protection.
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Induction and regulation of apoptosis of host cell in

vitro.

Antigenic Characters:
1. Antigenic Drift: (evolution): Antigenic Drift refers to the minor antigenic changes in either
HA or NA or both. These mutations accumulate with time. The human viruses appear to
accumulate more mutations than the equine, which may relate to the presence of more people
than horses in the world.
2. Antigenic shift: Major antigenic change in HA or NA is called Antigenic shift result in
emergence of a new sub type. Antigenic shift is the process by which two different strains of
influenza combine to form a new subtype having a mixture of the surface antigens of the two
original strains. Antigenic drift occurs in all types of influenza including influenza A, B and
C. Antigenic shift, however, occurs only in influenza A because it infects more than just
humans.
Source of transmission:

Manure, dust and feathers


Waterfowl and wild ducks are reservoirs
Wild Swans and wild birds
Nasal secretions
Swine and Turkey
Seal, Mink and whale fishes

Cage birds and game birds


Bird to Bird (Horizontal)
Poultry equipments vehicles, fed bags,
clothes, Egg tray
Feed and water
Rodents and insect

Pathogenesis: The incubation is highly variable and range from few days to week. The virus is
excreted prior to clinical signs. Aerosol virus infects the ciliated epithelium of the nasal mucosa and
then may extend to the bronchioles with resulting epithelial cell necrosis. The disease has high onset
and course.
Symptoms of Avian Flu in chickens: Inappetence, ruffled feathers, Cough, Lacrimation, Cyanosis of
head and skin, Edema of combs, wattles and face, Subcutaneous hemorrhage in legs, Profuse watery
diarrhea, Severe decrease in egg production

Dr. Rebanta Kumar Bhattarai, M.V.Sc. (Vety. Microbiology)

Page 53

Post mortem lesions in Poultry: Egg peritonitis. Petechial hemorrhage in proventriculus.


Necrotic foci in kidney, spleen, liver and lung, severe hemorrhagic enteritis, Mucopurulent
discharge in trachea
Diagnosis: Viruses replicate in a limited number of cell culture systems. Primary cultures of
chicken embryo fibroblasts (CEF) or kidney cells are most commonly. The chicken has been the
most frequently used animal in laboratory studies to determine pathogenicity and study
pathogenesis. Other commonly used laboratory species include the turkey, mice etc.
A definitive diagnosis of avian influenza is established by
Isolation of virus: direct detection of AI viral proteins or genes in specimens such as tissues,
swabs, cell cultures, or embrocating eggs (10 days); or Isolation and identification of AI
virus. A presumptive diagnosis can be made by detecting antibodies to AI virus.
Immunofluorescence: Viral antigen in the clinical specimens can be detected by
immunoflurescent technique, like ELISA and RIA.
Serology: HI is the widely used technique in detecting the AI virus. VN also be used.
Prevention of Bird Flu:

Disease awareness, early detection and notification are pre-requisites of Control


Bio-security is the first line of defense
Rapid diagnosis of the suspected flock
Strict quarantine in the borders
Disposal of manure and dead birds properly
Restricted movement of live chickens and its products
Depopulation or severe culling of the flocks
Rigorous disinfection with Formalin solution
Virkon - S is recognised worldwide as a disinfectant of choice for Emergency

Disease Control:
Biosecurity: The practice of accepted sanitation and biosecurity procedures in the rearing of
poultry is of utmost importance. In areas where waterfowl, shore birds, or sea bird is prevalent,
the rearing of poultry on open range is incompatible with a sound AI prevention program.
Appropriate biosecurity practices should be applied, including the control of human traffic and
introduction of birds of unknown disease status into the flock.
Quarantine
Quarantine should be strong in the borders between two countries.
Restricted movement of live chickens and its products.
Stop importation of parent stock and other poultry products from infected countries.
District quarantine office should be strengthening.

Vaccination: There are 3 types of vaccines:


Inactivated homologous vaccine:
o Inactivated vaccine prepared from H7N3 from Turkey in Utah 1995 protected from
challenge with H7N3 strain in chickens in Pakistan.
Inactivated Heterologous vaccine:
o It has been used successfully in Minnesota for LPAI not for HPAI. Vaccine based
on H5N8 virus isolated from Turkey in Ireland 1983 protected chicken against
challenge with H5N2 strain derived from chicken in Queretaro in 1995.
Recombinant vaccine:
o Recombinant vaccine Fowl poxvirus containing gene that code for the
production of H5 antigen has recently been licensed. It is good in those places
where Fowl pox disease does not occur. The use of this vaccine is also restricted
to the species in which the vector virus will replicate.
o Insect cell culture vaccine-H5 or H7
Disinfectant: Virkon - S is recognised worldwide as a disinfectant of choice for Emergency
Swine Influenza
(Swine flu, Hog flu)
It causes an acute respiratory illness in pigs. The disease causes influenza and pneumonia when
associated with bacterial infection. Morphology and replication is same as in general. No rigid
vaccine practice, although, formalinized vaccine have been attempted.
Cause
Influenza virus A. Subtypes H1N1 and H3N2 have been frequent causes of swine influenza.
More virulent variants of H1N1 have appeared in recent years. The H1N2 subtype has also been
implicated as a cause of acute swine influenza. It has been suggested that all porcine influenza
viruses were derived originally from birds.
Secondary infection with Haemophilus parasuis and other bacteria may contribute to a more
severe disease.
Occurrence
Swine influenza occurs worldwide. Influenza strains from swine may produce serious infections
in humans, other mammals, and birds.
Transmission
Swine influenza is widespread, occurring most commonly in the colder months. Aerosol
droplets, contact, and fomites are the means of spread. In swineherds where the virus is endemic,
young susceptible pigs are continually infected, thereby maintaining the virus. Explosive
outbreaks of acute disease occur when the virus is introduced into susceptible, naive herds.
Clinical & Pathologic Features
Morbidity is high but the mortality is usually no greater than 2%. Virus infection is mild without
secondary bacteria. In a typical outbreak, there is an incubation period of about three days

followed by an acute onset of respiratory distress with rapid respiration, coughing, anorexia, and
prostration. The clinical course is usually 2 - 6 days with rapid recovery in uncomplicated cases.
With secondary invaders and particularly Haemophilus suis the disease is much more serious and
some deaths may occur.
Necropsy in acute cases discloses edematous mediastinal lymph nodes and pneumonic lesions
usually limited to the apical and cardiac lobes. Affected areas are firm and purplish in color and
there is often a sharp line of demarcation between normal and affected tissue. Exudative
bronchitis and interstitial pneumonia are common microscopic findings.
Diagnosis

Clinical specimens: Nasal swabs and lungs, acute and convalescent sera.

Given the often mild character of the disease, laboratory diagnosis may not be sought.

A presumptive diagnosis is made on the basis of clinical and pathologic findings.


Confirmation requires isolation and identification of the virus, demonstration of
seroconversion or detection of viral infected cells in frozen sections of lung tissue by
immunofluorescence.

Swine influenza virus is most easily isolated by the inoculation of embryonated eggs via the
allantoic cavity.

Prevention

Swine influenza virus is usually introduced into herds via replacement stock or returning
show animals.
Vaccination is not practiced.

In the event the disease is particularly severe, antibiotics may be used to control
secondary bacteria.

Recovery from swine influenza infection confers immunity, but the duration is probably
less than a year.

13. Paramyxoviridae :
This is a family of large, negative-strand RNA viruses. The two subfamilies comprise several
genera with important veterinary and human pathogens, including canine distemper, rinderpest,
and Newcastle disease. Respiratory syncytial virus is the major cause of croup in babies and of

respiratory diseases in calves.

Morphologically similar to orthomyxovirus but lightly larger i.e. 100-250 nm in diameter.


Viruses of Paramyxoviridae are enveloped - many are pleomorphic - and have a helical
nucleocapsid.

Genomes are single-stranded, negative-sense, non-segmented RNA.

The diameter of the ribonucleoprotein helix (nucleocapsid) is


18
nm as compared with 6-9 nm of orthomyxovirus.
The molecules weight of this ve sense, ss RNA virus is at
least twice (i.e. 4-9106 daltons) as great as that of
orthomyxovirus.
The paramyxomirus are resistant to the action of
actinomycin-D.
Paramyxoviridae. The virions are
The envelop consists of two glycoprotein,
enveloped, with a helical nucleocapsid.
haemagglutin and in some species with
The virions are highly pleomorphic;
neuraminidase actively and fusion protein.
spherical and filamentous forms are
The viruses may also produce a soluble
observed.
haemolysin, are more active in cell cultures and
frequently produce intracytoplsamic inclusion and sometimes multiple intranuclear
bodies.

Replication takes place in the cytoplasm and envelopment at the surface (plasma
membrane) of infected cells.

The ssRNA (-) is used as a template for the production of mRNA (+) and progeny
genomes.

The viruses are generally sensitive to heat, drying, lipid solvents and many disinfectants.

Most can be propagated in embryonated eggs and in cell cultures with the production of
cytopathic effects including syncytia and cytoplasmic inclusions.

Genus : 3 genera
Family associated with the respiratory illness in animals, mans and birds. The family is divided
into 3 genera:
Paramyxovirus: Have both H & N antigens eg, New Castle Disease virus (NDV), mump
virus and parainflunza

Pneumovirus: Have No H & N. eg, Bovine Respiratory Syncytial Virus (BRSV),


pneumonia virus of mice and turkey rhinitractitis virus.
Morbillivirus: Have H but not N. CD Virus, RP virus, PPR virus.
Between the genera, they are antigenically distinct but within the species morbilivirus, they are
antigenically related.
13.1.

New castle disease virus:

ND is the highly contagious and destructive disease which attack mostly chicken, turkey, pigeons
etc. Water birds and sea birds act as carriers of the virus. It causes self limiting conjunction in
laboratory workers.
The severity of the disease depends upon the 3 factors,
1. Age and immune strains
2. Strains of virus
3. Present of concurrent infections
Morphology:
1. Generally, they are rounded and 100500 nm in diameter, although filamentous forms of
about 100 nm across and of variable length are often seen.
2. The surface of the virus particle is covered with projections about 8 nm in length.
3. The envelope has HN - haemagglutinin + neuraminidase activities
4. In most electron micrographs, the herring bone nucleocapsid, about 18 nm across and
showing helical symmetry, may be seen either free or emerging from disrupted virus
particles.
5. Non-segmented, (-) sense ssRNA, linear arrangement of six genes
6. Glycoproteins - Do not form such prominent spikes as on influenza virus:
Resistance to physical and chemical agents
Temperature: It is inactivated by temperatures of 56C for 3 hrs 60C for 30 min,
pH: Inactivated by acid pH
Chemicals: Ether sensitive
Survival: Survives for long periods at ambient temperature, especially in faeces and can
persist in houses (in faeces, dust etc) for up to 12 months. However it is quite sensitive to
disinfectants, fumigants and sunlight.
It is inactivated by UV light, oxidation and chemicals (lysol, detergents, and butylated
hydroxytoluene).
The rate of viral inactivation varies with the strain of NDV, quantity of virus initially
exposed, time of exposure, and presence of organic matter in the environment.
Infectious virus has been recovered from contaminated areas and eggshells several weeks
following and outbreak of ND, and can survive for long periods in eggs and in frozen
carcasses.

Replication:
The first step in the infection cycle involves attachment of the virus to host cell sialic acid
receptors, function served by the HN glycoprotein. Next, the F protein catalyzes fusion of the
viral envelope and host cell membrane, resulting in uncoating and release of the nucleocapsid
structure into the host cell cytoplasm. For transcription and protein synthesis to occur, first
mRNA is formed with the help of RNA-dependent RNA polymerase which must be supplied by
the virus. The genome is replicated by formation of a full-length positive sense RNA template
onto which a negative sense RNA is then transcribed. Assembly of the nucleocapsid occurs.
Mature virions are released from host cell membranes by budding.

Cultivation:
Chicken Embryo: the virus can be isolated in chicken embryos for about 10 passages to product
thickening of the CAM by virus. After about 100 passages in chicken embryos, the virus
becomes attenuated for dogs and ferrets and can be used as vaccine.
Cell Culture: The virus replicates in primary and continuous dog and ferrrt kidney cells and in
chicken embryo fibroblasts. The CPE includes granular degeneration, formation of giant cells
and syncytia with cytoplasmic and sometimes nuclear inclusion bodies.
NDV natural isolates: Based on severity of disease, NDV isolates are grouped into three
pathotypes: Lentogenic strains = Cause mild or inapparent respiratory disease, low virulent, low
pathogenicity and often used as vaccine, Mesogenic strains = Cause respiratory or nervous signs
with moderate mortality(less than 25%) and Velogenic strains = Cause severe intestinal and/or
neurologic disease resulting in high mortality (as high as 90% or more) Neurotropic,
Viscerotropic.
Pathogenesis: The infection may spread to the healthy birds by direct contact or indirectly by
ingestion of dust from the litter, food-stuffs, drinking water, faces etc. In natural condition, the
incubation is 5-6 days but in severe outbreaks the symptoms may appear within 3 days.
Under natural condition, virus enters via respiratory tracts or conjunctiva and less frequently via
digestive system.

Attach to the epithelial cell, penetration occurs and the virus replicate within 24 hours.
Initial replication of NDV occurs in the mucosa of the upper respiratory tract following aerosol
infection.
Viremia then disseminates the virus in cells of parenchymal organs leads to a secondary viremia,
which in some instances leads to the infection of the cells of the CNS.
Disease takes several different forms in chickens, depending on the virulence of the strain
involved.
Clinical symptoms: Incubation period =2 to 15 days (average 5 6 days). Young birds are
mostly affected. 3 different systems are involved. Generally velogenic form affects young ones
and accompanied by respiratory and nervous signs. & lentogenic and mesogenic form affects
adult ones and symptoms are;
Respiratory: Gasping, coughing, drooping wings, dragging legs,
Nervous: Twisting of the head and neck,chronic spasm, incoordination, opisthotonous
tremor and lameness.
Digestive; in appetence, Cessation of egg production, greenish diarrhea, chicks
huddled together
Morbidity and mortality depend on virulence of virus, degrees of vaccinate Immunity
and condition of flock.
PM lesion:
Velogenic: Prominent hemorrhages occur throughout the digestive tract especially in the mucosa
of the proventriculus and gut-associated lymphoid tissue. Severe tracheitis and pulmonary
congestion are evident in acute cases. These changes are not specific to vvNCD and may be
observed with highly pathogenic strains of avian influenza.
Lentogenic: Mild conjunctivitis and tracheitis are observed. Recovered flocks may show air
sacculitis due to secondary infection with E. coli.
Diagnosis:
1. On the basis of Clinical symptoms and PM lesion
2. Serology- by HA, HI and VNT Antigen detection: Radio-immunoasay, enzyme
immunoassay, fluoro-immunoassay, and immunofluoresence methods are used for
antigen detection. Antibody Detection: Serology uses HI to demonstrate antibody. A 4fold increase in antibody titers is considered positive.
3. Isolation of virus- Allointoic inoculation on 10 days old fertile eggs.
Eggs should be candled daily and tested for presence of hamagglutination

One drop of allontioc fluid +1% chicken RBC


Recheck by HI test as well showing +ve test on HA test.
Immunity and Control:
Young chicks received maternal immunity
By vaccination: Conventional programs = Systematic vaccination is the key of Newcastle
disease control. Two types of vaccines are available: 1. Newcastle disease live vaccine.
2. Newcastle disease inactivated vaccine.
Live vaccine: The immune response to a vaccine increases as the pathogenicity of live vaccine
increases. Therefore to obtain the desired level of protection without serious reaction,
vaccination programmes are needed.
Lentogenic strain- safe to use in young chicks because chance of spread and useful for primary
vaccination & booster vaccination eg, F1, Lasota. Newcastle disease live vaccine F. strain (ICPI
0.25) is used as primary dose in first few days (2 - 3 days) of the chick. It is a mild strain well
tolerated by a young chick. The subsequent live vaccines consist of NDV LaSota strain which
are administered 12 15 days, 20 25 days, 35 40 days (if necessary). The repetition of LaSota
vaccine will depend on MAb profile of the chick population. Newcastle disease mesogenic
vaccine (R2B strain) is administered when the birds are 8 to 10 weeks of age. Live velogenic is
not generally used and it is not safe too.
In inactivated vaccine the virus is inactivated without destroying antigenicity. It is safer to use in
young, laying but detection of immunity is short time. The standard program of vaccination is,
1st dose at -21th of days of age
2nd dose at 8-10 weeks
3rd dose at -16-20 weeks
Another at 5 months
ND killed vaccine can be used
13.2.
Canine Distemper Virus (CDV) /Hard Pad Disease
Definition: The canine distemper is a highly contagious disease of dog with a heavy death rate in
young susceptible dogs. It is characterized by biphasic fever, leukemia GI and respiratory catarrh
and frequenty pneumonia and neurological disorder. Prior to the development of vaccination, this
was the major pathogen of dogs. The disease was world wise in distribution.
Host affected: The disease occurs naturally in dogs (Canidae family) mink, ferrets and skunks.
The member of family felidae (cat, lions and tigers) are not susceptible.

Morphology: The morphology is similar to ND virus. The virus size is slightly smaller particle
measures 90-250 nm in diameter. In its outer envelope neuraminidase is absent. The central core
contains helical about 15-17 nm in diameter. Thermal inactivation of the virus is rapid. The virus
are rapidly destroyed if stored above 0 0C but survives for months at -10 0C. It is inactivated by
ether, 0.1% formalin and 1% Lysol. An irregular partial hecmagglutination of chicken and guinea
pig RBC has been reported in high concentration of adapted virus but this properly is variable.
The CDV forms a triad with measles and rinderpest virus; the last two viruses provide protection
against CDV. Currently, only one antigenic strain is recognized although it is well established
that strains vary in virulence. The pneumo, neutrotropic and epidermal strains (causing Hard
Pad) have been recognized. The variance in virulence exists in measles and rinderpest viruses as
well. In measles, virus antigenic variation between haemagglutinins of different isolates has been
detected by monoclonal antibodies. It is thus likely that antigenic variation also exist between
isolates of distemper virus while the immunodominant epitopes remain cross protective.
Replication: Same as ND virus
Cultivation: Sources, nasal discharge, nasopharyngeal and other lesion.
Animals: The virus can be adapted to grow in unweaned mice, baby hamsters and rabbits. The
virus can be adapted to grow in unweaned mice, baby hamsters and rabbits. The successive
passage of virus in ferrets was enhanced, this led to the discovery of live modified vaccine
against distemper in dogs.
Chicken Embryo: The ferret adapted strains can be further adapted in chicken embryos for about
10 passages to product thickening of the CAM by virus. After about 100 passages in chicken
embryos, the virus becomes attenuated for dogs and ferrets and can be used as vaccine.
Cell Culture: The virus replicates in primary and continuous dog and ferrrt kidney cells and in
chicken embryo fibroblasts. The CPE includes granular degeneration, formation of giant cells
and syncytia with cytoplasmic and sometimes nuclear inclusion bodies.
Pathogenesis:
Naturally, the disease attacks puppies and seldom older dogs, because of natural immunity. The
incubation period is 3-8 days.
Virus replication normally occurs in lymphatic tissue of respiratory tract.
The cell associated viraemia occurs in infection of all lymphatic tissue whoch is followed by
infection of respiratory, GI, urogenital epithelial as well as CNS.
The disease follows the further replication of the virus in these tissues.
Clinical signs:
The clinical signs are diphasic fever, vommition and diarrhea, coryza, conjunctivitis, viraemia,
vesicular and pustular dermatitis and hardened foot pads. Some dogs have nervous manifestation
like myalgia, incoordination, convulsion and coma. The nervous sign usually appear after the
second febrile phase and are generally followed shortly by death. The pattern of distemper is
largely dominated by the immune status of dog population in a given locality in one time.

PM lesion:
Haemorrhagic enteritis is common in puppies under 8 weeks of age.
The epithelium of bladder and pelvis is congested and large intestine has excessive
mucus.
The lungs are generally congested and pneumonic.
The spleen may be enlarged and necrotic areas seen in liver.
Perivascular cuffing, neuronal degeneration and demyelination occur in dogs with
neurological signs.
Intracytoplasmic and sometimes intranuclear inclusion bodies may be found in the
bladder, renal pelvis, epithelial cells of respiratory tract, intestine and brain.
Diagnosis:
1. On the basis of Clinical symptoms: A provisional diagnosis can be made from the clinical
symptoms which includes respiratory symptoms, diarrhea catarrhal discharges from eyes and
nose, hyperkeratosis of foot pad nervous signs. Intracytoplasmic inclusions may be found in
stained conjunctival smears.
2. On the basis of PM lesion: On postmortem examination inclusion bodies may be seen in
bronchial epithelium, bladder epithelium and lung macrophages.
3. By isolation of virus: A confirmatory diagnosis is made by isolation of virus from blood,
excretions and secretions, lympnodes, liver, spleen, lungs and brain of dogs with nervous
manifestation. Susceptible dogs and ferrets are used for isolations. Virus isolation in canine
or ferrrt kidney can be made.
4. Detection of specific distemper antigens using immunofluorescence, complement fixation or
gel diffusion test with hyperimmune anti-distemper or anti-rinderpest sera.
5. Serology- by immmunofluorescence, Complement fixation test (CFT), Gel diffusion test
(GFT). These tests can be used for detection of CD Antigen
6. CD virus can partially agglutinate th Rabbit RBC.
Immunity:
Active Immunity: In most animals long lasting immunity develops after recovery from natural
infection. The vaccines are usually given in puppies more then 12 weeks of age. The attenuated
measles vaccines have been used in puppies of 3-6 weeks of age. The measles virus stops
symptoms of distemper to develop but may not prevent the infection of CDV.
Combined CDV and infectious canine hepatitis vaccines are also available. Cell culture vaccines
are recommended for use.
Pulpy kidney cell culture vaccine
Modified measles virus vaccine- antigenically related
Multiple vaccines are also used.
Passive Immunity: Maternal antibodies are transferred to puppies via colostrums and usually
prevent infection upto 8 weeks or occasionally 12 weeks. The measles virus stops symptoms of
distemper to develop but may not prevent the infection of CDV.

13.3.

Rinderpest Virus/ Cattle plague disease

RP is the disease of four footed animals and characterized by fever, severe hemorrhage, catarrh
in mucus membrane with necrotic stomatitis & gastro-enteritis.
Morphology: Same as ND/CD virus. Generally, they are rounded and 100500 nm in diameter,
although filamentous forms of about 100 nm across and of variable length are often seen. The
surface of the virus particle is covered with projections about 8 nm in length.
Replication: same as other member of this group
Cultivation: The virus replicates in goat, sheep, rabbit and bovine kidney cells. The growth can
be detected by CPE within the 3-12 days of incubation at 37 0C. The CPE includes granular
degeneration, formation of giant cells and syncytia with cytoplasmic and sometimes nuclear
inclusion bodies. It can grow in the embryonated hens eggs s in CAM, yolk sac or allantioc
route.
Pathogenesis:
RP virus infection mainly through upper respiratory tract
Virus first replicates in the tonsils & lymph nodes & viremia occur.
The virus replicates in tissues (spleen, bone marrow, lymph nodes, muosa of alimentary &
upper respiratory track.
There is destruction lymphocytes in tissues & leukopenia.
Focal necrotic stomatitis &enteritis are characteristic of the disease.
Nasal &ocular secretion contain high titers of virus, then rise of fever up to 107 0F &
leucopenia occur prior to appearance of oral ulcer & unset of diarrhoea with blood (dysentery).
It causes pyknosis & fragmentation of the nuclei in lymphocytes.
The cells become shrivelled &necrotic with the cytoplasm
The virus produces lesions in the oral mucosa,oesophagus & severe affected.
Death is usually from severe dehydration & immunosupperessed due to destruction of
lymphoid organs by the virus.
Clinical findings: Sudden onset of fever, depression, restlessness, dry muzzle, serous and
lacrimal discharges, congested mucus membrane, anorexia, initially constipation etc.
Mucosal phase = begins after 2-5 days of prodermal phase & characterized by pinpoint necrotic
lesion in mucosa of mouth, nostrils, profuse salivation, and lacrimal discharges.
Diarrheic phase = starts when fever falls (2-3 days of mucosal phase), faces are dark with mucus
and streaks of blood (shooting diarrhea).
PM finding:
Erosive lesion on mouth, upper oesophagus and upper respiratory tract
Zebra-striping in rectum
Erosion can be seen in stomach and payers patches
Diagnosis:
On the basis of Clinical symptoms = Initial diagnosis from clinical sign, herd history of recent
introduce of animal from infected area. The morbidity &mortality rate in infected area.

Partial confirmatory Diagnosis = Examination of W.B.C. A marked leukopaenia occurs at the


peak of the infection. The total count comes to less than 400/ml. There is marked neutrophilia
after few days.
Examination of impression smear = the stained preparation may show the presence of
multinucleated eosinophilic intranuclear & basophilic intracytoplasmic inclusion bodies
Confirmatory Diagnosis = Animal inoculation: - 0.5ml of citrated blood is to be collected at the
peak infection & given I/V route to young calf. There will be high rise of temperature in 3-6days
& other symptoms. Besides blood, spleen & lymph node suspension may be used as in
inoculation.
Isolation & Identification of virus = Blood or lymph nodes or spleen from the affected animals
collected during the first 4 days of fever prior to unset of diarrhoea. The sample is inoculated in
bovine kidney cell culture. Cell cultures are examined daily for the cytopathic effect (CPE). CPE
develops within 18-48 hours. Complete destruction is noted in 10-12 days. Eosinophilic
intracytoplasmic & intranuclear inclusions are formed.
Serology = Serum Neutralization Test, Complement Fixation Test, Agar gel Diffusion test,
Counter immuno electrophoresis, Immunofluorescence test, ELISA Test etc.
Immunity:
Recovered animal develop long lasting immunity (usually)
Passive immunity from mother to calf for about 3-8 months
Vaccine = Caprinized vaccine, Lapined vaccine, Avianised vaccine can be used.

14. Rhabdoviridae :
(Gr: Rhabdos=rod)
The virus particle are enveloped, bullet shaped or bacilliform measuring about
18075nm.
The capsid has a helical symmetry which is closely attached to lipoprtotein envelop (the
outer layer or envelop which surrounds the axial nucleocapsid is typifies as a lipid layer
from which protein spikes (8nm) protrude.
All members of this group conatain ve sense, ss RNA ith a molecular weight of 3.54.5106 daltons.
Viruses are sensitive to ether and other lipid solvents.
Rabies and vesicular stomatitis virus posses a lipoprotein haemagglutinin which
agglutinates goose erythroscytes.
Rhabdoviruse resist freezing and thawing and some members will persist in soil for many
days at 4-6 0C.
Genus
1. Vesiculovirus sps: vesicular stomatitists virus causes sore mouth of cattle, pig and horse.
Lyssa virus sps : rabies virus
Unclassified sps Ephemeral fever virus