Application Of Immunohistochemistry In The Differential Diagnosis Of Three Chicken Viral Tumor Diseases

These years, the incidence of Avian Leukosis, Marek's disease and Retiucloendotheliosis is becoming bigger and bigger as the expanding raising. It harmed the breeding.And epidemiological study discovered polyinfection was very popular in clinical, so it is difficult to differentiate this three diseases. Moreover, there's a lot of insufficiencies to the detection of viral diseases for the laboratory diagnosis. Immunohistochemistry is fast and high specificity which aviod the abusment of traditional ways. This study challenged 1 day SPF chickens using ALV-J strain HN0001, MDV strain GX0101 and REV strain SNV, and developed the immunohistochemistry way of ALV-J, MDV and REV. Also this study coinfected 1day SPF chicken with these three strains, and get the liver, spleen, kidney and brusa to do HE staining. We studyed the dynamic distribution of pathogeny in different organ cells use the immunohistochemistry way and observed the infection againse the same cell of these three viruses. Then this study also vestigated these three diseases'infection rate of tumor chickens in Beijing, Shandong and Henan province using viral isolation and immunohistochemistry and compared the merit and demerit of these three ways.1. Development of immunohistochemistry for ALV-J, MDV and REV1.1 Development of immunohistochemistry for ALV-J1 day SPF birds were challenged through peritoneal cavity use ALV-J HN0001, they began to death after challenged 4 weeks later and tested ALV-J infected the birds successfully use IFA. Then, through the optimize of the way of antigen repair, H2O2 digestion, dilution radio of first antibody and chromogenic time of DAB, we developed immunohistochemistry for ALV-J. The way of antigen repair use heated for 15-20 minutes in citrate damping fluid within 92-98 centigrade. H2O2 digested 20-30min at room temperature. Dilution radio of first antibody is, monoclonal antibody of ALV-J is 1:150, Through research the antigen repair time, H2O2 digest time, dilution radio of first antibody, chromogenic time, this study developed immunohistochemistry successfully. It can detected the positive staining in karyolemma and hyalomitome of liver and brusa, in the hyalomitome of spleen, in the plasmalemma of kidney. 1.2 Development of immunohistochemistry for MDV1 day SPF birds were challenged through peritoneal cavity use MDV GX0101,they began to death after challenged 5 weeks later and tested MDV infected the birds successfully use IFA. Then, through the optimize of the way of antigen repair, H2O2 digestion, dilution radio of first antibody and chromogenic time of DAB, we developed immunohistochemistry for MDV. Dilution radio of monoclonal antibody of MDV is 1:150, the others are the same with ALV-J. It can detected the positive staining in karyolemma and hyalomitome of liver, spleen and kidney, in nucelus and hyalomitome of brusa.1.3 Development of immunohistochemistry for REV1 day SPF birds were challenged through peritoneal cavity use REV SNV, They began to death after challenged 4 weeks later and tested REV infected the birds successfully use IFA. Then, through the optimize of the way of antigen repair, H2O2 digestion, dilution radio of first antibody and chromogenic time of DAB, we developed immunohistochemistry for REV. Dilution radio of monoclonal antibody of REV is 1:300, the others are the same with ALV-J. It can detected the positive staining in hyalomitome of liver, spleen and kidney and brusa.2. Dynamic Pathology observation of SPF birds coinfected with ALV-J, MDV and REVThis study coinfected 1 day SPF birds use HN0001,GX0101 and SNV strains keeped in our lab. Using the orgens of different weeks challenged birds, we did the HE staining to understand dynamic pathology observation of the challenged birds. And in the same time we location the virus in the different orgens, study the dynamic distribution of these three virus in histiocytes and observed if the virus can infect the same histiocytesWe killed 3 birds after challenged, take the liver, spleen, kidney and bursa make paraffin section and did HE staining, we observed the pathology changing.Liver:prophase(1-42 days):liver cell cords was not dicovered, metaphase(42-120 days),parts of liver cell cords discovered, advanced stage(120-180 days )the liver cell cords were all discovered. Nucelus were dissolved.kidney: prophase(1-42 days): renal cell atrophy, metaphase(42-120 days),parts of renal cell discovered, advanced stage(120-180 days )the cell cords were all discovered.In the same time, we discovered using IHC, REV can be detected in 7 days after challenged in brusa, MDV and REV can be detected after 14 days after challenged.Positive signal of ALV-J can be detected first in the liver, as aging accretting, the amount of positive cells and dyeing was not challged evidently in liver, the amount of positive cells and dyeing was increased evidently in brusa, the amount of positive cells was not challged evidently and the dyeing was deepen in kidney as the age increasing.Positive signal of REV can be detected first in the brusa, as aging accretting, the dyeing in follicle cell and stromal cells was lighed, the amount of positive cells and dyeing was not changed evidently in brusa, the amount of positive cells and the dyeing was deepen in kidney as the age increasing.Positive signal of MDV can be detected first in the brusa, as aging accretting, the dyeing in follicle cell and stromal cells was lighed, the amount of positive cells and dyeing was not changed evidently in brusa, the amount of positive cells and the dyeing was deepen in liver as the age increasing, and there's no positive cells in kidney. Using continuous tissue section obvered the three virus in the same visual fields, the three virus can infect the same histiocytes. This tell us three virus can symbiosis in the same histiocytes3. Epidemiology investigation for the infection of ALV-J, MDV and REV in some fields of ChinaIn this study, we investigated the infection of AVL-J, MDV and REV in 18 chicken farms of Beijing, Shandong and Henan through IHC and use IFA detection, compared the differences of these two ways. Sepration of virus need longer time, and some inferior virus can missed in the course of virus seperation. Dotblot and IHC need shorter time, dotblot use nucleic acid probes hybridizate templates DNA, so the specificity is poored than IHC. Then IHC is the better detection way. The results showed that ALV-J infection was the gravest diseases and the positive rate for ALV-J alone infection in Beijing was the highest. The positive rate for MDV alone infection was the lowest. It showed the ALV-J is capital in the chicken virus diseases. In mixed infection, ALV-J and REV co-infection was the most prevalence, then it was MDV and REV co-infection and the detection rate of MDV and ALV-J co-infection was the lowest.According to the micro-pathology chanllge and IHC detection, it was suggested that the tumor types induced by ALV-J were more and more complex and multiplicity. In this study, ALV-J was detected from the chickens with haemangioma and fibrosarcoma.