Pathological Diagnosis and Genomic Characterization of ICP4 Gene of lnfectious Laryngotracheitis Virus (ILTV) Isolates in Clinically Infected Chicken in Tamil Nadu, India

Background: Infectious laryngotracheitis (ILT) is an economically important viral respiratory disease in poultry. Recently, re-emergence of Infectious laryngotracheitis virus (ILTV) has been reported in several countries including India. The current study aimed to evaluate the poultry flocks of Tamil Nadu with circulating GaHV-1 and to elucidate the origin of the virus involved in the outbreak. Methods: In this study, a molecular based survey on the overall occurrence of natural cases of Infectious laryngo-tracheitis in poultry flocks from Tamil Nadu, India were performed. Pathological findings in respiratory and secondary lymphoid organs like caecal tonsils and harderian gland was carried out. The PCR technique targeting Infected Cell Protein-4 (ICP4) gene along with molecular characterization was performed. Result: The overall prevalence rate in the outbreak was 42.86% with highest incidence in layer flocks (62.85%) than the broiler flocks (22.85%). The highest susceptible age groups were between 20-30 weeks old. Tracheal pathology revealed epithelial sloughing, syncytial cell formation, eosinophilic intranuclear inclusion bodies and heterophilic exudation microscopically. Partial genome sequencing and phylogenetic analysis of ICP4 gene revealed high genetic homology between field isolates and the virulent strains from Turkey, Germany, China and Brazil. In the present study, along with pathological findings, a rapid and sensitive PCR assay was used for detection of ILT virus specific ICP4 gene in commercial poultry farms in the region.

PATHOLOGICAL STUDY ON LARYNGOTRACHEITIS IN LAYERS

A clinical conditions resembling infectious laryngotracheitis were diagnosed amongst 20,000 , 18,000 , 16,000 and 17,500 respectively, 28-30 weeks old, ISA brown layers. The hens had nasal discharges, moist rales, coughing and gasping. Hemorrhagic mucous was ejected during sneezing, lacrirnation, conjunctivitis with facial swelling with eyes partially or completely" closed. Postmortum examination of dead and affected hens revealed hemorrhagic tracheitis with thin pseudomembrane formation. The larynx, congested with petechia on mucous membrane, Infraorbital sinus contained clear thick fluid. Histopathological examination of trachea showed hypertrophy, of epithelial pseudostratification of the mucosal cell surface, extensive hemorrhages and desquamative necrotizing tracheitis with mononuclear cells infiltration. Multinucleated gaint cells in theciliated epithelium containing round, oval shaped intranuclear A inclusion bodies. The lamina propiia shows edema, marked‘ congestion with lymphocytic infiltration.A presumptive diagnosis of laryngotracheitis was made.

Evaluation of Recombinant Herpesvirus of Turkey Laryngotracheitis (rHVT-LT) Vaccine against Genotype VI Canadian Wild-Type Infectious Laryngotracheitis Virus (ILTV) Infection

In Alberta, infectious laryngotracheitis virus (ILTV) infection is endemic in backyard poultry flocks; however, outbreaks are only sporadically observed in commercial flocks. In addition to ILTV vaccine revertant strains, wild-type strains are among the most common causes of infectious laryngotracheitis (ILT). Given the surge in live attenuated vaccine-related outbreaks, the goal of this study was to assess the efficacy of a recombinant herpesvirus of turkey (rHVT-LT) vaccine against a genotype VI Canadian wild-type ILTV infection. One-day-old specific pathogen-free (SPF) White Leghorn chickens were vaccinated with the rHVT-LT vaccine or mock vaccinated. At three weeks of age, half of the vaccinated and the mock-vaccinated animals were challenged. Throughout the experiment, weights were recorded, and feather tips, cloacal and oropharyngeal swabs were collected for ILTV genome quantification. Blood was collected to isolate peripheral blood mononuclear cells (PBMC) and quantify CD4+ and CD8+ T cells. At 14 dpi, the chickens were euthanized, and respiratory tissues were collected to quantify genome loads and histological examination. Results showed that the vaccine failed to decrease the clinical signs at 6 days post-infection. However, it was able to significantly reduce ILTV shedding through the oropharyngeal route. Overall, rHVT-LT produced a partial protection against genotype VI ILTV infection.

Molecular Detection of Infectious Laryngotracheitis Virus in Chickens with a Microfluidic Chip

Infectious laryngotracheitis (ILT) is a viral disease of chickens’ respiratory system that imposes considerable financial burdens on the chicken industry. Rapid, simple, and specific detection of this virus is crucial to enable proper control measures. Polymerase chain reaction (PCR)-based molecular tests require relatively expensive instruments and skilled personnel, confining their application to centralized laboratories. To enable chicken farms to take timely action and contain the spread of infection, we describe a rapid, simple, semi-quantitative benchtop isothermal amplification (LAMP) assay, and a field-deployable microfluidic device for the diagnosis of ILTV infection in chickens. Our assay performance was compared and favorably agreed with quantitative PCR (qPCR). The sensitivity of our real-time LAMP test is 250 genomic copies/reaction. Clinical performance of our microfluidic device using samples from diseased chickens showed 100% specificity and 100% sensitivity in comparison with benchtop LAMP assay and the gold-standard qPCR. Our method facilitates simple, specific, and rapid molecular ILTV detection in low-resource veterinary diagnostic laboratories and can be used for field molecular diagnosis of suspected ILT cases.

Prevalence and molecular detection of infectious laryngotracheitis virus in chickens in selected areas of Bangladesh

Infectious laryngotracheitis (ILT) is a viral disease of poultry species caused by infectious laryngotracheitis virus (ILTV) that shows high morbidity and mortality. The present study was under taken for ILTV prevalence in broiler and layer chickens from four different geographical areas including Bogura, Gazipur, Chattogram and Dhaka districts during 2017 to 2018. Total 350 tracheal swabs were collected and were evaluated by real time RT-PCR (rRT-PCR). The overall 5.14% (18/350) ILTV prevalence was found that included 6.5% (13/200) in layer and 3.33% (5/150) in broiler chickens. The prevalence of ILTV was highest (10%) in layer chickens under age below 20 weeks and broiler chicks showed ILTV (1. 42%) infection when they were 7-14 days old. Winter season showed highest 6.6% prevalence whereas 5% and 3% prevalence were noticed at summer and rainy seasons, respectively. Bang. J. Livs. Res. Vol. 27 (1&2), 2020: P. 113-117

Infectious laryngotracheitis of chickens: Pathologic and immunohistochemistry findings

Infectious laryngotracheitis (ILT) is an important upper respiratory disease of chickens. Gross and histologic lesions of ILT in chickens are compared to immunohistochemistry to evaluate the diagnostic test sensitivity. A total of 31 separate ILT-confirmed necropsy submissions (12 commercial meat-type flocks, 13 egg-type producers, and 6 backyard flocks) were arbitrarily selected. Each submission ranged from 1 to 18 birds, for a total of 246 chickens. Cases with available formalin-fixed tissues were selected to include a range of bird production types, ages, clinical histories, and severity of macroscopic and histologic lesions. Macroscopic findings in the respiratory tract varied from increased mucus (55.6%) to fibrinonecrotic exudate (20.3%) and hemorrhages in the larynx and trachea (13.0%). Syncytia with intranuclear inclusion bodies were present in the respiratory tract epithelium with or without hemorrhages. Sections of conjunctiva, sinus, larynx, trachea, lung, and air sac were analyzed by immunohistochemistry (IHC) to detect gallid alphaherpesvirus 1 (GaHV-1) antigen. Positive immunolabeling was detected in the cytoplasm and nuclei of syncytia and epithelial cells in 18/22 conjunctivae (82%), 12/13 sinuses (92%), 18/22 larynxes (82%), 23/25 tracheas (92%), 10/21 lungs (57%), and 3/8 air sacs (37%). Of the 34 tissues with no visible syncytia or inclusion bodies, 8 were positive by IHC. In conclusion, IHC was useful to study the viral antigen tissue distribution and support the diagnosis of ILT when the histopathologic interpretation was doubtful.