| Fowl adenovirus serotype 4 (FAdV-4) mainly causes the sudden death of 3-to-5-week-old broiler chicks,characterized by symptoms of Hydropericardium and hepatitis(HHS).August 1987 FAdV-4 was first reported in Pakistan Angara Region,and in China in July 2015 for the first time reported in the scientific literature. FAdV-4 Guangxi strian (FAdV-4 GX2013) was isolated in 2013 from a broiler farm in Guangxi.China, which has strong pathogenicity in clinical signs. To further investigate the biological characteristics of FAdV-4 GX2013 strain, cytopathic effect, pathogenic of SPF chicken embryos and chicks were studied, and the whole genome was determined and analyzed with other sources of avian adenovirus strain’s genome for comparison. In addition, the strain’s structural protein fiber protein II (Fiberll) was expressed through prokaryotic expression system, then the establishment of a specific method for detecting antibodies against FAdV-C was done, laid the foundation for FAdV-4 infection diagnosis and monitoring.1 Identification of biological characteristics of FAdV-4 GX2013 strainAfter being inoculated at 9-day-old through chorioallantoic membrane by using FAdV-4 GX2013 strain, the SPF chicken embryos began to die after 5 days. Then, the dead and survived embryos with being artificially infected with FAdV-4 GX2013 strain were autopsied, thickened chorioallantoic membrane and dysontogenesis could be observed. The characteristic like pectoralis and crureus distributed with extensive needle-like hemorrhagic spots also exist. Most embryos were with pericardial effusion, and there were different degrees of myocardial dotted hemorrhage. The other embryos without hydropericardium could have hepatosplenomegaly, hemorrhage, marginal necrosis. Spleen was also hyperemia. Kidney was edema and hemorrhage,with renal tubular degeneration and protrusion. Proventriculus was edema and hemorrhage. Ulcerations occurred in the gizzards near the junction between the proventriculus and gizzard.To observe FAdV-4 GX2013 strain’s pathogenicity to chickens, the viruses were inoculated to day-old SPF chickens through intranasal inoculation and subcutaneous injection at neck respectively, within 7 days, the mortality in injection group and intranasal inoculation was 100% and 80% respectively.The changes of autopsy were mainly hydropericardium, hepatosplenomegaly with multiple necrosis, ulceration syndrome of the koilin layer of the gizzard,Swelling in the kidney.To investigate FAdV-4 GX2013 strain’s multiplication on cells, the viruses were infected with DF-1, Vero,293T, CEF, CEL, and LMH cells. The results showed that the virus could not reproduce on theCEF, DF-1,293T and Vero cells, but could multiply and cause cytopathic effects on the CEL,and LMH cells.Primers were synthetised to amplify the genome of strain GX2013, then sequenced and analyzed. The results showed that FAdV-4 GX2013 strain’s genome size was 43752bp, the characteristic of the genome was almost consistent with the reported FAdV-4 strain HB1510 in China recently.2 Establishment of indirect ELISA method for detecting FAdV-CIn this study, the gene encoding FiberⅡ was amplified by PCR and cloned into pET-32a (+) expression vector, then the recombinant plasmid was transformed into E. coli BL21 (DE3), and induced by IPTG to obtain the recombinant fiberⅡ protein, which is about 40KD and expressed in supernatant form. Then we developed an indirect ELISA method based on the recombinant of fiberⅡ to detect antibodies against FAdV-C. The indirect ELISA method has good specificity, repeatability.Using this method to detect 16 clinical samples, the positive rate was 18.75%.The establishment of this method provided a simple and quick serological diagnosis method for the detection and epidemiological investigation of FAdV-C. |
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