Isolation And Identification Of Infectious Laryngotracheitis Virus From Chizhou City And Sequence Analysis Of The GD Gene

Chicken Infectious Laryngotracheitis(Infectious Laryngotracheitis,ILT)a kind of chicken viral respiratory disease caused by chicken Infectious Laryngotracheitis Virus(Infectious Laryngotracheitis Virus,ILTV),causing egg production decrease or chicken death serious which may leads to serious economic loss.Laying ihens of 120 days on a farm of Anhui province in 2015 were suspected having had ILT.In order to make a definite diagnosis of this infection situation,this study collected diseased chickens throat tracheal tissue and its secretion.After cutting that up,mix it with 3 times the volume of sterile 0.2 M PBS and fully grind.After freezing and thawing three times,centrifuge it,get supernatant and add green-streptomycin(500U/mL).Then get it incubate at 4℃ overnight and keep it in-80℃ refrigerator as a standby.After extract the sick chicken throat tracheal,total DNA of its secretion,get amplification of 1134 bp size fragments from infectious chicken’s throats and secretion according to the published GenBank chicken ILT virus gD gene synthesizing and designing a pair of specific primers.Prepare tissue fluid supernatant and vaccinate SPF chicken embryos of 10 days by CAM.120 hours later,a typical acne marks were found in chicken embryos CAM after opening the chicken embryos.So collect lesions chicken embryo acne marks and allantoic fluid,fully grind them,centrifuge it,get supernatant after freezing and thawing repeatedly.Then vaccinate the chicken embryos in the same way three times continuously.After that,take the third generation of chicken embryos separation poison as poison seed to be saved at-80℃.Extract virus DNA from the third generation of chicken embryos separated poison and extend gD gene.After recycling,purifying plastic and cloning it to pMD18-T carrier,transform such as DH5a cells and sort out monoclonal to be sequenced after being identified right.Then it turned out be of sequence homology as GenBank gD gene by above 99%.Genetic evolution tree analysis results showed that separated poison is in the independent branch,and has close affinity with Serva plants.Then vaccinate health MaYu broilers of 5 days with 0.2 mL poison seed for each one by dripping nose or eyes as experimental group,and compare it with the control group being vaccinate the same dose of sterile negative PBS.After continuous observation,finding that three days later poison attacked group have symptoms of drop in food intake,difficulty breathing,eye conjunctiva necrosis.4 to 5 days later,the symptom aggravated and two chicks died and after 9 days we began the prognosis.At the same time the control group had no symptoms;After the autopsy of the dead chicken,we found their throat trachea hemorrhage,caecum tonsil swollen,and virus can be detected from the ill chicken throat tracheal and secretion.The results confirm that a chicken infectious laryngotracheitis virus strain was isolated,named CZ-ILTV16 strains.