Dynamic Distribution Of Virulent GPV In Vivo And Molecular Analysis On Structure Of The Intestinal Flora After Infected GPV

Goose Parvovirus,which also known as Gosling Plague Virus(Goose Parvovirus,GPV),is the causative agent of Gosling plague(GP),an acute,contagious,and fatal disease, variously known as Derzsy's disease.The virus was classified as an autonomous parvovirus belonging to the family parvovirdae and it was more closely related to the human parvovirus HPV,bovine parvovirus BPV,canine parvovirus CPV and porcine parvovirus PPV.While qualitative PCR was useful for laboratory diagnosis of GPV infection,it still had some problems,it had the processes of electrophoresis and dyeing which had the shortcomings such as time consuming and easy to contaminate,poor quantification and unsuitable for large-scale investigations.Moreover,the amount of virus in different tissues and cells,was very useful for exploring the nosogenesis,virus replication,host-virus interactions,tropism,and effective for screening anti-viral drug,it couldn't be determined by qualitative PCRIt should not be ignored that the occurrence and development and turnover of GP with the changes in the structure of intestinal flora of goose.The following results were obtained by using different research methods and from different perspectives on health and infected GPV1.In this study,7-day-old goslings were chosen as animal model of the virus disease to research the pathogens of GPV CHv strain.To provide the theoretical basis of prevention and treatment of the disease and understanding the relationgship between the distribution of virus in vivo and the clinical manifestations and the development of the disease,the TagMan MGB probes were the first time used for real-time fluorescence quantitative-PCR (FQ - PCR) to amplificate the VP3 gene of GPV whth different routes of infection of the virus.The results showed that:(1) Optimize the FQ-PCR method can be achieved in 2 h for the rapid quantitative detection of GPV,the minimum detection limit was 28 copies /μL,are ordinary PCR sensitivity of 1000 times.(2) 4 h after oral administration of the esophagus can be detected gosling CH_V strain,4 h after intranasal infection of the trachea gosling CHV strain could be detected;injection 4 h after infection of the intestine in addition to Gosling,esophagus,trachea,brain,lung,other organizations can detect CHV strain;12 h after the different organs and tissue can be detected CHV strain,2 - 9 days gradually reached a peak,followed by gradually reduced;viral load Most of the liver, spleen,brain,Harderian gland and other organs.It is suggested that it should be based on different time of infection with different organizations which containning more virus when prepare or detect the antigen,it should be preferred with liver,spleen,brain,Harderian gland when do not know the exact time of infection.2.To reveal the overall regularity of the structure of intestinal flora in the gastro-intestinal virus and to analyze the dynamic of intestinal flora by the variation infection ways with GPV,DGGE technology was used based on the success of the quantitative detection of GPV.The results showed that:the structure of intestinal microflora was relatively stable in healthy control goslings,it is at most of the number of PCR-DGGE band with cecum, followed with the rectum,jejunum and ileum,and it is at least in duodenum.PCR-DGGE bands of hypodermically inoculated goslings gradually decreased and then gradually increased to normal with the extension of time after inoculation.PCR-DGGE bands of orally inoculated goslings decreased after inoculation and then gradually increased to normal.The changes of microbial community of nasally infected goslings were the same as other two inoculated way only delay the time.It was the least in dead goslings.The maladjusted degree of intestinal microbial community was correlative with the infected routes of GPV. 3.The 16S rDNA library of goose fecal bacterial was set up based on successful analysis the overall situation of intestinal flora.Five major bands from 16S rDNA library of gosling feces were sequenced.It was revealed that four major bands which has more than 96% homology with Bacillus,Lactobacillus,Enterococcus members and coliform respectively by BLAST analysis,the dominant microorganisms in intestinal flora was increased with Salmonella after infected GPV and had fundamental changed.4.ERIC-PCR is one of the molecular biology techniques on study the change of structure and flora species in recent years.It has characteristic such as fast and simple and not rely on pure culture with the change of structure and the species of intestinal microflora in the sample by analysis directly from the information such as distribution and quantity and brightness of ERIC strip.Compared with other methods,it has the advantege such as sensitivity,better repeatability and reliability,and saving time and labor.ERIC-PCR was used to reveal the overall variation of the structure of intestinal bacteria flora in the gastro-intestinal virus with analysis of intestinal bacteria flora dynamic structures by different ways of GPV infection.With the similar to DGGE results:it is relatively stable of bacillus flora of clinical healthy controls goslings,The band numbers formed by ERIC-PCR of duodenum were the least and were the most of cecal,followed by the rectum, jejunum and ileum.ERIC-PCR bands of hypodermically inoculated goslings gradually decreased and then gradually increased to normal with the extension of time after inoculation.ERIC-PCR bands of orally inoculated goslings decreased after inoculation and then gradually increased to normal.The changes of microbial community of nasally infected goslings were the same as other two inoculated way only delay the time.It was the least in dead goslings.The maladjusted degree of intestinal microbial community was correlative with the infected routes of GPV and the study plays an important role to elucidate the pathogenesis of GPV infection.5.There is no systematic research data on the dynamics distribution and changes of normal flora in vivo of healthy goslings and infected with GPV.It is very necessary to set up the detection method which with the advantage of fast,accurate and quantitative detection to some normal flora for the adjuvant treatment of GP.ERIC-PCR or DGGE-PCR was a good fingerprint technique to analysis the structure of the flora but with the accurate number of bacteria.In view of this,Taqman-MGB probe real-time fluorescence quantitative PCR(FQ-PCR) was adopted for detect E.coli,Bifidobacterium genus Bacillus and Lactobacillus in normal gosling flora infected with GPV.The results indicated that the four kind of bacteria of all infected goslings with GPV were varying degrees of changing. The chronologicaol order of Escherichia coli's quantity crest value is hypodermically inoculated goslings＞orally inoculated goslings＞nasally inoculated goslings.The quantity of Lactobacillus is nasally inoculated goslings＞orally inoculated goslings＞hypodermically inoculated goslings.The dynamic changes of the other two kind of bacterium are also hypodermically inoculated goslings＞orally inoculated goslings＞nasally inoculated goslings.So the maladjusted degree of microbial community in respiratory tract was correlative with the infected routes.This study will provide valuable insight into fully understand the pathogenesis of GPV infection.