| Fowl adenovirus(FAd V), a non-enveloped, linear double stranded DNA virus, belongs to the family Adenoviridae. The virus can infect chickens, turkeys and other birds. It caused inclusion body hepatitis, hydropericardium syndrome, gizzard erosions and other diseases in infected chicken flocks. FAd V contains 8 species, Respectively as FAd V A~E, goose adenovirus A, falcon adenovirus A, turkey adenovirus B. Generally, 3~5 week-old chickens are more susceptible to FAd V infection, including laying hens and broilers. FAd V usually infects chickens together with other infectious agent of avian diseases. Infected chickens suffered from aplastic anemia, hepatitis, egg production decreasing, etc. FAd V distributed worldwide in chicken flocks, and was transmitted through the fecal-oral route horizontally and also transmitted vertically, causing huge economic losses for poultry industry. In this study, we established a rapid and sensitive real-time PCR method for the detection of fowl adenovirus. At the same time, it provides a technical platform for the disease erasion, which is of great significance to healthy development of poultry industry.By multiple comparison of the Hexon gene of FAd V-A, B, C, D and E reference strains published by Gen Bank, we selected the conserve region to design primers for amplifing the Hexon-1 gene fragment from FAd V-D genome by conventional PCR method. The fragment was cloned into p MD18-T vector to construct standard product. The standard product was used as a template for the amplification of real-time PCR and established the standard curve. The standard samples, of which the plasmid concentration detection range is from 1×1010copies/μL to 1×104copies/μL, result in error rate of 0.0599, amplification efficiency of 1.904 and slope of-3.575, which is a good linear relationship. The concentration of the standard products was 1×1010copies/μL~1×104copies/μL was calculated by real-time PCR, The sensitivity of the standard product is 1×103copies/μL. The mean Cp values were 0.22%、0.24%、0.11%、0.27%、0.18%、0.35% and 0.37%, coefficients of variation CV% respectively were 0.50% 、0.86%、0.73%、1.32%、1.06%、0.56% and 0.78%. Therefore, the real-time PCR detection method constructed in this study for FAd V has a good repeatability and stability. The minimum detectable concentration of FAd V-D and FAd V-C species was evaluated by using the real-time PCR method to determine the sensitivity of the detection method. The lower detection limitation of FAd V-C and FAd V-D was 102 TCID50/ml, which is higher than the conventional PCR method nearly 10 times. It was showed that the real-time PCR detection method for FAd V established in this study has a good sensitivity.SPF chicken embryos were used for inoculating with the same dose and titer(100EID50) of FAd V-D strain. The incoculated route was performed via yolk sac(6.5 days), chorioallantoic membrane(9 days) and chorioallantoic antrum inoculation(9 days) method. Acquisition of chick embryo tissues including the brain, heart, intestine, lung, thymus, liver, glandular stomach, kidney, spleen, bursa of Fabricius, chick embryo allantoic fluid of chick and embryo chorioallantoic membrane(CAM). It was showed that different inoculation route affected the propagation of FAd V-D in the SPF chicken embryos. FAdV-D had better propagation capability via the yolk sac inoculation route, the viral load was the highest in the liver tissue of chicken embryo egg. The viral DNA copies in allantoic fluid and chicken chorioallantoic membrane of SPF chic ken embryos inoculated via yolk sac were higher than that of inoculated via chorioallantoic membrane and chorioallantoic antrum inoculation routes. In the yolk sac and chorioallantoic membrane(CAM), the allantoic cavity inoculation of SPF chicken, chick embryo chorioallantoic membrane(CAM) in DNA copy numbers were 1.32×108copies/mg, 2.89×105copies/mg and 9.07×104copies/mg. After embryo allantoic fluid in DNA copy numbers were 4.99×104copies/mg, 3.28×104copies/mg and 1.32×104 copies/mg. Therefore, yolk sac inoculation route in SPF chicken embryo was the optimal route for FAd V isolation, chicken allantoic fluid and chorioallantoic membrane were could be collected as virus stock. In addition, a real-time PCR detection method reported by Günes was performed to compare with the results of the real-time PCR method established in this study, the viral distribution in chicken embryo tissues and the viral load in the tissues were consistent.FAdV-D was used to infect 1-day-old SPF chicks through subcutaneous injection. The oropharyngeal and cloacal swabs were collected every 4 days post infection and was detected by using the real-time PCR detection method for FAd V established in this study to evaluate the virus shedding. The virus could still be detected thorugh oropharyngeal and cloacal swabs 72 days post infection. The positive rate of oropharyngeal swab was 7/10(70%) and 8/10(80%) through cloacal swabs at 72 days post infection. While, there was no virus shedding detected by conventional PCR through oropharyngeal and cloacal swabs at 72 days post infection. Three birds died in the group infected by FAd V-D at the 8th day post infection, tissues were collected for viral load detection. Real-time PCR was performedto detecte the virus distribution in infected chi ckens, it was showed that the virus has an extensive tropism in the majority of tissues in infected chickens. Rectum and liver were the main troposim tissue of FAd V, the viral load in the rectum was the highest and the spleen contained the lowest viral load of FAd V.In this study, we used the real-time PCR method to detect the clinical samples. 143 samples were collected from 10 provinces in China for detecting the FAd V infection by using the real-time PCR method. In addition the conventional PCR was performed to detect the samples for comparing with the result of real-time PCR. 91 out of 143 samples were detected to be FAd V positive by real-time PCR detection and the positive rate was 63.6%, the positive rate was 40.5%(58/143) detected by conventional PCR method. It was indicated that the real-time PCR established in this study had better sensitivity for FAd V detection. |
PDF Full Text Request