FTA liver impressions as DNA template for detecting and genotyping fowl adenovirus.
The feasibility of using liver impressions on Flinders Technology Associates (FTA filter paper for the collection, inactivation, and molecular analysis of fowl adenovirus (FAV) was evaluated. FAV I European Union (EU) serotype 1 spotted on FTA was shown to be inactivated using specific-pathogen-free (SPF) primary chicken embryo liver cell culture as indicated by absence of cytopathic effect. Sensitivity of the polymerase chain reaction (PCR) test using tenfold dilutions of allantoic fluid from 100 to 10-4 for the detection of adenovirus serotype 1 on FTA cards was determined to be 0.0005 mean tissue culture infectious dose per FTA spot. The stability of the DNA from liver impressions on the FTA was found to be 198 days when stored at -20 degrees C. In a trial, inclusion body hepatitis (IBH) was experimentally reproduced in SPF chickens inoculated with FAV I EU serogroup 1, 4, 8, or 11, which presented weakness, pallor, depression, dehydration, and mortality within 6 days after inoculation. PCR performed on FTA liver impressions from the inoculated birds was able to detect all four viruses, and the nucleotide sequence analysis of the amplified PCR products (1219 bp of the hexone gene) revealed the expected serotypes. In addition to the trial, 55 clinical samples were analyzed from liver impressions on FTA cards, and FAV was detected in 11 of 55 (20%). Sequencing analysis showed that the viruses were EU serotypes 4, 5, 9, and 10. The results demonstrate that FTA filter paper inactivates the FAV I and maintains the DNA template for molecular analysis.
Moscoso
Hugo
H
Poultry Diagnostic and Research Center, Department of Population Health, The University of Georgia, Athens, GA 30602-4875, USA.
Bruzual
Jose J
JJ
Sellers
Holly
H
Hofacre
Charles L
CL
D000257:Adenoviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D000818:Animals; D000259:Aviadenovirus / Q000235:genetics* / Q000302:isolation & purification; D002645:Chickens / Q000821:virology*; D003588:Cytopathogenic Effect, Viral; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D005838:Genotype; D006524:Hepatitis, Viral, Animal / Q000821:virology; D007181:Inclusion Bodies, Viral; D008099:Liver / Q000166:cytology / Q000821:virology*; D008851:Micropore Filters; D010209:Paper; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary; D011201:Poultry Diseases / Q000175:diagnosis / Q000821:virology*; D012680:Sensitivity and Specificity; D013047:Specific Pathogen-Free Organisms; D013698:Templates, Genetic
D004279:DNA, Viral