Chicken Anemia Virus in Europe: From qPCR Validation to Molecular Epidemiology

The intensification of the global poultry industry has led to increased economic output while also increasing the chances of outbreak of infectious diseases. Chicken Anemia Virus (CAV) stands out among them in terms of threat in virtue of the immunosuppression that it causes which endangers the health of the birds, lowers productivity and predisposes them to secondary infection. This thesis describes development and verification of a real time PCR assay of very high sensitivity for the detection and measurement of CAV, paying particular attention to clinical and subclinical infection cases. The design of the assay was carried out by meticulous selection of primers and probes using Primer3 and then silico validation against GenBank sequences to guarantee the sensitivity and specificity of the diagnosis. The positive control was a synthetic plasmid called the pCAV which contained the targeted genomic region. Limit of detection was determined as 1 copy per microliter based on a tenfold dilution series from 10⁸ copies per microliter to 10² copies per microliter. Extensive optimization of reaction conditions as well as primer concentrations and annealing temperature provided robust performance of the assay. Its repeatability was proven by GLM statistical analysis through days and replicates confirming the result from the CT values independent of day-to-day variation apart from some fluctuation at lower viral concentrations. Field validation included assessing 80 archival bursa of Fabricius samples gathered from eight European countries during the years 2019-2024. Among these, a sample from a broiler farm in the United Kingdom was positive, with a Ct of 41.7, suggestive of a low viral titer. Follow-up full-genome amplification and Sanger sequencing with overlapping primers directed at the variable region of the phylogenetic VP1 gene provided comprehensive analysis of the VP1 gene. This analysis integrated the previously classified strain into Geno group IIIa, which confirms the great variation and global spread of CAV along with the reference sequences from Poland, Taiwan, China, and Egypt. The findings underline the test’s ability to detect low levels of viral presence and suggest its use for routine diagnostics and epidemiological surveillance. These findings also strengthen the argument for regular assessment of CAV genetic variability to construct new vaccines and directed biosecurity measures. In any case, this work represents a border between modern molecular diagnostic and phylogeny, which lean towards improved disease control in poultry and strived productivity sustainability.