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INFECTIOUS LARYNGEOTRACHEITIS
MOUSUMI BORADIVISION OF VIROLOGY
INDIAN VETERINARY RESEARCH INSTITUTE
CONTENTS
INTRODUCTION
HISTORY
EPIDEMIOLOGY
ETIOLOGY
VIRUS REPLICATION
STRAIN CLASSIFICATION
PATHOGENESIS
PATHOLOGY
IMMUNITY
DIAGNOSIS
PREVENTION AND CONTROL
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)
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
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
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
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 fusionafter virion uptake by endocytosis
VIRAL REPLICATION
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
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
PATHOGENESIS
RESPIRATORY TRACT EPITHELIUM
MULTIPLIES IN RESPIRATORY EPITHELIUM
LARYNX, TRACHEA, RESPIRATORY SINUSES
, AIR SACS, LUNGSCONJUNCTIVA
EPITHELIAL DAMAGE AND HAEMORRHAGE
Swollen , watery eyes
IP 6-12 days
Nasal discharge, Coughing, Gasping ,
Blood stained mucus
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
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
CLINICAL SIGNS
Sub-acute form Nasal and ocular discharge Tracheitis Conjunctivitis Mild ralesAcute form ( Severe epizootic form) Nasal discharge Moist rales Gasping (Pump handle respiration) Dyspnea Expectoration of blood stained mucusMild enzootic form Decreased egg production Watery eyes, conjunctivitis, swelling of infra-orbital sinuses, Mild tracheitis, persistent nasal discharge and hemorrhagic conjunctivitis
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%)
PATHOLOGY
Gross lesions
• Edema and congestionConjunctiva
• CongestionInfra orbital sinuses
• Blood stained mucus exudates• Mucoid cast Trachea and Larynx
• BronchitisBronchi
• Inflammation Air sacs and lungs
PATHOLOGY
Microscopic lesions• Loss of goblet cells• Infiltration of mucosa with
inflammatory cellsTracheal mucosa
• Cells enlarge, lose cilia, and become oedematous
• Syncytia are formedRespiratory 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 3days PI
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
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
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
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
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
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
5. ELISA Antigen capture ELISA using monoclonal antibodies to LTV
B. DETECTION OF VIRAL DNA
1. Dot-blot hybridization2. Polymerase chain reaction (PCR)3. Real time PCR
C. DETECTION OF ANTIBODY AGAINST LTV
4. Agar-gel immunodiffusion (AGID)5. Virus neutralization (VN)6. Indirect fluorescent7. Antibody (IFA) test8. ELISA
DIAGNOSIS
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
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
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
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
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