الفهرس 
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Abstract
The present study was undertaken with a view to assess the prevalence of CAV in 11 commercial broiler flocks selected from 6 different localities in El-Behera province, Egypt. The study particularly focused on detection of CAV in clinical samples by advanced technique like PCR with assessment of the efficacy of different PCR primers for detecting CAV DNA as well as molecular and biological characterization of virus isolates after being inoculated in one-day-old SPF chicks and various uncommonly used cell cultures for CAV (VERO, CEF, MDBK and BHK-21).
For this purpose, the following experiments were done:
1. A total of 22 clinical samples (11 spleens and 11 thymi) were screened for the presence of CAV DNA by PCR using two sets of primers (VP1-F/VP1-R and VP2-F/VP2-R). Results revealed that at least one sample from each flock was turned out to be positive by PCR with at least one primer set. This established 100% relationship between the suspected flocks and detection of CAV. In most of infected cases, CAV DNA was detected in both spleen and thymus samples. VP1 gene-based PCR was able to detect CAV DNA in 86.36% of the tested samples (11 spleens and 8 thymi) while VP2 gene-based PCR was able to detect CAV DNA in 77.27% (9 spleens and 8 thymi). The positivity of spleen samples was found to be 100% and 81.81% with VP1 and VP2 PCR assays, respectively; however, positivity of thymus samples was 72.72% with both PCR assays.
2. The amplified PCR products of VP1 and VP2 gene sequences of the 11 CAV field isolates and Cux-1 strain of Avipro thymovac vaccine were digested with HaeIII, MobI, HindIII and EcoRI enzymes. Results revealed the same restriction pattern for all CAV field isolates. RE results of HaeIII enzyme revealed that CAV field isolates have a unique restriction profile combining that of the Cux-1 strain with two additional bands of about 182 bp and 440 bp in the RE digestion pattern of VP1 and VP2 regions, respectively. These findings suggesting that the field isolates contain a mixtures of CAV strains that have different RE profiles. Also analysis of the VP2 region after digestion with HindIII, revealed that the enzyme had no cutting sites in the VP2 gene product of the Cux-1 strain, whereas the VP2 region of the field isolates retained the specific cutting site for this enzyme, yielding two products of size 459 and 253 bp. EcoR1 enzyme had no restiction sites in the VP1 and VP2 regions for both field isolates and Cux-1 strain. No restriction site differences were recorded between Cux-1 strain and the field isolates by RE results of MboI enzyme digestion on the VP1 and VP2 regions.
3. Primary isolation of CAV field isolate was carried out in one-day-old SPF chicks for pathogenicity studies and virus seed stock preparation. Three chicks in the inoculated group were died between days 5 and 13 post-inoculation and the remainig chicks exhibited the typical symptoms of CAV infection. The mean PCV value of the inoculated group was 12.14% while in the control group was 25.11%. The electrophoresis of the genomic DNA from the tissues (spleen, thymus, bursa of Fabricius and bone marrow) of inoculated SPF chicks showed typical DNA laddering pattern of 180 bp and its multiplication indicating large scale apoptosis in these tissues due to CIAV infection. CAV distribution in the various organs (spleen, thymus, liver, bursa of Fabricius and bone marrow) of inoculated chicks was determined by VP1 and VP2 PCR assays. Results revealed that CAV DNA could be detected at days 5, 10 and 15 post-inoculation in all tested samples.
4. Four cell cultures (VERO, CEF, MDBK and BHK-21) were evaluated for their permissiveness to CAV replication. Three successive passages of CAV field isolate and Cux-1 strain of Avipro thymovac vaccine were conducted on each type of cells. No obvious CPE was recorded in the inoculated monolayers of CEF, MDBK and BHK-21 cells. whereas cell rounding, cell detachment and subsequent vacculation of the infected monolayers were observed. The characteristic pattern of nucleosomal laddering during agarose gel electrophoresis was not detected in the extracted the DNA from any of the inoculated cell cultures. CAV DNA was detected by both VP1 and VP2 PCR assays in the cell culture fluids obtained from every viral passage conducted in VERO, CEF, MDBK and BHK-21 cells. Results of semi quantitative VP1 PCR assay for evaluation of viral growth revealed that the infected cell culture fluids obtained by the first viral passage on MDBK, BHK-21 and CEF cells having a relatively high intensity value of CAV in comparison to that obtained from the second viral passage. In case of VERO cells, the relative intensity value of CAV was found to be increased by the second viral passage.
5. VP1 PCR assay was found to be more efficient when compared with VP2 PCR assay in detection of CAV DNA from tissues of naturally infected birds but both PCR assays were equal in their efficacy for detection of CAV DNA from organs of experimentally infected chicks and infected cell culture fluids. The overall efficacy of VP1 PCR assay was about (93.87%), however, VP2 PCR assay was less efficient (89.79%). The K value of kappa statistics for both PCR assays was found to be (0.97) and this indicates that there is perfect agreement between them in detection of CAV DNA in the tested samples.
Conclusions:
The analysis of the findings from the present study implies the following conclusions:
1. Exposure of broiler chickens to CAV was more common than expected (100%) in El-Behera province, Egypt.
2. A higher percentage of spleen field samples were found positive for CAV DNA when being compared with thymus samples.
3. PCR worked well, can be adopted easily for rapid, economical and efficient detection of CAV from affected poultry tissues as well as infected cell culture fluids. PCR can be automated or run in batches and is capable of generating a simple ”yes/no” answer. PCR not only works for giving diagnosis, but also provides the amplified product for its subsequent molecular characterization by other technique like RFLP.
4. VP1 gene-based PCR assay was found to be more efficient than VP2 gene-based PCR assay in detection of CAV DNA.
5. RFLP profiles using REs HaeIII, MboI, HindIII and EcoR1 for VP1 and VP2 gene segments did not reveal any genetic variations among the field CAV isolates.
6. RE analysis of VP1 and VP2 gene segments using HaeIII indicated that the field isolate contain a mixtures of CAV strains that have different RE profiles.
7. Out of the four RE enzymes used, HaeIII and HindIII were found useful to differentiate CAV field isolate from Cux-1 strain of Avipro thymovac vaccine.
8. The pathogenicity study indicated that CAV field isolate was able to produce clinical disease and consistent low PCV values in one-day-old SPF chicks.
9. The electrophoresis of genomic DNA showed large scale apoptosis in tissues of experimentally infected SPF chicks, indicating apoptosis as one of the important phenomenon during CAV infection.
10. Semi-quantitative VP1 PCR assay was found to be very useful in determining the relative intensity value of CAV in the infected cell culture fluids.
11. Impaired pattern of viral growth was obtained by attempted isolation of CAV in MDBK, BHK-21 and CEF cell cultures.