The Detection Of Related Toxins Of Proteus Mirabilis From Chicken And The Establishment Of The PCR Detection Methods

Proteus mirabilis disease is newly discovered in recent years. Features of the disease are difficulty breathing, cough, high temperature, and diarrhea. Sick chicken are mainly performance for bacteremia, sepsis, one side or both side paralysis or watery diarrhea. Chickens can be infected in any ages, and less than 7 weeks is the most susceptible time. It also can be passed to generation through the eggs, the result of which is a large number of chickens deadly. So, Proteus mirabilis is a potential threat to farming industry.The pathogenesis of Proteus mirabilis is related to its virulence factors. Presumably, these factors may play an important role in pathology and immunity. In order to solve problems mentioned above, the study select pure endotoxin, enterotoxin, tracheal cytotoxin of Proteus mirabilis, preliminary understand the pathogenesis of Proteus mirabilis, and provide important basis for researching protective antigen of Proteus mirabilis. At present, the detection of Proteus mirabilis usually adopts the traditional bacteria separation cultivation, biochemical analysis and Serologic test, et al. Its long cycle, cockamamie operation, low sensitivity does not meet the fast detection of proteus mirabilis. In recent years, the methods of molecular biology overcome the deficiency of traditional testing method. Therefore, this study built proteus mirabilis PCR and multiple PCR detection method, provide technical support for the rapid detection, and better control the occurrence and spread of this disease.The research divides three parts:Part One: Extraction and detection of biological characteristics of Proteus mirabilis related toxinsPure endotoxin and heat-stable enterotoxin of Proteus mirabilis were prepared to study pathogenicity by animal test At the same time, chicken embryo tracheal ring model was established to verify the existence of tracheal cytotoxin and its pathogenic effects on tracheal cilia epithelium. The results showed that: endotoxin of Proteus mirabilis can effectively lead to acute septicemia lesions of animals. One of eight strains Proteus mirabilis produces ST, the little mousse injected ST are sluggishness, in appetence, cutting inspection can see bowel oedema, fluctuate effusion, etc. When the strains (Q3, Q5) Proteus mirabilis were diluted 10-8.5 and 10-8.3 times , abscission and injury of 50% of chicken embryo tracheal rings cilia could be caused by inoculating 0.1mL Proteus mirabilis. This result preliminarily verified the existence of tracheal cytotoxin and revealed pathogenic mechanism of Proteus mirabilis on tracheal cilia. The biological characteristics of these toxins played an important role for studying Proteus mirabilis vaccines.Part Two: Sequence analysis of 23S rRNA genes of Proteus mirabilisEstablish the sequence analysis of 23S rRNA of Proteus mirabilis for the identification of Proteus mirabilis strains. The fragments (1045bp) of 23S rRNA genes of seven chicken Proteus mirabilis strains and one rabbit Proteus mirabilis strains were amplified by PCR and were sequenced. The sequences obtained in this paper were compared with the Proteus mirabilis 23S rRNA sequence reported in GenBank and other similar generas by the software of DNAStar analyses. Construct the Phylogenetic tree of Proteus mirabilis strains. Results showed the homologies between seven chicken Proteus mirabilis strains and the reported one strain in GenBank were 99.4%-99.8%, and were 98.8%-99.3% identical to the rabbit Proteus mirabilis strains, were 95.4%-96.2% identical to Proteus vulgaris, were 92.9%-93.4% identical to other similar generas. The result shown that 23S rRNA sequence analyses may be a reliable and rapid way for identification of Proteus mirabilis.Part Three:Establishment and application of multiple PCR for diagnosing Proteus mirabilis,Salmonella and Listeria monocytogenesBased on the gene sequences of positive adjustment factor R gene of Urease (ureR) in Proteus mirabilis, conservative invasive antigen gene (invA) in Salmonella and the hly gene in Listeria monoytogenes, a three pairs of primer were designed. The specificity and sensitivity of PCR were analyzed for single gene. Multiple PCR method has been developed after analysis and optimization reaction condition by orthogonal experimental design L16 (43). The result shows, under the optimized conditions, the anticipated PCR of Proteus mirabilis, Salmonella and Listeria monocytogenes were specificity highly, and the minimum detection limit was 105 cfu/ml, which are low a dilute degrees in the sensitivity of single PCR. The results were stable in simulated examination and in marketing examination. A rapid, specific and sensitive multiplex PCR system has been established and it is valuable for diagnosis and monitoring for Proteus mirabilis, Salmonella and Listeria monocytogenes.