Detection Of Reticuloendotheliosis Virus And Establishment Of Animal Infective Model

REVs are a group of avian retroviruses that infect chicks, turkeys and ducks. REV causes an acute neoplastic syndrome characterized as lymphopoiesis and reticuloendotheliosis.The objection of this paper was to set up methods of detecting REV by polymerase chain reaction(PCR) and immunohistochemical technique(IHC). as well as to establish animal infective model of REV.REV itself can little make the poultry leathal. Horizontal spread of REV is caused by touching with ill animals. Verticaltransmission of REV is induced by infected egg embryo. Vaccine contaminated REV is an important reason that causes REV infection and prevalence in practice. Firstly, genomic DNA extracted from SPF chick embryo fibroblasts(CEFs) infected with REV-T and spleen necrosis virus(SNV) individually according to the routine test method. The primers were designed and synthesized in the long terminal repeats(LTR) sequence of REV provirus. Purified proviral genomic DNA as template were amplified with these pairs of primers by polymerase chain reaction(PCR). Nothing amplified from REV-T was detected on the agarose gel by electrophoresis. However, a clear band sized about 300bp was showed after amplification following to electrophoresis on the agarose gel. Simultaneously, DNA extracted from MDV, Adenovirus I and III, Rouse sarcoma virus(RSV) type A and B. as well as ILTV. There was no band showed by PCR, which expressed that amplification with these pairs of primers from LTR could come out specific electrophoresis band. On the other hand, the cells infected by REV were fixed with the mixture of acetone and 100% ethanol. Then to add anti-serum to REV and conjugate, followed to color with substrate DAB. Cells all could be detected to infect with REV-T and SNV.Secondly, 60 one-day-old SPFchicks were divided into 4 groups, three groups were administered with REV-T, SNV and REV-T(SNV) by subcutaneous injection individually. The fourth group was negative control. The chicks were fed in the negative pressure isolators and observed the clinical symptoms daily. All chicks from each group were bled every week. From the fourth week after inoculation, all chicks were weighed. Then 2 chicks were collected from the testing groups and negative control group individually.The chicks were dissected and examined RE pathological changes, simultaneously were plucked heart, lung, liver, spleen, fabricius bursa, thymus gland, glandular stomach and kidney in the biological safety cabinet. Parts of the whole tissues were extracted DNA to be amplified by PCR technique. The others were fixed into 10% neutral formalin solution. The tissue slices were made by routine tissue slice preparative method, which were not only stained with haematoxylin and eosin(HE), but also detected by aid of antibody to REV. The results showed that all the chicks of testing groups were less weighed than that of control group. The chicks infected REV appeared stunted symptom. The chicks showed extinctly clinical symptom, such as inspirited, poor appetite, slow growth, feather defects. Both the thymus gland and the fabricius bursa were severely atrophic in testing groups in post mortem. The heads and claws of some chicks showed cyanosis;The adenous stomach of some chicks were swell, a few came out erosion and haemorrhage in the inner surface of adenous stomach; Some chicks were heamorrhage in legs; There was obviously haemorrhage and grey-white necrosis in the heart; Livers were swell and scattered nubbles. The two chicks from the testing group administered both REV-T and SNV manifested distinct tumor on the surface and inside of livers. The band was not detected from the tissues infected with REV-T. But the specific bands were amplified from the tissues infected with SNV and REV-T(SNV) by PCR. The tumor tissues, configuration and distribution of tumor cells were observed by HE stain and immunohistochemical technique. Although the PCR method was rapid and specific, the band was not amplified in the LTR region of REV-T. Nevertheless, REV infection could be examined by immunohistochemical technique. In addition, the antibody to REV could not be detected by immune agar diffusion method, which clarified the chicks presented durative viraemia. Hence, the antibody detetion can not be regared as an index monitoring whether the chickens infected REV, especially for SPFchicks. The chicks should be considered to test pathogen. It is suggested that PCR technique be used to monitor SPF chicks as routine test. This paper was the first report that SNV provided the helper virus functions required for REV-T replication, co-infection 1-day-old chicks. The chicks were dispirited and the fether was fluffy. The body weigh was obviously lower. There were clear tumor foci formed by neoplastic cells observed in internal organs of the chick bodies by H.E. and IHC technique. Moreover, the specific bands were amplified by PCR.At last, the study is to set up a method of duplicate PCR for REV-MDV. Not only cells infected with MDV and REV were amplified, but also the clinical samples could be detected by duplicate PCR. Two chicks infected with REV and MDV simultaneously were found by duplicate PCR. The duplicate PCR can be used to identify REV and MDV.In this study, REV was detected by IHC and PCR combined with pathological diagnostic method. It is helpful to diagnosis tumor disease prevail in the poultry industry, as well as to test SPF chicks fed in our centre periodically. It is very usefully and effectively test way to purify breeding poultry. Rapidly diagnosis for immunosuppressive disease by specific methods is a practical means to prevent the neoplastic disease. REV animal infective model was established by inoculating REV-T, SNV and REV-T(SNV) into chicks individually. All of these above provided scientific reference for understanding the development of REV popular strain, and further materials for followed work of REV research, particularly for studying immune pathology and prevention of REV.