Molecular Epidemiological Investigation And Establishment Of Diagnostic Technique On Duck Circovirus

Duck circovirus (DuCV) is listed as a tentative member of the Genus circovirus by the most recent 8th ICTV Report. Ducks infected by DuCV exhibit disordered feather, retarded growth, reduced body weight. Pathological examination of the sick ducks revealed necrosis in lymphoreticular tissue, reduction in the lymphocyte count or proliferation of lymphoid tissue. Circovirus infection is characterized by lesion in lymphoid tissue that leads to the reduction or suppression in immunity (Soike et al., 2004) and secondary infection. Coinfection by DuCV and other viral or bacterial pathogens is very frequently examined. In the present study, we conducted and discussed epidemiological investigation, molecular gentic variation, Preliminary Pathologic Observations, serological methods and culturing in vitro.1,An epidemiological investigation on duck circovirus in Shandong, ChinaCoinfection of duck circovirus (DuCV), Riemerella anatipestifer (RA), Escherichia coli (E. coli) and duck hepatitis virus I (DHV-I) in Cherry Valley ducks in Shandong, China was investigated by PCR-based methods. 742 ducks sampled during 2006-2007 from 70 duck farms were examined by PCR and dot blot analysis. Overall DuCV-infection rate was 33.29%. Ducks at 3-4 weeks of age were more susceptible to DuCV infection, comparing with those at 2 weeks of age. The DuCV-positive ducks had a higher rate of infection by duck hepatitis virus I (25.5% vs 7.475), RA (23.48% vs 8.28%) and E. coli, comparing with DuCV-negative ones. This investigation showed that DuCV infection is common in Cherry Valley ducks.2,Sequence Analysis for the Complete Genome of Duck Circovirus IsolatesThe complete genome of two strain Duck circovirus (DuCV), LY0701 and WF0701, were gained from cherry valley ducks in mainland of China for the first time. The numbers of Genbank accession database are EU022374 and EU022375, respectively. Sequences analysis shows that the two stains DuCV exhibited the four genetic features characteristic of other circovirus: a stem-loop structure; four intergenic repeats; the presence of the ORFs rep and cap; the conserved motifs for the rolling circle replication and for the dNTP binding domain observed in the Rep protein. Comparisons of the two strains with that of representative DuCV strains demonstrated that the two DuCV strains were grouped in two main branches. LY0701, including the DuCV T1/2002, T2/2002, T3/2002 from Taiwan of China and FJ0601 from mainland of China were in one branch. The identity was as high as 96.7%. WF0701, including the German DuCV, the USA DuCV, and the DuCV T4/2002 from Taiwan were another branch. The results of the study showed the variety of DuCV in China.3,Preliminary Pathologic Observations of Cherry Valley Duck Artificially Infected with Duck CircovirusThe whole genome of duck circovirus (DuCV)was obtained from Cherry Valley ducks from a diseased duck farm located in China's Shandong Province and named LY0701 strain, with its Genbank login no. EU022374. Artificially inoculate DuCV negative duckling at three weeks of age by using positive sample infected with LY0701strain, and conduct pathological observation separately after 2, 4, 6, 8 weeks. Compared with the control group, the clinical symptoms of the ducklings infected with positive LY0701 all showed stunted, thin, messy feathers, and so on, autopsy showed that there was bleeding and swell of different degrees in thymus, bursa, spleen and liver, other internal organs with unobvious lesions. Pathological section showed bleeding in thymus, bursa, spleen and liver, lymphocyte necrosis and/or collapse.4,Study on DuCV Cap Gene Expression Products and ImmunogenicityAccording to the published sequence of Cap gene of (DuCV) LY0701 strains, design a pair of primers for the each gene, amplify the corresponding gene fragment based on the plasmid gene of Cap of DuCV LY0701 strains of correct sequence as a template, then connect respectively taking pGEX-6P-1 as the carrier, transform coli strain BL21 and clone the positive ones, finally carry out prokaryotic expression when the sequence is correct and verified. The SDS-PAGE electrophoresis shows that the band of expression products meets the theoretical values in size. To immunize the BALB / c mouse respectively by using the prokaryotic expression products, and prepare the serum of anti-Cap, marked as Ab-Cap. Establish the indirect immunofluorescence staining assay (IFA) by using goat anti-mouse IgG-FITC as secondary antibody. The results of Ab-Cap assay for diseased samples of artificial infection show that the entire staining cells can be detected in frozen sections of spleen, bursa, thymus, and liver. But the Ab-Cap has not detected the typically staining cells in samples of heart, lung, and kidney.In order to further determine the phagocytosis of DuCV on organs and tissues, bursa, spleen, liver, and bone marrow of 96 suspected DuCV infected ducks were collected and examined using IFA, the results showed that the detection rate of spleen of DuCV infected ducks was the highest, up to 18.75%, followed by Bursa, thymus, liver.5,Study on Culture of DuCV In Different tissues and cells5 samples (LQ76,LQ66,LQ14,LY5,WS23)were inoculated by different ways: Duck embryonal yolk cavity, allantocherion, allantoic cavity, embryonal body, duck embryo fibroblast, hepatic cell, renal cells, spleen cell. Tissue and cell cultures were continued 10 generations by blind passage. All cultures were identified by PCR, electron microscope, IFA. The results showed that all the above-mentioned methods were not success.