| The piglet diarrhea is one of the common diseases in pig production, which hindering the development of the pig industry. The cause of diarrhea is complex and diverse, such as non-communicable disease and infectious pathogen infection. The bacterial and viral infections are more common, and other kinds of pathogens are variety, complex, and in the high incidence. To understand the pathogen prevalence of the diarrhea in Beijing area,400diarrhea samples were collected from August2010to December2013were detected.We isolated132strains of bacteria from400diarrhea samples. These isolates were assigned to16species by biochemical identification and16SrRNA gene sequence analysis. Virus gene specific PCR detection results showed that, there were23sample were positive for porcine epidemic diarrhea virus (PEDV),6sample were positive for transmissible gastroenteritis virus (TGEV) and58for rotavirus (RV), accounted for5.75%(23/400),1.5%(6/400) and14.5%(58/400), respectively. Using the colloidal gold method adopted for the rotavirus antigen, RV infection positive rate is17.7%, the rates of incidence was more higher in December and January, the infection rate was40%,0-7day age piglets were the most susceptible to infection,that the rate was30.6%.One strain of rotavirus was isolated from the rotavirus antigen-positive samples by Marc-145cell culture. The cytopathic effect (CPE) could be observed. Using specific anti-rotavirus serum for indirect immune fluorescence staining, the positive signal could be found in the cytoplasm of infected cell. The typical rotavirus particles can be detected in the infected cells by electron microscopy of ultrathin sections. Amplification and sequencing of VP6and VP7genes showed that the isolate belonged to subtype G in group A rotavirus. Experiment infection of newborn reproduced the clinical symptoms and pathological histology similar to the field infections.Using the standard O-antigen specific antisera,42strains of E.coli could be classified as8serotypes:0101(18.8%)、08(11.0%)、O20(11.0%)、064(12.5%)、O45(4.6%)、0149(4.6%)、02(1.5%),089(1.5%). Twenty two isolates were non-typable.To further investigate the pathogenesis of the E.coli isolates, virulence genes were detected by PCR.Result showed the major virulence factor were sta, stx2e, astA and eaeA. Fifty percent of the bacteria carried both astA and eaeA, while more than30%of the strains carried both sta and stx2e. We selected two strains of E. coli which carried the most virulence genes to sequence and analysis the full-length genome. The genome of strain A and B was approximately5.5Mb and5.6Mb, containing5743and5829ORFs, respectively. The ORF sequences for GO and KEGG annotation revealed that A and B was quite different about membrane transport protein genes. The number of ORFs of strain A was241, B was342.Further analysis revealed that differences in two strains of the bacterial membrane protein gene was mainly reflected in the type IV combined transport systems (lncll type). In the IV-type transport system, B carries VirB3 protein-coding genes that the bacteria may be involved in the invasion of the host cell membrane cilia course, which was the difference between A strain.In summary, our results provided a more comprehensive understanding of the prevalence of causative agents for piglet diarrhea around Beijing. The isolation, identification, and biological characterization of the major pathogens will provide the scientific data for the prevention and control of diarrhea diseases in pig production, as well as the further development of diarrhea disease vaccine. |
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