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Detection and analysis of Infectious Laryngotracheitis (ILT) Virus field isolates in domestic poultry from Laser Capture Microdissection (LCM) tissues

Melanie N. WarthanA,John El-AttracheA, Maricarmen GarciaB, Jordan BrodC. ATexas A&M University, College of Veterinary Medicine, Department of Pathobiology. BUniversity of Georgia, College of Veterinary Medicine, Department of Avian Medicine.  CTexas Veterinary Medical Diagnostic Laboratory


Background - Infectious Laryngotracheitis Virus (ILT) is an important economical disease in the U.S. and State of Texas commercial poultry industry.  Diagnosis of infected flocks in the State of Texas is reportable to the Texas Animal Health Commission and often results in a quarantine mandate.  Clinical signs associated with severe forms of ILT include gasping, coughing, nasal discharge, conjunctivitis and hemorrhagic blockages in the trachea.  Milder forms of ILT have been reported and clinical signs associated with these cases include a mild tracheitis, conjunctivitis and swelling of the infraorbital sinuses along with closed eyes and persistent nasal discharge.  Virus isolation and PCR are routinely utilized for the detection of ILT with histopathologic examination remaining the standard method for the rapid confirmation and diagnosis of ILT. However, syncytial cell formation along with intranuclear inclusion bodies may not be detected by histopathologic examination in less severe forms of the disease.

Objective - Therefore, the objective of this project was to examine a new technique for the early detection of ILT and to obtain more sensitive results than had been achieved using previously described methods.

Procedures - Formalin-fixed-paraffin-embedded (FFPE) tracheas, eyelids, and trigeminal ganglia were obtained from the University of Georgia Department of Avian Medicine.  The specific pathogen free birds had been inoculated with the USDA ILTV challenge strain, and the tissue collected at both 7 and 15 days post inoculation.

Experimental Design - We evaluated and analyzed Laser Capture Microdissection (LCM) for the detection of the less severe form of ILT field isolates.  Histopathologic analysis was performed and samples were either labeled as ILT-positive, negative, or indeterminate tracheitis, based on the presence or absence of characteristic microscopic lesions of ILT on hematoxylin and eosin stained slides from FFPE tissue (tracheas, eyelids, trigeminal ganglia).  LCM was then performed on these slides and 100 - 500 cells were obtained from each slide for further molecular analysis. DNA extraction was done on each set of cells, followed by nested PCR - RFLP.

Results - Preliminary results show that ILT can be detected in FFPE samples via the LCM method, however the sensitivity of the procedure has yet to be determined.

Conclusions - In conclusion, further studies need to be done in order to fine tune the LCM procedure specifically for ILT detection and to arrive at greater sensitivity for detection.

[Supported by: Texas Veterinary Medical Association]