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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