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Avian Influenza virus-H9N2 (AIV), Newcastle disease virus (NDV), Infectious bronchitis virus (IBV), Avian Pneumovirus viruses (aMPV), Escherichia coli (E. coli ), Ornithobacterium rhinotracheale (ORT) and Mycoplasma gallisepticum,(M.G) is a common organism that can cause severe respiratory symptoms of pathogens. These respiratory pathogens are of major importance because they can cause disease independently, in association with each other, or in associ-ation with bacterial or viral agents. But when two or more these pathogens infect avian, whatever at the same time or one after another, both can cause collaborative pathogenic.And the result is more serious than single pathogen. In recent years,there are more and more reports about the co-infection of espiratory pathogen .The multiple infection of respiratory pathogen are already very commen in avian,especially AIV H9N2、NDV、IBV、aMPV、MG、ORT.And this is one of the most important reasons why it is so hard to the preventive treatment of respiratory pathogen at present.In order to establish a rapid and accurate diagnostic method of co-infection of respiratory pathogens, we labeled the template DNA, which is gene fragment of AIV H9N2, NDV, IBV, aMPV, MG, ORT and E. coli. The sensitivity experimental results show that the labeled probe showed good specificity and the specificity experimental results show that the labeled probe can be detected in the lowest 1pg specific nucleic acid.In order to investigate the infections of respiratory diseases in different flocks, we did the following research. In this test, the simples of respiratory diseases were detected by specifically nucleic acid probes. The simples were isolated by ?ocks of nature disease and healthy broilers and dead chick-embryo from Dongying, Rizhao, Weifang and Liaocheng of Shandong province. Then dot blot hybridization was used to detect respiratory pathogens, as for provide epidemiological data to the prevention and control of respiratory diseases.The 421 lung samples of broilers with respiratory symptoms and 160 lung samples of clinically healthy broilers are collected from broiler farm at Jinan, Weifang, and Qingdao in Shandong Province, and then dot blot hybridization was used to detect Avian Influenza virus(AIV), Newcastle disease virus(NDV) , Infectious bronchitis virus(IBV) , Avian Pneumovirus viruses(aMPV) , Escherichia coli ( E.coli), Ornithobacterium rhinotracheale( ORT), and Mycoplasma gallisepticum(M.G). The results show that the detection rates of the seven pathogens of the 421 lung samples of broilers with respiratory symptoms are 53.68%(H9N2),68.41%(NDV), 33.49%(IBV), 54.87%(aMPV), 57.25% (MG), 9.02%(ORT), 49.17%(E. coli) and the co-infection is very serious. The ratio of co-infection is up to 90.26% and single infection and negative is 7.36% and 2.38% respectively. The detection rate of dual infection, triple infection, quadruple infection , quintuple infection, sextuple infection and sevenfold infection is19.48%, 26.12%, 27.32%, 11.88%, 4.51%, and 0.95% respectively, and among which the detection rates of the dual infection, triple infection, quadruple infection are higher than others. The results show that the detection rates of the seven pathogens of 160 lung samples of clinically healthy broilers are 12.50% (H9N2), 23.13% (NDV), 5.63% (IBV), 36.87% (aMPV), 21.88% (MG), 0%(ORT), 16.88% (E. coli) and single infection and negative is 25.00%and 43.75% respectively, the ratio of co-infection is only to 31.25%. The co-infection of the respiratory pathogen of broiler has already been universal , but the co-infection is more serious in broilers with respiratory symptoms. It is one of the most important epidemiologic factors of the rising incidence in respiratory disease.The liver samples of 297 dead chick-embryo were collected from ?ocks with respiratory symptoms of broiler breeder at Jinan, Weifang, and Qingdao in Shandong Province, and then dot blot hybridization was used to detect Avian Influenza virus(AIV), Newcastle disease virus(NDV) , Infectious bronchitis virus(IBV) , Avian Pneumovirus viruses(aMPV) , Escherichia coli ( E.coli), Ornithobacterium rhinotracheale( ORT), and Mycoplasma gallisepticum(M.G). The results show the samples of broilers with respiratory symptoms are 33.33% (H9N2), 44.44% (NDV), 26.11% (IBV), 35.56% (aMPV), 48.33% (MG), 0% (ORT), 52.22% (E. coli) and the co-infection is very serious. The ratio of co-infection is up to 77.72% and single infection and negative is 2.59% and 19.69% respectively. The detection rate of dual infection, triple infection, quadruple infection , quintuple infection, sextuple infection and sevenfold infection is 30.56%, 31.61%, 11.40%, 4.15%, 0%and 0% respectively, and among which the detection rates of the dual infection, triple infection, are higher than others. The co-infection of the dead chick-embryo has already been universal. It is one of the most important epidemiologic factors of the rising incidence in the of dead chick-embryo.A strain virus isolated from the ill chicken in Shandong province was identified as New castle disease virus by HA , HI and neutralization of virus. The virus was named LVCX strain. The MDT, ICPI and IVPI for LVCX strain were 47.4 h, 1.975 and 2.85, respectively. ELD50 of chicken embryos is 10-9.8. Amino acid sequence analyzing showed that the isolate was gene typeⅦ. The FPCS of LVCX strain between positions 112 and 119 were 112-RRQKRF-117. The results indicated that LVCX isolate was virulent strain. The nucleotide homology of F gene is 93.5%-98.3% between LVCX strain and those of gene typeⅦviruses isolated from chicken. And the nucleotide homology of F gene is 95.2%-96.5% between LVCX strain and those of ND viruses isolated from ducks and gooses. The nucleotide homologies of F gene are respectively 85.8% and 83.3% between LVCX strain and F48E9 and Lasota strain. And the amino acid homologies of F gene are respectively 91.3% and 88.4% between LVCX strain and F48E9 and Lasota strain.
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