| Duck-origin Newcastle disease is an acute septic infectious disease that caused by duck-origin Newcastle disease virus. The disease is mainly characterized by diarrhea, neurological symptoms, focal glandular gastric bleeding or ulcers, intestinal mucosal bleeding and necrosis of the pancreas or the spleen. It is generally believed that NDV is disseminated by horizontal transmission via alimentary canal, respiratory tract and conjunctiva in direct or indirect ways. But the study our laboratory had done shows that duck-origin Newcastle disease virus exist vertical infection which had lead to the neurosis phenomenon such as strophocephaly and circling of ducklings. In order to verify this phenomenon, duck-origin Newcastle disease virus, stain SDFCH, which were isolated from ducklings'brain of neurosis phenomenon, was made use of to challeng 28-week-old Cherry Valley ducks which were without Newcastle disease infection, then animal model of vertical infection of duck-origin NDV was established. This study contains four parts:Part 1:Establishment and application of RT-PCR diagnostic method for duck-origin Newcastle diseaseThe fusion (F) gene fragment (727 bp) of Newcastle disease was amplified by RT-PCR diagnostic method using a pair of primers, which was designed according to F gene sequence of chicken Newcastle disease virus, followed by specificity test and sensitivity test. Then 20 suspected strains of duck paramyxovirus and 150 samples of infectious or diseased tissues of ducks paramyxovirus were detected by the RT-PCR method. The specificity test showed that the method could specially amplify the 727bp gene-specific fragment of duck paramyxovirus SDFCH strain, while the amplification results of duck plague virus, H9N2 subtype avian influenza virus, duck hepatitis virus were negative. Sensitivity test showed that the minimum detectable amount of cDNA concentration was 3 pg/μL.15 from the 20 isolated strains were paramyxovirus (positive) while 125 from the 150 tissue samples were positive. The established RT-PCR diagnostic method appeared to be fast, accurate, highly sensitive and specific for the purpose.Part 2:Establishment of the indirect immuno-fluorescence assay for Newcastle disease virusFirst of all, monoclonal antibody anti-fusion protein was used as 1st antibody, immunoglobulin G (IgG) labeled by fluorescein isothiocyanate (FITC) was used as 2nd antibody, at the same time, experimental condition, such as stationary liquid, stiky liquid, eluant, dilution and incubation time of antibody, mounting liquid were optimized, followed by a series of specificity tests, then the indirect fluorescence assay (IFA) was established. The results showed that histological section of NDV infected organs were IFA positive, including parts of fallopian tube, liver, kidney, lung, spleen, abdominal salivary gland, brain and heart, while the histological section of control group of healthy ducks were IFA negative. Meantime, histological section of other virus were IFA negative, duck plague virus, avain influenza virus, riemerella anatipestifer and duck hepatitis virus-I included, which showed that the established IFA method was highly specific for the purpose.Part 3:Detection of Newcastle disease virus in the reproductive system of ducks by artificial infectionArtificial infection experiment was done to 42 twenty-eight weeks old Cherry Valley ducks which were without Newcastle disease infection. The ducks were divided into three groups namely A, B and C. In group A, female ducks were challenged by duck-origin Newcastle disease virus, strain SDFCH, In group B female ducks were challenged by duck-origin Newcastle disease virus, strain SDFCH, group C was the normal control group. After being challenged for 4 days, two ducks were killed every other day. And ovarian tissue, follicles and fallopian tube were collected from female ducks while testis vas deferens and semen were collected from drakes. Then the virus was re-isolated and F gene was amplified and analyzed by established RT-PCR method to verify the existence of the Newcastle disease virus SDFCH in these organs. And the distribution of the virus in oviduct was detected with the indirect immunofluorescence assay that was established by anti-Newcastle disease virus F protein monoclonal antibody. The result showed that strain SDFCH of the Newcastle disease virus had been detected from tissues of reproductive system of the duck and drake with RT-PCR method. Positive signal had been detected in ciliated cells of the ciliated columnar epithelium of oviduct and secretory cells by IFA.Part 4:Animal experiment of Newcastle disease virus'vertical infectionArtificial infection test was done to 68 twenty-eight weeks old Cherry Valley ducks which were without Newcastle disease infection. After being challenged for 4 days, two ducks were killed every other day. And ovarian tissue, follicles, fallopian tube, eggs not having outputted and eggs disinfected promptly after outputting were collected from female ducks. Meconium, brain and lung were collected from ducklings. Testis vas deferens and semen were collected from drakes. Then the virus was re-isolated and F gene was amplified and analyzed by established RT-PCR method to verify the existence of the Newcastle disease virus SDFCH in these organs. And the indirect immuno-fluorescence assay (IFA) to locate antigen of Newcastle disease virus in the oviduct. Positive signal had been detected in ciliated cells of the ciliated columnar epithelium of oviduct and secretory cells by IFA. Strain SDFCH of the Newcastle disease virus had been detected from tissues of genital system of the duck and drake and 80% ducklings with RT-PCR method. The results suggest that some strains of duck-origin Newcastle disease virus could be found in oviduct epithelial cells of ducks and semen of drakes, and they might enter into the egg with protein secretion, yolk formation and sperm excretion to the occurrence of vertical infection. It might be one of the reasons of causing embryo death and outbreak of Newcastle disease in one day old ducklings, but the exact mechanism of the vertical transmission needs further research. |
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