In this study, a SYBR Green Ⅰ real-time RT-PCR assay was developed for detection of new-type duck reovirus(NDRV) with the specific primers designed according to the conserved sequence of S3 genes of NDRV in GenBank. The results showed that recombinant plasmids were used to generate standard curve with correlation coefficient(R2) of 0.9996, and the melting curve showed one specific peak. No cross-reaction was detected for other duck viruses and the limit detection of real-time RT-PCR was about 29 copies/μL for the target gene, which was 100 times higher sensitivity than conventional PCR. The reproducibility tests in inter-assay and intra-assay indicated that the coefficients of variation were less than 2%. The method established in this study could be useful for earlier rapid laboratory diagnosis and quantitative analysis of the infection of NDRV.

从广东地区某养鸭场的患病雏番鸭群中分离到1株病毒,该病毒能凝集鸡红细胞,且这种特性可被ND标准阳性血清所抑制,但不能被减蛋综合征-76阳性血清以及禽流感H5、H7和H9阳性血清抑制.运用特异引物进行PCR扩增,能扩增出大约520bp长度的目的片段.毒力测定结果表明,MDT、ICPI、IVPI分别为56.0 h、1.64、2.29,符合新城疫病毒强毒株的毒力判断标准.

A method to detect porcine reproductive and respiratory syndrome virus (PRRSV) was developed using fluorescent RTPCR method. Based on the published PRRSV genomic sequence in GenBank,a pair of primers F,R and a TaqMan fluorescent probe P were designed. This method was more sensitive than conventional RT-PCR by 100-fold. It can detect cell culture material of PRRSV as little as 2 TCID 50 . With high specificity,it could well differentiate variant and classical PRRSV strains. Besides,it could relatively quantificate PRRSV RNA according to the established standard curve of sensitivity experiment.

Newcastle disease virus strain GD1002,isolated from the diseased ducks in Guangdong province in 2010,was velogenic according to the pathogenetic tests and highly pathogenicity to duck.The results of virulence determination showed that the MDT,ICPI and IVPI were 56.0h,1.64and 2.29,respectively,belonged to velogenic strain virulence criterion.The Fgene of GD1002strain was amplified by reverse transcription polymerase chain reaction(RT-PCR).The cleavage motif 112 RR-Q-R-R-F117of F protein was identical to the characteristic of velogenic strain.For genotyping,phylogenetic tree based on the nucleotide 47to 420nt of the Fgene were performed using the isolate and other NDV sequences obtained from GenBank.The results revealed that the isolate belonged to genotypeⅨ.And the results of nucleotide sequence homology and evolutionary processes analyses showed that the isolate had a close relationship with F48E9,JS/1/97/Ch and FJ/1/85/Ch strains.

为了研究清远麻鸡禽白血病的净化效果,试验选用某清远麻鸡祖代鸡场所有祖代鸡,于8周龄和20周龄时进行连续3个世代的泄殖腔拭子采样,使用BioChek公司禽白血病抗原检测试剂盒进行禽白血病抗原ELISA检测,及时淘汰阳性鸡,同时加强清远麻鸡祖代鸡群禽白血病净化的饲养管理和种蛋孵化过程管理,并比较3个世代禽白血病的净化效果.结果表明:该场清远麻鸡祖代鸡在第1世代(G1)阳性率较高,20周检测的阳性率为14.01％,其中公鸡的阳性率为19.04％,母鸡阳性率为8.16％；第2世代(G2)20周检测的阳性率为9.05％,其中公鸡的阳性率为15.52％,母鸡的阳性率为8.16％；第3世代(G3)20周检测的阳性率为4.15％,其中公鸡的阳性率为6.93％,母鸡的阳性率为3.40％.说明清远麻鸡祖代鸡群经过3个世代的净化取得显著效果.

In this study,GX-08 strain was selected to perform virus infection test on 26 d chickens and 20 d muscovy ducks at the dose of 105.5EID50.The morbility and mortality of chickens infected with viruses through intramuscular route were 100% and 100%,70% and 70% for muscovy ducks.The morbility and mortality of chicken infected with viruses through both oral and oculonasal routes were 80% and 30%,20% and 20% in muscovy ducks.Pathological changes of digestive tract and some respiratory tract of the infected chicken were severe,which was characterized as the infection of typical visceratonia ND.In comparison with slight pathological changes in digestive tract and some respiratory tract,obvious pathological change were observed in liver,spleen,pancreas etc.of muscovy ducks.It indicated that GX-08 strain had highly pathogenicity in ducks and chickens,causing systemic infections involving many organs and tissues in duck and chicken,and resulting in the invasion and death.In in vitro tests,cell lesions could be observed in both CEF monolayers and DEF monolayers inoculated with GX-08 strain at the dose of 200 TCID50,and the cells were destroyed completely at about 96 and 84 h respectively.In comparison with CEFs,the cytopathic effect on DEFs infected with virus developed more rapidly,the number of syncytial cells and its nuclei were more,and the HA titer in the culture supernatants was higher.The results indicated that GX-08 strain could cause cytopathic effects to CEFs and DEFs,and induced stronger syncytium formation in DEFs than CEFs.

15 Qingyuan MA chickens which appeared obviously clinical symptom and were tested to be avian leukosis positive were selected.The tumor organs,such as liver,spleen,kidney and so on,were carried on histopathology and PCR diagnosis.The pathology tissue slices from organs of liver,kidney,spleen,marrow,gland stomach,heart,lung,pancreatic gland ovary and so on,were observed,results showed that the proliferation of massive lymphoblasts or scatter and of small lymphomyelocytes were observed.The PCR identification indicated that all 15 samples can amplify a fragment about a length of 545 bp,which consisted with the expected size.The results suggested that all 15 isolates were the ALV-J avian Leukosis viruses.

In order to demonstrate the optimal time of detecting avian Leukosis in Qingyuan MA grandparent breeding stocks,500 samples were separately collected from the same Qingyuan MA chicken group at the age of 8 weeks,12 weeks,20 weeks and 26 weeks,using the BioChek kit and tested for infecting avian Leukosis virus or not.The results indicated that the positive rate of leukosis in the chicken group was 5.2%,9.4%,12.6% and 8.6% respectively,and the positive peak for assay was at 20 weeks old.It demonstrated that the optimal elimination time was 20 weeks old for detecting avian Leukosis virus.

Newcastle disease virus strain GX-08,isolated from the invasive immunized chicken flocks in Guangxi province in 2008,was velogenic according to the pathogenetic tests and highly pathogenicity to duck.According to the sequences of NDV strains published in GenBank,ten pairs of primers were designed and complete genome cDNA of the isolate was successfully amplified by PT-PCR.Sequence analysis showed that the genome length of GX-08 strain was 15192 nt which contained six ORFs,encoding 6 proteins of NP,P,M,F,HN and L,respectively.The cleavage motif 112R-R-Q-K-R-F117 of F protein was identical to the characteristic of velogenic strain.The evolution analysis of the 47 to 420 nt sequence including the cleavage site of F gene indicated that GX-08 strain belonged to genotype Ⅶ.

Using the genome RNA of NDV-SS strain of Newcastle disease virus (NDV) isolated from duck as a template,the F gene of NDV-SS strain was amplified by reverse transcription polymerase chain reaction (RT-PCR). Then its gene cloning was completed,and its nucleotide sequence was determined. Nucleotide sequence homology and evolutionary processes analyses on amino acids sequence of F gene between SS strain and other NDV reference strains published in GenBank was executed using the sequence analysis soft. The F gene sequence of SS strain showed that NDV-SS strain was determined with its full-length of 1662 bp coding 553 amino acids. Amino acids sequence of F protein proteolytic cleavage position was 112R-R-Q-K-R-F117,indicated it is the virulent NDV strain.And the results of nucleotide sequence homology and evolutionary processes analyses showed that this strain belonged to the gene type Ⅵ,and had a close relationship with IT-227 and Italy-2736 strain. The presumable result was that this strain evolved from the NDV of pigeon origin.