Infectious laryngotracheitis is a highly contagious respiratory disease of chickens and pheasants caused by gallid herpesvirus 1. The virus infects the upper respiratory tract, causing swelling and the production of bloody mucus. Clinical signs range from mild illness to high mortality depending on the strain. The virus establishes latency in trigeminal ganglia. Diagnosis involves detecting viral antigens or DNA in samples. Vaccines include live-attenuated and inactivated versions. Prevention relies on vaccination programs, biosecurity, and cleaning between flocks.

1. INFECTIOUS LARYNGEOTRACHEITIS
MOUSUMI BORA
DIVISION OF VIROLOGY
INDIAN VETERINARY RESEARCH INSTITUTE

2. CONTENTS
INTRODUCTION
HISTORY
EPIDEMIOLOGY
ETIOLOGY
VIRUS REPLICATION
STRAIN CLASSIFICATION
PATHOGENESIS
PATHOLOGY
IMMUNITY
DIAGNOSIS
PREVENTION AND CONTROL

3. INTRODUCTION
 Infectious laryngotracheitis (ILT) is an acute, highly contagious infection of chickens
and pheasants
 Result in severe production losses due to mortality and/or decreased egg production
 Severe epizootic forms of infection are characterized by signs of respiratory
depression, gasping, expectoration of bloody mucus and high mortality
 Mild enzootic forms of infection are encountered increasingly in developed poultry
industries and manifest variously as mucoid tracheitis, sinusitis, conjunctivitis, general
unthriftiness and low mortality
 Included within List B of the World Organization for Animal Health (OIE)

4. HISTORY
 The disease was first described in 1925 in Canada
 Followed by United States in 1926
 Given several different names including laryngotracheitis, infectious laryngotracheitis,
and avian diphtheria
 The name infectious laryngotracheitis was adopted in 1931 by the Special
Committee on Poultry Diseases of the American Veterinary Medical Association
 The cause of LT was first shown to be a filterable virus by Beaudette
 Laryngotracheitis was the first major avian viral disease for which an effective vaccine
was developed

5. EPIDEMIOLOGY
 Distributed world-wide
 May be present only in certain localities within a country or geographic region
 The greatest incidence of disease is generally seen in areas of highly intensive
poultry production
 Route of entry : upper respiratory and ocular routes
 Transmission occurs : acutely infected bird , mechanical transmission (contaminated
equipment and litter)
 No evidence for vertical ILTV transmission to the egg or for shedding ILTV on shells
of eggs laid by infected hens
 Incubation period : 6-12 days, following natural exposure
: 2-4 days, following experimental inoculation

6. ETIOLOGY
 Gallid herpesvirus 1
 Genus Iltovirus ; Subfamily Alphaherpesvirinae ; Family Herpesviridae
 Symmetry Icosahedral
 Complete virus particle has a diameter of 195–250 nm and consists of an irregular
envelope surrounding the nucleocapsid
 DNA genome consists of a linear 155-kb ds molecule composed of unique long
(UL) and unique short (US) regions flanked by inverted repeats
 Nucleic acid of LTV is composed of DNA with a buoyant density of 1.704 g/mL

7. VIRAL REPLICATION
 Initiation of infection begins with receptor binding through the initial interactions of the
virus genome with the host transcriptional machinery in the nucleus
 Herpesviruses enter cells by two major pathways
 Fusion of virion and cellular membranes is mediated by gB and the gH /gL complex
 Three classes of mRNA- α, ß and ɤ are transcribed in sequence by cellular RNA
polymerase II
 α RNA (immediate early) - processed appropriately to become mRNAs, are
translated to form α proteins
 ß RNA ( early RNA) - the translation of which produces ß (early) proteins
 ɤ RNA (late) mRNAs, which are transcribed from sequences situated throughout the
genome are translated into ɤ proteins
 Viral DNA replication then commences, utilizing some of the viral α and ß proteins, in
• addition to host-cell proteins.
1. Fusion of the virion
envelope with the
plasma membrane
2. Membrane fusion
after virion uptake by
endocytosis

8. VIRAL REPLICATION

9. VIRAL REPLICATION
 Maturation involves the completion of encapsidation of virion DNA into nucleocapsids
and the association of nucleocapsids with altered patches of the inner layer of the
nuclear envelope
 Complete envelopment occurs by budding through the nuclear membrane
 Mature virions accumulate within vacuoles in the cytoplasm and are released by
exocytosis or cytolysis
 Virus-specific proteins found in the plasma membrane, where they are
involved in cell fusion

10. STRAIN CLASSIFICATION
Antigenicity
 Appear to be antigenically homogenous based on virus-neutralization,
immunofluorescence tests, and cross-protection studies
Molecular classification
 Restriction endonuclease analyses of viral DNA
 DNA hybridization assays
 Polymerase chain reaction (PCR) procedures combined with restriction fragment
length polymorphism (RFLP) analyses of amplified DNA (PCR-RFLP)
 PCR-RFLP combined with gene sequencing
 Gene sequencing alone
Pathogenicity
 Laryngotracheitis virus strains also were shown to differ based on -
 Virulence for chicken embryos
 Plaque size and morphology in cell culture
 Plaque size and morphology on chorioallantoic membrane (CAM) of embryonated
chicken eggs

11. PATHOGENESIS
RESPIRATORY
TRACT EPITHELIUM
MULTIPLIES IN
RESPIRATORY
EPITHELIUM
LARYNX, TRACHEA,
RESPIRATORY
SINUSES , AIR SACS,
LUNGS
CONJUNCTIVA
EPITHELIAL DAMAGE
AND HAEMORRHAGE
Swollen , watery
eyes
IP
6-12 days
Nasal discharge,
Coughing, Gasping ,
Blood stained mucus

12. PATHOGENESIS cont…
 Infectious virus usually is present in tracheal tissues and tracheal secretions
for 6–8 days PI
 Extratracheal spread of LTV to trigeminal ganglia after 4-7 days of tracheal
exposure
 Trigeminal ganglion is the principal site of LTV latency
 Reactivation of latent LTV from the trigeminal ganglia 15 months after
vaccination of a flock has been reported from Germany

13. IMPORTANCE OF LATENT INFECTION
 A landmark study which helped to explain how apparently spontaneous outbreaks of
ILT can occur in field situations showed that rates of shedding of ILTV into the trachea
could be significantly increased by the stresses of either the onset of lay or mixing
with unfamiliar birds
 In this case, the latently infected chicken can act as an unsuspected reservoir host
and enable ILTV to infect further susceptible chickens

14. CLINICAL SIGNS
Sub-acute form
 Nasal and ocular discharge
 Tracheitis
 Conjunctivitis
 Mild rales
Acute form ( Severe epizootic form)
 Nasal discharge
 Moist rales
 Gasping (Pump handle respiration)
 Dyspnea
 Expectoration of blood stained mucus
Mild enzootic form
 Decreased egg production
 Watery eyes, conjunctivitis, swelling of infra-orbital sinuses,
 Mild tracheitis, persistent nasal discharge and hemorrhagic conjunctivitis

15. CLINICAL SIGNS
 Course of the infection varies with the severity of lesions
 Generally, most chickens recover in 10–14 days
 Epizootic forms of the disease cause high morbidity (90–100%) and variable mortality
 Mortality can vary from 5% to 70% but usually is in the range of 10–20%
 Mild enzootic forms of the disease result in morbidity as low as 5% and very low
mortality (0.1–2%)

16. PATHOLOGY
Gross lesions
• Edema and congestion
Conjunctiva
• Congestion
Infra orbital sinuses
• Blood stained mucus exudates
• Mucoid cast
Trachea and Larynx
• Bronchitis
Bronchi
• Inflammation
Air sacs and lungs

17. PATHOLOGY
Microscopic lesions
• Loss of goblet cells
• Infiltration of mucosa with
inflammatory cells
Tracheal mucosa
• Cells enlarge, lose cilia, and become
oedematous
• Syncytia are formed
Respiratory epithelium
• Blood vessels within the lamina
propria may protrude into the
tracheal lumen
Lamina propria
• Rupture of capillariesBlood capillaries
Intranuclear
inclusion bodies are
found in epithelial
cells by 3
days PI

18. Ultrastructural
 Electron microscopic studies – the first cellular changes occur in the
nucleus of epithelial cells during formation of viral capsids
 Viral capsids bud through the nuclear membrane, acquiring lipid envelopes,
and aggregate into large masses within vacuoles in the cytoplasm
 The cloudy swelling observed in light microscopic studies of early cellular
changes has been associated with the presence of these large masses of
viral particles in the cytoplasm
PATHOLOGY

19.  Virus-neutralizing antibodies become detectable within 5-7 days PI
 The numbers of IgA- and IgG synthesizing cells in the trachea increased
substantially in experimentally infected chickens between days 3 and 7 PI
 Delayed-type hypersensitivity responses to LTV
 The principal mediator of LT resistance is the local cell-mediated immune
response in the trachea
 Bursectomized and cyclophosphamide treated chickens fail to mount
humoral immune responses following LT vaccination but develop full
immunity
IMMUNITY

20. A. DETECTION OF VIRAL ANTIGENS
1. Histopathology
 Laryngotracheitis is characterized by the development of pathognomonic intranuclear
inclusion bodies in respiratory and conjunctival epithelial cells
 Intranuclear inclusion bodies may be detected in tissues stained with Giemsa or
Hematoxylin and Eosin
2. Isolation and identification of the causative agent
 Sample of choice : suspensions of respiratory exudate, conjunctival exudate, or
homogenates of appropriate tissues
 Route of inoculation : CAM of 9–12 day-old embryonated chicken eggs
 Chorio-allantoic membrane plaques can be observed as early as 2 days PI;
 Opaque edges and a central depressed area of necrosis
DIAGNOSIS

21.  Cell culture
 Cell lines : Chicken embryo liver cells and Chicken Kidney cells
 Viral cytopathology may be observed in cell culture within 24 hr PI
 Cytopathic effects :
 Increased refractiveness
 Swelling of cells
 Chromatin displacement
 Rounding of the nucleoli
 Formation of multinucleated giant cells (syncytia)
 A maximum of two serial passages in CEL and CK cell cultures are
required to ensure detection of LTV in clinical samples
DIAGNOSIS

22. Chicken embryo kidney cell monolayer.
72 hr after inoculation with
laryngotracheitis virus. A multinucleated
giant cell (syncytium) has formed with
many nuclei containing inclusion bodies.
DIAGNOSIS

23. 3. Electron microscopy
 Electron microscopy has been utilized to detect LTV in tracheal scrapings
 This approach is successful only when large numbers of virus particles are present in
clinical samples
 Virus particles were observed only when clinical samples contained a minimum of
10 3.5 infectious virus/ 0.1 mL
4. Fluorescent antibody (FA) and Immuno peroxidase test (IPT)
 LTV antigens can be detected in tracheal tissue from day 2 through day 14 PI by
using FA
 LTV antigens in frozen sections of tracheal tissues can be detected from day 1 to day
9 PI using immuno peroxidase test
DIAGNOSIS

24. 5. ELISA
 Antigen capture ELISA using monoclonal antibodies to LTV
B. DETECTION OF VIRAL DNA
1. Dot-blot hybridization
2. Polymerase chain reaction (PCR)
3. Real time PCR
C. DETECTION OF ANTIBODY AGAINST LTV
1. Agar-gel immunodiffusion (AGID)
2. Virus neutralization (VN)
3. Indirect fluorescent
4. Antibody (IFA) test
5. ELISA
DIAGNOSIS

25.  No drug has been shown to be effective in reducing the severity of lesions
or relieving disease signs. If a diagnosis of LT is obtained early in an
outbreak, vaccination of unaffected birds may induce adequate protection
before they become exposed
TREATMENT

26. Immunization
PREVENTION
Modified-Live
Virus Vaccines
Infraorbital sinuses / Intranasal instillation/
Feather follicles/ Eye drop and orally
through drinking water
˃10 2 plaque-forming
units/mL
10 5 embryo infective
doses ( oral vaccination)
Inactivated
Vaccines
Prepared from inactivated whole LTV or
affinity-purified preparations of LTV
glycoproteins
Practical use is very less
due to high cost
Vaccines Based
on Recombinant
DNA Technology
constructed by insertion of LTV genes
into virus vectors and by alteration or
deletion of viral genes
Capable of inducing
protective immunity

27.  Primary vaccination with current modified-live ILT vaccine strains will confer:
 Partial protection against challenge by 3-4 days post exposure.
 Complete protection after one week
 Revaccination with live vaccines may or may not assist in maintaining
protection levels, as the infectivity of any vaccine virus may be neutralized
and replication prevented at the portal of entry into the host chicken
PREVENTION

28.  Purchase birds from a source known to be free of ILT
 Should be isolated on your farm for 21 days before being mixed with your resident
birds
 Establish a vaccination program that protects the flock from ILT and other important
poultry diseases
 Early detection of the disease is essential to minimize the impact of severity
 Prevent contamination of feed and water sources with particular attention to wild birds
and animals
 Store dead carcasses in a closed container until they can be disposed of according to
the requirements of the Destruction and Disposal of Dead Animals Regulation
 Thorough cleanout and disinfection between flocks
MANAGEMENT