Bacteriology Survey And Establishment Of Molecular Testing Method To Main Pathogenic Bacteria On Mattess Of Fermentation Bed

Since the promotion of the mattess of fermentation bed in the South area, people focus on the categorization and distribution of bacteria in the mattess of fermentation bed, the function of the fermentation bed and because of non-adapting of local climatic conditions of this raising model. Especially, whether the number and sorts of the bacteria in the mattress will be reduced by the fermentation technique have been involved people's attention. To estimate this model of the bio-ferment techniques in Sichuan district, the research conducted the type and the number of bacteria, the detection methods of the pathogens and molecular indetifying pathogen in the mattress.This experiment collected 45 samples of three types of litter pigs, weaned pigleits and finishers from traditional farm,15 samples from each type.And the other 45 samples collected from the corresponding mattess of fermentation bed. After isolating all bacteria will be identified by biochemical test,16S rDNA PCR and sequencing, and pathogenicity test. Also, to meet the need of clinical treatment, drug sensibility test would be conducted.Meanwhile, based on the heat-labile enterotoxin LT of ETEC and shiga toxin Stx2e of STEC of Escherichia coli, two pairs of detected primers were designed and diagnosis detection method of double PCR were established. The purpose of the double PCR was to detect whether there were the presence of the Escherichia coli of producing the heat-labile enterotoxin LT of ETEC and shiga toxin Stx2e of STEC. All the results were described as following:1. Microbial population analysis of bacteria showed that the bacteria types from the pigs and mattess of fermentation bed were almost the same, including mainly Escherichia coli, Staphylococcus, Streptococcus, Salmonella and Bacillus subtilis etc, but Bacillus only from the the mattess of fermentation bed. Analysis of significance of difference displayed that difference of the number of Escherichia coli, Staphylococcus, Streptococcus from the mattress and the pigs is not significant (P>0.05), while the number of Salmonella and Bacillus subtilis is significant in the mattress(P<0.05), especially the number of Bacillus (P<0.01). And the order of magnitude of each bacteria in litter pigs,weaned piglets and finishers were that (1)the number of E.coli were 1.6×106cfu/g,7.0×105cfu/g and 1.3×106cfu/g, respectively;(2)the number of Staphylococcus were 2×104cfu/g,2×105cfu/g and 2×105cfu/g, respectively; (3)the number of Streptococcus were 2×104cfu/g. Besides, Salmonella and Bacillus in mattess of fermentation bed have a content of 2×102cfu/g, which just content a little in air.In the mattress, the content of E.coli in litter pigs, weaned pigleits and finishers were 4×106cfu/g,1.6×106cfu/g and 2×106cfu/g of respectively. Analysis of significance of difference displayed that difference of the number of Escherichia coli is extreme significance(P<0.01) compared with that in fecal of tranditional farm. The content of Staphylococcus in the mattress keeps at 1.0×106cfu/g,2×107cfu/g in litter shed, and also extreme significance(P<0.01) than that in litter(P<0.01). The concentration of Streptococcus keeps at 4×104cfu/g, and it is prominent lower than that in the litter(P<0.01). And beneficial bacillus was not found in litter, but kept at 2×106cfu/g in the mattress.2. Pathogenicity experiments confirmed that E. coli and Salmonella from the manure and litter can kill all mice when injecting 0.5ml(5×108cfu/mL) of isolates culture broth, and death rates were 100%, respectively. The causative effect pathogenic Staphylococcus is similar to the corresponding pigpen. However, strains isolated from pig feces is 50% lethal of mice, and litter homes are 0%. Bacillus are non-pathogenic to Mice.3. Drug sensitivity tests show that all the bacteria have drug resistance to some extent.E. coli is sensitive to Cefepinme and Rocephin, Salmonella is sensitive to Amikacin, Enoxacin etc, the golden Staphylococcus is sensitive to Rocephin, Cefepime etc, and Streptococcus is extremely sensitive to Cotrim, sensitive to Rocephim, Cefepime etc, and moderately sensitive to Gentamicin and Amoxicillin.4. Having founded fast and precise PCR molecular detect method of 16S rDNA, with which we detected the main separate bacteria in the mattress. And we found that the homology of E.coli SC-E and E.coli ATCC43895 is 99.5%, Salmonella SC-S and Salmonella AF227869 is 98.4%, golden Staphylococcus SC-sa and golden Staphylococcus bacteria DQ997837 is 99.8%, Streptococcus SC-ss and swine Streptococcus AF009487 is 99.8%, and the mucolurulent bacillus SC-B and starch amylase is 99.2～99.7%.5. Establishment of the duplex PCR detection method based on two main pathogenic gene LT toxin gene and Stx2e of E. coli. The method is specific, sensitive, simple and fast etc, which can be used for clinical detection of pathogenic E. coli.15 isolated strains of E. coli were detected by the method of litter homes and piglet feces litter, feces were detected in two enterotoxigenic E. coli, litter homes were not detected in enterotoxigenic Escherichia coli, production toxin Stx2e of E. coli were not detected.