Isolation And Identification Of Fowl Adenovirus Type 4 And Development Of Loop Mediated Isothermal Amplification Technique For Diagnosis

Avian adenovirus serotype 4 can cause severe pericardial effusion hepatitis syndrome,which also known as "Ankara" disease.Since 2013 this disease that causes huge losses in the poultry industry has been reported in China's Henan,Jiangsu,Hebei,Shaanxi and other places.In this study,a virus was isolated from the pathogenesis of suspected pericardial effusion syndrome.Based on the fowl adenovirus Hexon gene sequence published on GenBank,we designed specific PCR primers and obtained PCR positive results.Nucleic acid sequence alignment and genetic phylogenetic analysis showed that the isolated strain was fowl adenovirus serotype 4(FAdV-4).According to GenBank published FAdV-4 Hexon gene sequence,two pairs of LAMP primers were designed,and LAMP detection method for fowl adenovirus serotype 4 was established according to LAMP operation guideline,and compared with ordinary PCR method.The obtained results are as follows:1.The liver tissues collected form suspected FAdV-4 infected chickens were homogenize and were inoculated with 9-day-old non-immunized chicken embryos.The infected chick embryo began to die at 3 to 4 days after inoculation.The dead chick embryo exhibit curled,dysplasia,significantly smaller than the matched age chick embryo.Autopsy changes showed enlargement and bleeding of liver,gray necrosis or yellow soil on the liver surface.The 20-day-old non-immune healthy white feather fowl broilers were inoculated with allantoic fluid of infected chicken embryos by intramuscular injection route.The infected birds showed symptom and death at 24 and 72 hours after infection,respectively.Autopsy of died birds showed pericardium filled with a large number of pale yellow liquid,liver enlargement,the surface of bleeding or koji necrotic plaque.Similar autopsy changes were observed at natural infected chiks.The control group did not show any clinical pathological changes and abnormal autopsy changes.2.A pair of PCR primers were designed according to the Hexon gene sequence of the fowl adenovirus and PCR assay was performed to detecte allantoic fluid of infected chicken embryos.The FAdV positive PCR product was ligated and the partial Hexon gene nucleic acid sequence was cloned.The results of sequence alignment showed that the homology of the isolate was 100% with that of the fowl adenovirus serotype 4(KU569296.1),31.5% withthat of the turkey hemorrhagic enteritis virus(AY849321.1)The homology of the virus(Y09598.1)was 30.8%.The phylogenetic tree analysis showed that the isolated strain was in the same branch as FAdV-4.3.The LAMP detection method for FAdV-4 was established based on two pairs of LAMP detection primers.The amplification products were added to 1000 × SYBR GreenI 1uL,and the FAdV-4 positive showed yellow-green fluorescence,the FAdV-4 negative showed no changes for color.The optimum reaction temperature was 61 ? and the reaction time was 30 min.Compared with the common PCR LAMP has a higher sensitivity,the detection limit was 6.4 fg/uL.Twenty-one copies of FAdV-4 were detected by routine PCR,and 6copies of FAdV-4 were detected by LAMP method.The detection rate by LAMP method was higher than that of conventional PCR method.