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Isolation, Identification and Molecular Characterization of Infectious Laryngotracheitis Virus in Chicken

KrishiKosh

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Title Isolation, Identification and Molecular Characterization of Infectious Laryngotracheitis Virus in Chicken
 
Creator Puvarajan, B.
 
Contributor Sukumar, K.
 
Description A study was undertaken to isolate, identify and characterize the causative agent of
Infectious laryngotracheitis (ILT) in layers from field outbreaks in Namakkal area, the leading
poultry layer belt and second largest egg bowl of the nation.
The prevalence of ILT was observed in the age groups of 7 to 42 weeks in layer
chicken. The maximum prevalence was noticed in the age group of 10 to 20 weeks (40.47
%) followed by 20 to 30 weeks (26.19 %). The maximum mortality was observed in 10 to 20
weeks age group (41 %) followed by 20 to 30 weeks (35 %) and the average mortality per
cent recorded in layer was 28.60.
The season-wise pattern of occurrence of ILT was studied as the incidence peaked
during mid-summer of 2011 where the mortality rate increased from 5 to 25 per cent in birds
raised under intensive system and decreased in winter.
The field isolates were confirmed by Agar Gel Precipitation Test and histopathology.
Histopathological examination of affected trachea revealed presence of intranuclear
inclusions.
Twenty tracheal samples collected from suspected outbreaks of ILT from 34 layer
farms were propagated in embryonated chicken eggs via chorioallantoic membrane (CAM)
route showed typical pock lesions and thickening of CAM.
Molecular characterization of the field ILTV isolates were carried out by
amplification of ICP4 gene and Tk gene by polymerase chain reaction and produced
amplicons size of 635 bp and 649 bp respectively.
Restriction endonuclease analysis of ILTV field isolates were carried out for the Tk
gene amplicons size of 649 bp with endonuclease enzymes viz. HaeIII, Sau 96I and NciI and
two bands of sizes of 420 and 226 bp for HaeIII, three bands of sizes of 410, 176 and 60 bp
with Sau96I and there was no cleavage with NciI were observed. The same RFLP pattern
could be observed for the vaccine strain also.
The PCR products of Tk gene and ICP4 gene of the twenty field ILTV isolates were
sequenced and compared with reference and vaccine strains. The sequence analysis
showed that the homology of nucleotide sequences between the twenty field isolates was
99.2 – 100 per cent for ICP4 gene and 96.5 – 100 per cent for Tk gene and between the
isolates, reference and vaccine strain were 99.2 - 100 per cent for ICP4 gene and 96.5 -100
per cent for the Tk gene.
Multiple sequence alignment analysis of the nucleotide sequences with that of
vaccine strain of ILTV showed that nucleotide mutation at two different positions in ICP4
gene and six different positions in Tk gene of all the isolates.
Phylogenetic analysis on the ICP4 gene and Tk gene sequence of the twenty
isolates, vaccine strain and three other reference strains revealed that they were
indistinguable.
The protein profile of field ILTV isolates and vaccine strain were studied by Sodium
Dodecyl sulphate – Polyacrylamide Gel Electrophoresis and which yielded four polypeptides
of molecular weights viz., 60, 85,160 and 205 kDa.
A dot-ELISA technique was developed for detection of ILTV from field samples.
 
Date 2016-07-26T16:19:28Z
2016-07-26T16:19:28Z
2013
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/69970
 
Language en
 
Format application/pdf
 
Publisher Tamil Nadu Veterinary and Animal Sciences University