Development And Epidemiological Survey Of Duplex PCR For Duck Circovirus Genotype 1 And 2

Duck circovirus(Duck circovirus, DuCV) is a member of the genus circovirus(Circovirus)of the Circoviridae family(Circoviridae).It’s genome is a single strand covalently closed circular DNA, sized about 2kb. Duck circovirus predominantly infects the thymus, bursa and other immune organs. Ducks show feathering disorder, poor body condition and low body weight when infected by DuCV. In recent years, many kinds of detecting methods for duck circovirus has been developed, such as PCR methods, ELISA methods and iELISA methods.but so far there is no quick duck circovirus type detection method to identify DuCV-1 and DuCV-2 separately at the same time. In this study, we have established a duplex PCR method to detect separately two types of duck circovirus based on Cap protein gene sequence.This study consists of three parts as below:1. Development of the duplex PCR for duck circovirus genotype 1 and 2.DuCV can be classified into two genotypes according to Capsid protein sequence.Sequence analysis shows that the sequence similarity is about 78% and amino-acid sequence similarity is 77.6%. The classification results based on genome sequence analysis is almost the same as the result which is based on capsid protein sequence. Thus, we choose capsid protein sequence, on which we design two primers to classify DuCV into two types. These results showed this method of high specificity and sensitivity. The epidemiological survey results indicated that there are more serious DuCV-1 DuCV-2 mixed infection in duck flocks in Shandong province.2. Molecular epidemic survey of DuCV in Weifang, Shandong province.Tracking a DuCV positive farm in Weifang and we had collected 120 dead young ducks.We dissected the carcass and collected liver, thymus, spleen, bursa, kidney and heart. We applied the duplex PCR method to detect Du CV in the organs. Results showed that: 3(37.5%)flocks were identified as mixed infection of DuCV-1 and DuCV-2, and 3(37.5%) flocks were DuCV-2 single infection. Among them, 3.33%(4/120) of ducklings were detected as mixed infection of DuCV-1 and DuCV-2, and 9.17%(11/120) of ducklings were DuCV-1 andDuCV-2 single infection. These results indicated that there are more serious DuCV-1 DuCV-2mixed infection in duck flocks in Shandong province.3. Preliminary inquiry on vertical transmission of duck circovirus.Wang et al. had detected the 11 duck farms were DuCV-positive, and the positive ratios of the duck farms were 27.32% and 42.81% for DuCV-1 and DuCV-2, respectively. We had collected 30 duck embryos died in the final stage of incubation and 80 non-enbryonated eggs were detected in this experiment. Results show that4 of 30(13.33%) samples were found to be DuCV positive. As a result, one was positive for both DuCV-1 and DuCV-2 DNAs, one for DuCV-1 DNA, and two for DuCV-2 DNA. In the case of glair,3 of 80(3.75%) samples were found to be DuCV positive,one positive for both DuCV-1 and DuCV-2 DNAs, and two for DuCV-2 DNA. In the case of yolk, all samples were negative. It indicated that embryos can carry the viruses and is an indirect evidence of vertical transmissiom of duck circovirus.