AVIAN INFECTIOUS LARYNGOTRACHEITIS
Poultry industry is one of the country’s rapidly developing sectors and contributes its great share to the country’s economy, however, the production is hampered by several disease conditions. The consequence of globalization, climate change and rapidly expanding poultry population results in the emergence of several diseases of major public health concern. (Gowthaman et al., 2020).
Avian infectious laryngotracheitis (ILT) is a respiratory disease of chickens caused by an alpha herpesvirus, Gallid herpesvirus 1 (GAHV-1) leading to upper respiratory tract infection. It can also affect pheasants, partridges and peafowl. In the virulent form, the history, clinical signs and severe tracheal lesions are indicative of the disease, however, the mild form may remain indistinguishable from other mild respiratory illnesses (OIE, 2018). The infection can be noted as catarrhal haemorrhagic to fibrinous inflammation of the respiratory tract (Dinev, 2007). It can be identified by marked dyspnoea, gasping, coughing and expectoration of bloody exudate (Boulianne, 2012).
The disease was first described in 1925 by May and Thittsler and it has been identified in many countries as a serious disease affecting mainly the areas of intensive production and high density population of chickens in countries like North America, South America, Europe, China, Southeast Asia and Australia (Hidalgo, 2003).
The respiratory tract and conjunctiva are affected by the virus and strains vary in virulence. Fowls of all ages are susceptible. Broilers and very young chicks and are most susceptible (Boulianne, 2012). the disease is mostly seen in the birds aged 3-9 months old while the older birds are usually immune. Additionally, males are more susceptible than females; and the heavier breeds more susceptible than lighter ones (Vegad and Katiyar, 2008).
Avian infectious laryngotracheitis is caused by the infectious laryngotracheitis virus, also known as Gallid herpesvirus 1 (GaHV-1), belonging to the Genus Iltovirus, subfamily Alphaherpesvirinae of the family Herpesviridae (Davison et al., 2009).
VIRAL MORPHOLOGY AND CHEMICAL COMPOSITION
The viral envelope contains viral glycoprotein spikes on its surface as fine projections. Replication of ILT virus is similar to other alpha-herpesviruses like pseudorabies virus and herpes simplex virus (Vegad and Katiyar, 2008). The GaHV‐1 virion has a hexagonal nucleocapsid (80-100 nm in diameter) of icosahedral symmetry surrounded by a protein tegument layer, encapsulated by an outer envelope (Garcia and Spatz, 2020). The nucleocapsid are composed of 162 elongated hollow capsomeres (Cruickshank et al., 1963; Watrach et al., 1963). The virus particle including the irregular envelope surrounding the nucleocapsid has a diameter of 195- 250 nm. A guanine plus cytosine ratio of 45% has been reported in the Laryngotracheitis virus DNA (Plummer et. al., 1969). The viral genome consists of a linear 155-kb double-stranded molecule (Johnson et al., 1991). The glycoproteins of the virus are responsible for stimulating humoral and cell-mediated immune responses and act as major immunogens of ILT virus. (York et al., 1987; 1990).
The incubation period of the disease may be variable depending on the viral dose, route of inoculation, degree of viral virulence and age of birds. Under experimental conditions, the onset of clinical signs can range from 3 to 7 days post-inoculation (dpi) (Vagnozzi et al., 2015). Virus replication in the trachea can be detected as early as 2 to 7 dpi (Oldoni et al., 2009). The clinical signs of the disease commonly appear between 6 to 14 days post infection. The incubation period of ILTV varies between 6 to 14 days (Kernohan 1931; Seddon, 1935).
REPLICATION OF VIRUS
Gallid Herpes Virus‐1 infection is initiated by its attachment to the cell membrane. Following attachment, the viral envelope fuses with the cell plasma membrane, the nucleocapsid enters the cytoplasm, and the DNA–protein complex is then freed from the nucleocapsid and enters the nucleus through nuclear pores (MacLachlan and Dubovi, 2011). Transcription and replication of viral DNA occur within the nucleus (Guo et al., 1993). The transcription of herpes simplex virus (HSV‐1) genes occurs in an ordered pattern of gene expression namely immediate–early (IE), early (E), early–late (E/L), and late (L) (Garcia and Spatz, 2020). Transcription of GaHV‐1 genes classified the ICP4 as the only immediate–early gene, 30 genes are classified as early, 28 as late, while the transcription kinetics of around 15 genes appeared quite “leaky” because these genes have features of both early and late genes subjecting the virus to a more complex pattern of regulation (Mahmoudian et al., 2012). Following DNA replication, cleavage of viral DNA occurs. Maturation involves the encapsidation of virion DNA into nucleocapsids and nucleocapsids combine with altered patches of the inner lamellae of nuclear envelope. (Fuchs et al., 2007; Guo et al., 1993). Enveloped particles then migrate through the endoplasmic reticulum and accumulate within vacuoles in the cytoplasm where mature capsids are formed by the incorporation of the tegument material and a secondary envelopment step. Mature virions are then released from cells by exocytosis (Garcia and Spatz, 2020).
Latency refers to persistent life-long infection of a host with restricted but recurrent virus replication which causes shedding, transmission and the regular maintenance of detectable antiviral immune responses. Therefore, latent infections in clinically normal hosts leads to occurrence of undetectable reservoir for virus transmission (MacLachlan and Dubovi, 2011).
Like other herpesviruses, ILTV establishes latent infections, which was identified by the re-isolation of virus by repeated tracheal swabbing (Bagust, 1986) and tracheal organ cultures (Adair et al., 1985). Trigeminal nerve ganglion is the main site of ILT virus latency. The virus remains dormant in the ganglion and trigeminal ganglion provides the main sensory innervation to the tissues of the upper respiratory tract which then leads to neural viral migration. Extratracheal spread of LTV to trigeminal ganglia 4-7 days after tracheal exposure has been reported (Bagust et al., 1986). Latent laryngotracheitis virus also got reactivated from the trigeminal ganglia after vaccination of bird flock (Kaleta et al., 1986).
Viral infection of the upper respiratory tract is followed by intense viral replication. The virus infects tracheal tissues and causes secretions for 6-10 days. Virus multiplies in the respiratory tissues, however there is no evidence of viraemia. From trachea the virus spreads to trigeminal ganglia 4-7 days after tracheal exposure. Reactivation of latent virus from the trigeminal ganglia after long periods has been recorded (Vegad and Katiyar, 2008).
The viral entry is through respiratory and ocular routes. Virus initially replicates in the epithelium of the conjunctiva, respiratory sinuses, larynx and upper respiratory tract (MacLachlan and Dubovi, 2011). The virus can be found in the latent infection sites (Trigeminal ganglion) from two of cytolytic infections onwards (Bagust et al., 1986; Kirkpatrick et al., 2006a; Oldoni et al., 2009). Severe damage to tracheal and conjunctival epithelial lining leading to haemorrhages and other clinicopathological manifestations in birds is caused by active cytolytic infection (Bang and Bang, 1967; Tully 1995). Later the virus infects the underlying lamina propria of the tracheal epithelium after invading through the basement membrane with the help of up-regulated cellular proteases (Glorieux et al., 2009; Steukers et al., 2012, Reddy et al., 2014) and disseminated to the liver, caecal tonsils and cloaca (Bagust et al., 1986; Oldoni et al., 2009). Up-regulation of cell growth and proliferation related genes is caused by the virus replication. The infected cells produce cytokines and other inflammatory mediators leading to immune responses such as elevated body temperature, intensive oedema and infiltration of lymphocytes (Purcell, 1971a). The viral infection also leads to scattering of CD4+ and CD8+ cells, as well as clustering of B lymphocytes in the mucosa (Devlin et al., 2010). Effective adaptive immunity is induced following the lytic phase of an infection and causes establishment of latency (Williams et al., 1992). Reactivation of the virus and its latency is mediated by thymidine kinase and infected-cell polypeptide 4 (ICP4) (Johnson et al., 1995; Schnitzlein et al., 1995; Han et al., 2002).
The disease occurs in laryngotracheal and conjunctival form where suffocation, rales and cough are observed in the laryngotracheal form. The head and neck are extended upward and forward during inspiration. (Dinev, 2007). In severe cases, the neck is raised and the head extended during inspiration- “pump handle respiration.” Wet eyes, tear secretion and oedema of infraorbital sinuses are observed in the conjunctival form of the disease.
The clinical signs are characterized by a sudden increase in mortality among affected flock (Aziz, 2009). The factors affecting the severity of the disease comprise the virulence of the virus, stress conditions, co-infections with other pathogens, immune status of the flock and age of the birds (Vasudevan et al., 2016).
The disease may appear in different forms, namely, per acute, acute subacute and chronic or mild form.
- Per acute form- In the per acute form, onset of disease is sudden with a rapid spread. The morbidity is high and mortality may exceed 50% (OIE, 2018). The birds may die in good body condition before the appearance of signs (Preis et al., 2013) such as dyspnoea with extension of the neck and gasping in an attempt to inhale. Due to obstructions in the trachea birds express gurgling, rattling and coughing of clots of blood. The birds are lethargic with moderate-to-severe conjunctivitis, swelling of eyelids and lacrimation (Blakey et al., 2019)
- Acute form- It includes dyspnoea, lethargy and anorexia (Garcia and Spatz, 2020). Fever commences between 4 to 6 days post infection and the total leukocyte count (TLC) indicates lymphopenia and heterophilia (Chang et al., 1997). Obstruction of trachea due to clotted blood and presence of exudate causes gasping with open-mouthed breathing, high-pitched squawk sound and moist rales (Kernohan, 1931; Jordan, 1966). Purulent conjunctivitis with frothy exudates in eye, sinusitis and nasal discharge are observed (Beach, 1926). The morbidity reaches 100% while mortality varies from 10 to 30%. Some flocks may face reduced to deceased egg production (Lohr, 1977; Creelan et al., 2006).
- Subacute form- In the subacute form, the onset of illness is slower and respiratory signs may extend over some days before deaths are seen. There is high morbidity but lower mortality between 10% and 30% (OIE, 2018).
- Chronic or mild form- This form is seen among survivors of the infection. Incidence may be 1–2% and severely affected birds die of suffocation. Signs coughing and gasping, unthriftiness, moist rales, head shaking, squinting eyes, swelling of the infraorbital sinuses causes almond-shaped eyes), nasal and oral discharge, decrease in body weight and reduced egg production (up to 10%). (Hinshaw et al., 1931; Ou et al., 2012). The morbidity may go up to 5% and mortality usually restricted. Recovered birds act as a potential means of transmission of the virus (Hughes et al., 1987).
The gross lesions are usually noted in the sinuses and upper respiratory tract and vary with the severity of the disease (Seifried, 1931; Gough et al., 1977). Mucoid rhinitis and haemorrhagic tracheitis is noted in the per acute form. In concurrent infection, muco-fibrino acute rhinitis and sinusitis, occlusion of paranasal sinuses by caseous exudate, facial swelling, and muco-fibrino tracheitis have been observed (de Macedo Couto et al., 2015).
Blood casts or blood-stained mucus is present in the trachea causing respiratory obstruction (Barhoom and Dalab, 2012). In the acute form, caseous and diphtheritic exudate along with mucus in the trachea. Haemorrhages, congestion and cyanosis are present in the trachea. Yellow caseous diphtheritic membranes adherent to the larynx, syrinx and mucosa of the upper trachea with or without haemorrhages are noticed and the obturation caused by it leads to asphyxiation and death (Gowthaman et al., 2014). Yellow caseous cheesy plug can be present in primary bronchi in deeply extended lesions (OIE, 2018). Milder form of the disease leads to excess mucus with or without diphtheritic fibrino-necrotic exudation in trachea (Linares et al., 1994). There may be congestion of the lungs, thickened wall of air sacs and presence of caseous exudate in the lumen however involvement of lungs and air sacs is rare (Aziz, 2009). Heterophilic exudation accompanies the inflammatory response in nares (Gowthaman et al., 2014). Ocular lesion includes conjunctivitis along with lacrimation, oedema and congestion of infraorbital sinuses (Hinshaw et al., 1931; Kirkpatrick et al., 2006a).
The microscopic lesions are present in the conjunctiva, sinuses, trachea and lungs (Linares et al., 1994). Conjunctiva shows signs of early hyperaemia, swelling, inflammatory cell infiltration including red and white blood cells and fibrino-cellular debris and destruction of epithelium due to sloughing (Aziz, 2009). Tracheal mucosa is affected due to the loss of goblet cells and infiltration of mucosa with inflammatory cells. As the infection progresses, the cells enlarge, lose cilia and become oedematous. Multinucleated cells (syncytia) are formed and lymphocytes, histiocytes, and plasma cells migrate into the mucosa and submucosa The epithelial cells undergo hyperplasia (Russell, 1983). This is followed by necrosis and diffuse denudation of the tracheal epithelial cells. Further, severe laryngitis and tracheitis occurs due to protrusion and rupture of blood vessels of lamina propria into tracheal lumen (Sary et al., 2017).
Multinucleated cells (syncytia) are produced predominantly in ciliated cells of the epithelium. Small syncytia, often of circular shape, can be also found beneath the normal ciliated epithelium (Purcell, 1971b). Nuclear inclusion bodies in syncytia cells are characterized by strong eosinophilic staining surrounded by clear haloes (2 to 5 days) and disappear later due to necrosis and denudation of epithelial cells (Seifried, 1931; Guy et al., 1992; Vanderkop, 1993). The microscopic changes in primary and secondary bronchi includes epithelial degeneration and denudation with infiltration of mononuclear cells (Preis et al., 2013). Presence of syncytial cells with the intranuclear inclusion bodies may observed in the lesions (Purcell, 1971b; Timurkaan et al., 2003)
The diagnosis is confirmed with viral isolation, detection of intranuclear inclusion bodies in the trachea, molecular diagnosis, serological tests etc.
- Virus isolation- The samples are taken from live birds for virus isolation including tracheal, oropharyngeal or conjunctival swabs. Tracheas are transported in an antibiotic broth. Exudate and epithelial cells can also be scrapped from the trachea (OIE, 2018). Laryngotracheitis virus can be isolated in embryonated chicken eggs and cell culture lines. Formation of opaque plaques seen on the chorioallantois membrane CAM in embryonated chicken eggs resulting from necrosis and proliferation of tissue (Brandly, 1937; Burnet, 1934). The virus can be propagated in a variety of avian cell culture lines such as chicken embryo liver (CEL), chicken embryo lung, chicken embryo kidney (CEK) and chicken kidney (CK) cell cultures (Chang et al., 1977; Meulemans and Halen, 1978a; McNulty et al., 1985, Hughes and Jones, 1988; Schnitzlein et al., 1995). Viral cytopathic effects reflected as increased refractiveness and swelling of cells, chromatin displacement and rounding of the nucleoli. Cytoplasmic fusion leads to formation of multinucleated giant cells. Intranuclear inclusion bodies can be detected within 12 hr post-infection. Large cytoplasmic vesicles develop in the multinucleated cells and become more basophilic (Reynolds et al., 1968).
- Histopathology- Microscopic examination remains the standard method for the rapid diagnosis of the disease. Tracheas for histopathological examination can be placed in 10% neutral buffered formalin or Bouin’s fixative (preferable for detection of intranuclear inclusion bodies) immediately after removal from the birds and then followed by embedding (OIE, 2018). Characteristic lesions in the tracheal epithelial cells include syncytial cell formation and development of pathognomonic Cowdry type A intranuclear inclusion bodies, necrosis and haemorrhage (Cover and Benton, 1958; Pirozok et al, 1957). Inclusion bodies are detected in the early infection and then disappear as infection progresses due to necrosis and epithelial cell desquamation (Garcia and Spatz, 2020).
- Immunofluorescence- Immunoprobes can be used for identification of ILTV and detection of viral antigens. Fluorescent-labeled polyclonal antibodies can be used as immunoprobes for virus detection in tracheal and conjunctival smears (Braune and Gentry, 1965; Goodwin et al., 1991; Ide, 1978; Wilks and Kogan, 1979). Immunoperoxidase-labeled monoclonal antibodies have been used in frozen tissue sections to detect viral antigens (Guy et al., 1992). detect detection of viral antigens can also be done using monoclonal antibodies in suspensions of tracheal scrapings by ELISA (York and Fahey, 1988).
- Electron microscopic examination- Direct electron microscopic examination of tracheal scrapings can be used for detection of virus (Hughes and Jones, 1988; Van Kammen and Spradbrow, 1976). Diagnosis can be done by visualizing and morphologic identification of herpesviruses and succeeds only in cases of heavy load of virus particles in clinical samples.
- Serology. LTV antibodies can be demonstrated in serum using different tests like: agar gel immunodiffusion (AGID), virus neutralization (VN), indirect fluorescent antibody (IFA) test and ELISA (Hidalgo, 2003). Heavily infected cell cultures at the time of maximum CPE can provide the antigen for ELISA. Viral Neutralization tests may be performed using the dropped CAMs of embryonated chicken eggs (incubated for 9–11 days), where antibody specifically neutralises pock formation caused by the virus. The tests can also be performed using cell cultures, where antibody specifically neutralises the infectious laryngotracheitis virus and thus prevent cytopathic effect.
- Molecular detection- The viral DNA in clinical samples can be detected using several molecular methods and PCR has proved to be more sensitive than virus isolation for clinical samples, especially when mixed infection of other contaminant viruses such as adenoviruses is present (Williams et al., 1994).
- Conventional PCR- The DNA virus can be isolated from clinical samples such as swabs samples, tissues, chorioallantoic membrane plaques, cell culture supernatants or vaccines. (Kirkpatrick et al., 2006b).
- Real-time PCR- Real-time PCR assays are also conducted for ILTV detection (Mahmoudian et al., 2011). The procedure is less time consuming and can be conducted in less than 2 hours and hence becomes a very rapid method of ILT diagnosis. (Callison et al., 2007).
- Nucleotide sequence analysis- ILTV genes can be targeted for PCR and then the resultant amplicons may be subjected to nucleotide sequence analysis for identification of viral strains. For example, ICP4 may be amplified by PCR using the primers described (Chacon and Ferreira, 2009) and the disposable mini column method used for purification of resultant amplicons and then bi-directional DNA sequencing using the PCR primers as sequencing primers are often performed.
- Restriction fragment length polymorphism (RFLP)- RFLP analysis of ILTV PCR products are often done employing a sort of restriction endonucleases (RE) and a number of other genes are targeted for digestion such as ICP4, TK, UL15, UL47 glycoprotein G and ORF-BTK genes. The combination of PCR and RFLP are often used for differentiation of field strains of ILTV from vaccine strains (Han and Kim, 2001; Creelan et al., 2006; Kirkpatrick et al., 2006a; Ojkic et al., 2006; Oldoni and Garcia, 2007).
- ILTV DNA detection- Techniques for detection of viral DNA using dot-blot hybridization assay and cloned LTV DNA fragments labelled with digoxigenin have been described by Keam et al. (1991) and Key et al. (1994). These procedures were highly sensitive for detection of virus in acutely infected birds and convalescent chickens. These assays also useful for rapid detection of virus in chickens latently infected with LTV.
Avian infectious laryngotracheitis should be differentiated from infectious bronchitis, Swollen head syndrome, mycoplasmosis etc. the pathogen must be distinguished from other respiratory pathogens of poultry having similar clinical signs and lesions which include tracheal lesions due to fowl pox virus (Tripathy and Reed, 2013) and infections by Mycoplasma gallisepticum (de Macedo Couto et al., 2015), Newcastle disease virus, avian influenza virus, infectious bronchitis virus and fowl adenovirus infections (Garcia and Spatz, 2020).
PREVENTION AND CONTROL
No treatment has been found to be effective for reducing the severity of infection or relieving clinical signs. Infectious laryngotracheitis is a communicable disease that encourages the need for effective biosecurity measures such as cleaning and disinfection of equipment and vehicles, litter management, appropriate dead bird disposal, rodent and insect control measures, and discouraging visitors from visiting other production sites (Halvorson, 2011). Densely populated areas of poultry, vaccination, movement of birds, human involvement and litter disposal management are some of the aspects to manage ILT. Special precautions should be taken about vaccination during introduction of “spiking males” into the flock (Giambrone et al., 2008). Laryngotracheitis virus infectivity is readily inactivated outside the host chicken by disinfectants and warm temperatures, thus carryover between successive flocks in a house can be prevented by adequate clean up (Hidalgo, 2003).
Quarantine and hygiene are salient features in the poultry farm premises for preventing the spread of contaminated feed, utensils, infected birds and human movement for optimum prevention and control of the disease (Kingsbury & Jungherr, 1958). For controlling the outbreak of ILT, proper diagnosis of the infection is crucial to establish a vaccination program and prevent further spread of the virus.
Vaccination of ILTV is provided to the birds at 6 to 8 weeks of age and later a booster follows 12 to 15 weeks in layers and breeders (Gowthaman et al., 2020). ILTV vaccination is not performed in broilers due to the economical aspect (Giambrone et al., 2008). Live attenuated vaccines lead to effective protection against the disease (Garcia and Spatz, 2020). Vaccines produced via embryonated eggs are known as chicken embryo origin (CEO) vaccines delivered via water lines, spray, or eye drop (Menendez et al., 2014). Tissue Culture Origin (TCO) vaccines are also instilled by eye drop. They are generated by multiple passages in tissue culture (Gelenczei and Marty, 1964); however, they offer less protection as they are more highly attenuated and less immunogenic. TCO vaccines are commonly used in breeders and layer flocks. Genetic immunization using plasmid DNA is another approach to induce protective immunity to infectious diseases. DNA vaccines can be relatively quick and easy to generate (Gowthaman et al., 2020). vHVT-ILT and vPox-ILT vaccines are applied via wing web or injection and prepared using ILT gene fragments which can be vectored into either pox virus or HVT virus.
With the TCO vaccine, vaccination of breeders is done via eye drop at 4-6 weeks of age and repeated after 10 weeks. Vaccination occurs twice in breeder flocks located in endemic regions and with a high population density of broiler and commercial layers with the CEO vaccine in drinking water at 4–5 weeks and 10-12 weeks of age. With an HVT or FPV vector vaccine (vHVT-ILT), layers are vaccinated subcutaneously on the day of hatch followed by eye drop method with CEO/TCO or CEO can be used in drinking water between 8–12 weeks of age. Alternatively, vPox-ILT vaccine is instilled via the wing web between 5–12 weeks of age. (Dufour‐Zavala, 2008).
ILT appears as a major threat to the poultry industry and the apprehension of the virus morphology, epidemiology and pathogenesis and application of strict biosecurity measures are critical to control the disease dissemination. Presence of faeces, dust, feathers and beetles in the contaminated poultry environments often carry high loads of virus. No evidence of GaHV‐1 transmission to human beings or other mammals has been reported. GaHV‐1‐induced infection leads to mortality, reduced egg production, vaccination expenditure and lowered performance of birds due to vaccination response specially in broilers may cause heavy economic losses to the poultry farmers.
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