Pathological Observation Of An Experimental Infection Of Geese With Goose Circovirus

Goose circovirus (GoCV) was a member of Circovirudae Circovirus. As other circoviruses, it often causes subclinical disease rather than clinical disease. However, circoviruses invade lymphoid tissue and lead to immunosuppression, growth retardation and an increased probability of secondary infection. To date, GoCV was studied mostly with natural infected cases. It is necessary to establish the animal model of GoCV experimental infection to study the pathogenicity.The BF tissue sample collected from a goose of natural case, which was positive for GoCV DNA confirmed by PCR, was homogenized and used as the inoculation for the experimental infection. Forty GoCV-free geese were randomly assigned into inoculated group (n=30) and uninoculated group (n=10). Each goose of the inoculated group received the inoculation, administered by the combination of oral, intranasal and intraocular routes. For control purpose, each goose of uninoculated group received GoCV-free physiological saline using similar procedures. The geese of the two groups were housed separately. The health status of all individual geese was recorded throughout the entire experimental period. Body weight was measured and blood samples were collected every week until euthanised. Blood samples were centrifuged and serum was frozen at-70℃until tested. Five inoculated geese were euthanised at 7,14,21,28,35 and 49 days post inoculation (DPI) respectively, while five uninoculated geese were euthanised at 28 DPI and 49 DPI. A complete necropsy was performed on all birds. Samples of BF, spleen, thymus, cecal tonsil, duodenum, jejunum, ileum, pancreas, brain, heart, lung, liver and kidney were collected and then detected by methods including PCR, histological examination, immunohistochemistry and apoptosis assay.The bursa of Fabricius in natural cases was confirmed for GoCV positive by PCR Forty GoCV-free geese were randomly devided into two groups, the infected group and the negative group. The geese in the infected group were infected with tissue homorage GoCV-positive bursa of Fabricius by oral, intranasal and intraocular routes.The killed group was used to study the pathogenicity of the virus was studies by the observation of clinical signs, PCR detection, histological examination, immunohistochemical detection and apoptosis method. The geese in the group were devided into positive group and negative group. Five geese in positive group were killed at 7,14,28,35, and 49 days after infection respectively, while five geese in negative group were killed at 28 and 49 days afer infection respectively.The clinical signs observed included that three geese showed feather disorders, almost all geese developed diarrhea between 17 and 30 days after infection, and there was difference growth rate among experimental geese. At necropsy, endocardium, epicardium and liver showed hemorrhage, thymus enlargement and hemorrhage, and individual bursa of Fabricius hemorrhage and splenomegaly. Histological examination showed lymphocytic depletion and histiocytosis in BF, inflammation and lymphatic infiltration in lung, liver and kidney.Regular PCR results showed that viruses were detected in serum only at 14 days after infection, and viruses were also detected in bursa of Fabricius, spleen, thymus, liver and intestine. However, the positive rate differed in different tissues and timepoits. Regular PCR positive samples were then detected by real-time PCR to determine the viral copies. Results showed that The viral copies were lower in serum (～103 copies/μl), higher in bursa of Fabricius (～105-6 copies/mg), moderate in other tissues (～103-5 copies/mg)。The distribution of GoCV antigen in tissues was detected in PCR positive samples by immunohistochemical method. Results showed only 3 bursa of Fabricius appeared positive signals (3/8), suspecting that it might be related to the lower sensitivity of the method established。Positive cells were observed in cortex and medulla region of bursa of Fabricius. GoCV positive signals mainly occurred in cytoplasm of infected cells, less in nuclei.Apoptosis was detected in bursa of Fabricius of all geese by TUNEL method. Resutls showed that apoptosis occurred in cells of cortex, medulla and epithelial cells. Geese in negative group showed lower apoptosis index and less intensity. The apoptosis index of geese in positive group increased from 14 days after infection, highest at 21 days after infection, and kept the level till the experiment end. There was a significant statistics difference in the apoptosis index of geese between infected group and negative group at 28 and 49 days after infection (p<0.05)。Thirty-seven GoCV-free geese were randomly assigned to infected immunized group, uninfected immunized group and uninfected nonimmunized group. There were 26 geese in infected immunized group, which were inoculated with virus suspension separated with GoCV positive bursa of Fabricius at 21 days of age. Among those geese,6 geese were immunized with H5N1 inactivated vaccine and Newcastle Disease virus (NDV) live vaccine, while six, seven and seven geese were immunized at 1,2 and 3 weeks after inoculation respectively. Five geese in uninfected immunized group were inoculated physiological saline, and at the same day immunized with vaccines. Geese in uninfected nonimmunized group were only inoculated physiological saline. The geese in infected group and uninfected group were housed separately. Serum were collected weekly to examine the corresponding antibody levels to study the effect of GoCV to vaccine immunization. Results showed that AIV and NDV vaccine immunization were both affected after GoCV infection, showing lower hemagglutinating titers and shorter crest time.